Yeasts

Mol Cell Biol, 1999 Feb, 19(2), 1325 - 33
Antagonistic interactions between yeast chaperones Hsp104 and Hsp70 in prion curing; Newnam GP et al.; The maintenance of {PSI}, a prion-like form of the yeast release factor Sup35, requires a specific concentration of the chaperone protein Hsp104: either deletion or overexpression of Hsp104 will cure cells of {PSI} . A major puzzle of these studies was that overexpression of Hsp104 alone, from a heterologous promoter, cures cells of {PSI} very efficiently, yet the natural induction of Hsp104 with heat shock, stationary-phase growth, or sporulation does not . These observations pointed to a mechanism for protecting the genetic information carried by the {PSI} element from vicissitudes of the environment . Here, we show that simultaneous overexpression of Ssa1, a protein of the Hsp70 family, protects {PSI} from curing by overexpression of Hsp104 . Ssa1 protein belongs to the Ssa subfamily, members of which are normally induced with Hsp104 during heat shock, stationary-phase growth, and sporulation . At the molecular level, excess Ssa1 prevents a shift of Sup35 protein from the insoluble (prion) to the soluble (cellular) state in the presence of excess Hsp104 . Overexpression of Ssa1 also increases nonsense suppression by {PSI} when Hsp104 is expressed at its normal level . In contrast, hsp104 deletion strains lose {PSI} even in the presence of overproduced Ssa1 . Overproduction of the unrelated chaperone protein Hsp82 (Hsp90) neither cured {PSI} nor antagonized the {PSI}-curing effect of overproduced Hsp104 . Our results suggest it is the interplay between Hsp104 and Hsp70 that allows the maintenance of {PSI} under natural growth conditions.

Mol Cell Biol, 1999 Feb, 19(2), 1271 - 8
Identification of a new sea urchin ets protein, SpEts4, by yeast one-hybrid screening with the hatching enzyme promoter; Wei Z et al.; We report the use of a yeast one-hybrid system to isolate a transcriptional regulator of the sea urchin embryo hatching enzyme gene, SpHE . This gene is asymmetrically expressed along the animal-vegetal axis of sea urchin embryos under the cell-autonomous control of maternal regulatory activities and therefore provides an excellent entry point for understanding the mechanism that establishes animal-vegetal developmental polarity . To search for transcriptional regulators, we used a fragment of the SpHE promoter containing several individual elements instead of the conventional bait that contains a multimerized cis element . This screen yielded a number of positive clones that encode a new member of the Ets family, named SpEts4 . This protein contains transcriptional activation activity, since expression of reporter genes in yeast does not depend on the presence of the yeast GAL4 activation domain . Sequences in the N-terminal region of SpEts4 mediate the activation activity, as shown by deletion or domain-swapping experiments . The newly identified DNA binding protein binds with a high degree of specificity to a SpHE promoter Ets element and forms a complex with a mobility identical to that obtained with 9-h sea urchin embryo nuclear extracts . SpEts4 positively regulates SpHE transcription, since mutation of the SpEts4 site in SpHE promoter transgenes reduces promoter activity in vivo while SpEts4 mRNA coinjection increases its output . As expected for a positive SpHE transcriptional regulator, the timing of SpEts4 gene expression precedes the transient expression of SpHE in the very early sea urchin blastula.

Mol Cell Biol, 1999 Feb, 19(2), 1226 - 41
Cyclin B-cdk1 kinase stimulates ORC- and Cdc6-independent steps of semiconservative plasmid replication in yeast nuclear extracts; Duncker BP et al.; Nuclear extracts from Saccharomyces cerevisiae cells synchronized in S phase support the semiconservative replication of supercoiled plasmids in vitro . We examined the dependence of this reaction on the prereplicative complex that assembles at yeast origins and on S-phase kinases that trigger initiation in vivo . We found that replication in nuclear extracts initiates independently of the origin recognition complex (ORC), Cdc6p, and an autonomously replicating sequence (ARS) consensus . Nonetheless, quantitative density gradient analysis showed that S- and M-phase nuclear extracts consistently promote semiconservative DNA replication more efficiently than G1-phase extracts . The observed semiconservative replication is compromised in S-phase nuclear extracts deficient for the Cdk1 kinase (Cdc28p) but not in extracts deficient for the Cdc7p kinase . In a cdc4-1 G1-phase extract, which accumulates high levels of the specific Clb-Cdk1 inhibitor p40(SIC1), very low levels of semiconservative DNA replication were detected . Recombinant Clb5-Cdc28 restores replication in a cdc28-4 S-phase extract yet fails to do so in the cdc4-1 G1-phase extract . In contrast, the addition of recombinant Xenopus CycB-Cdc2, which is not sensitive to inhibition by p40(SIC1), restores efficient replication to both extracts . Our results suggest that in addition to its well-characterized role in regulating the origin-specific prereplication complex, the Clb-Cdk1 complex modulates the efficiency of the replication machinery itself.

Mol Cell Biol, 1999 Feb, 19(2), 1144 - 58
Seven novel methylation guide small nucleolar RNAs are processed from a common polycistronic transcript by Rat1p and RNase III in yeast; Qu LH et al.; Through a computer search of the genome of the yeast Saccharomyces cerevisiae, the coding sequences of seven different box C/D antisense small nucleolar RNAs (snoRNAs) with the structural hallmarks of guides for rRNA ribose methylation have been detected clustered over a 1.4-kb tract in an inter-open reading frame region of chromosome XIII . The corresponding snoRNAs have been positively identified in yeast cells . Disruption of the nonessential snoRNA gene cluster specifically suppressed the seven cognate rRNA ribose methylations but did not result in any growth delay under the conditions of yeast culture tested . The seven snoRNAs are processed from a common polycistronic transcript synthesized from an independent promoter, similar to some plant snoRNAs but in marked contrast with their vertebrate functional homologues processed from pre-mRNA introns containing a single snoRNA . Processing of the polycistronic precursor requires nucleases also involved in rRNA processing, i.e., Rnt1p and Rat1p . After disruption of the RNT1 gene, the yeast ortholog of bacterial RNase III, production of the seven mature snoRNAs was abolished, while the polycistronic snoRNA precursor accumulated . In cells lacking functional Rat1p, an exonuclease involved in the processing of both pre-rRNA and intron-encoded snoRNAs, several processing intermediates of the polycistronic precursor accumulated . This allowed for the mapping in the precursor of the presumptive Rnt1p endonucleolytic cuts which provide entry sites for subsequent exonucleolytic trimming of the pre-snoRNAs . In line with known properties of double-stranded RNA-specific RNase III, pairs of Rnt1p cuts map next to each other on opposite strands of long double-helical stems in the secondary structure predicted for the polycistronic snoRNA precursor.

Mol Cell Biol, 1999 Feb, 19(2), 1136 - 43
Role of a complex containing Rad17, Mec3, and Ddc1 in the yeast DNA damage checkpoint pathway; Kondo T et al.; Genetic analysis has suggested that RAD17, RAD24, MEC3, and DDC1 play similar roles in the DNA damage checkpoint control in budding yeast . These genes are required for DNA damage-induced Rad53 phosphorylation and considered to function upstream of RAD53 in the DNA damage checkpoint pathway . Here we identify Mec3 as a protein that associates with Rad17 in a two-hybrid screen and demonstrate that Rad17 and Mec3 interact physically in vivo . The amino terminus of Rad17 is required for its interaction with Mec3, and the protein encoded by the rad17-1 allele, containing a missense mutation at the amino terminus, is defective for its interaction with Mec3 in vivo . Ddc1 interacts physically and cosediments with both Rad17 and Mec3, indicating that these three proteins form a complex . On the other hand, Rad24 is not found to associate with Rad17, Mec3, and Ddc1 . DDC1 overexpression can partially suppress the phenotypes of the rad24Delta mutation: sensitivity to DNA damage, defect in the DNA damage checkpoint and decrease in DNA damage-induced phosphorylation of Rad53 . Taken together, our results suggest that Rad17, Mec3, and Ddc1 form a complex which functions downstream of Rad24 in the DNA damage checkpoint pathway.

Mol Cell Biol, 1999 Feb, 19(2), 1038 - 48
Fission yeast cdc24 is a replication factor C- and proliferating cell nuclear antigen-interacting factor essential for S-phase completion; Tanaka H et al.; At the nonpermissive temperature the fission yeast cdc24-M38 mutant arrests in the cell cycle with incomplete DNA replication as indicated by pulsed-field gel electrophoresis . The cdc24(+) gene encodes a 501-amino-acid protein with no significant homology to any known proteins . The temperature-sensitive cdc24 mutant is effectively rescued by pcn1(+), rfc1(+) (a fission yeast homologue of RFC1), and hhp1(+), which encode the proliferating cell nuclear antigen (PCNA), the large subunit of replication factor C (RFC), and a casein kinase I involved in DNA damage repair, respectively . The Cdc24 protein binds PCNA and RFC1 in vivo, and the domains essential for Cdc24 function and for RFC1 and PCNA binding colocalize in the N-terminal two-thirds of the molecule . In addition, cdc24(+) genetically interacts with the gene encoding the catalytic subunit of DNA polymerase epsilon, which is stimulated by PCNA and RFC, and with those encoding the fission yeast counterparts of Mcm2, Mcm4, and Mcm10 . These results indicate that Cdc24 is an RFC- and PCNA-interacting factor required for DNA replication and might serve as a target for regulation.

Mol Cell Biol, 1999 Feb, 19(2), 979 - 88
Activator-specific requirement of yeast mediator proteins for RNA polymerase II transcriptional activation; Han SJ et al.; The multisubunit Mediator complex of Saccharomyces cerevisiae is required for most RNA polymerase II (Pol II) transcription . The Mediator complex is composed of two subcomplexes, the Rgr1 and Srb4 subcomplexes, which appear to function in the reception of activator signals and the subsequent modulation of Pol II activity, respectively . In order to determine the precise composition of the Mediator complex and to explore the specific role of each Mediator protein, our goal was to identify all of the Mediator components . To this end, we cloned three previously unidentified Mediator subunits, Med9/Cse2, Med10/Nut2, and Med11, and isolated mutant forms of each of them to analyze their transcriptional defects . Differential display and Northern analyses of mRNAs from wild-type and Mediator mutant cells demonstrated an activator-specific requirement for each Mediator subunit . Med9/Cse2 and Med10/Nut2 were required, respectively, for Bas1/Bas2- and Gcn4-mediated transcription of amino acid biosynthetic genes . Gal11 was required for Gal4- and Rap1-mediated transcriptional activation . Med11 was also required specifically for MFalpha1 transcription . On the other hand, Med6 was required for all of these transcriptional activation processes . These results suggest that distinct Mediator proteins in the Rgr1 subcomplex are required for activator-specific transcriptional activation and that the activation signals mediated by these Mediator proteins converge on Med6 (or the Srb4 subcomplex) to modulate Pol II activity.

J Biol Chem, 1999 Jan 22, 274(4), 2539 - 48
Domain organization and functional properties of yeast transcription factor IIIA species with different zinc stoichiometries; Pizzi S et al.; Transcription factor IIIA (TFIIIA) binds to the 5 S rRNA gene through its zinc finger domain and directs the assembly of a multiprotein complex that promotes transcription initiation by RNA polymerase III . Limited proteolysis of TFIIIA forms with different zinc stoichiometries, in combination with DNA binding and in vitro transcription analyses, have been used herein to investigate the domain organization and zinc requirements of Saccharomyces cerevisiae TFIIIA . Species containing either nine, six, or three zinc equivalents were produced by reductive resaturation and controlled metal depletion of recombinant TFIIIA . Partial digestion of the metal-saturated, 9 Zn2+-liganded factor yields a stable intermediate comprising the eight N-terminal zinc fingers, and a less stable fragment corresponding to a C-terminal portion including the ninth finger . Proteolyzed TFIIIA has the same 5 S DNA binding ability of the intact protein yet no longer supports in vitro 5 S rRNA synthesis . Both the structural compactness and the 5 S DNA binding ability of the TFIIIA form only containing 3 zinc ions are severely compromised . In contrast, the 6 Zn2+-liganded species was found to be indistinguishable from metal-saturated TFIIIA . By demonstrating the existence of three classes of zinc-binding sites contributing differently to yeast TFIIIA structure and function, the present study provides new evidence for the remarkable flexibility built into this complex transcription factor.

J Biol Chem, 1999 Jan 22, 274(4), 1949 - 56
The CDK-activating kinase (Cak1p) from budding yeast has an unusual ATP-binding pocket; Enke DA et al.; Cak1p is an essential protein kinase that phosphorylates and thereby activates the major cyclin-dependent kinase in budding yeast, Cdc28p . The sequence of Cak1p differs from other members of the protein kinase superfamily in several conserved regions . Cak1p lacks the highly conserved glycine loop motif (GXGXXG) that is found in the nucleotide binding fold of virtually all protein kinases and also lacks a number of conserved amino acids found at sites throughout the protein kinase core sequence . We have used kinetic and mutagenic analyses to investigate whether these sequence differences affect the nucleotide-binding properties of Cak1p . Although Cak1p differs dramatically from other protein kinases, it binds ATP with a reasonable affinity, with a KM of 4.8 microM . Mutations of the putative invariant lysine in Cak1p (Lys-31), homologous to a residue required for activity in virtually all protein kinases and that interacts with the ATP phosphates, moderately reduced the ability of Cak1p to bind ATP but did not dramatically affect the catalytic rate of the kinase . Similarly, Cak1p is insensitive to the ATP analog 5'-fluorosulfonylbenzoyladenosine, which inhibits most protein kinases through covalent modification of the invariant lysine . We found that Cak1p is tolerant of mutations within its glycine loop region . Remarkably, Cak1p remains functional even following truncation of its first 31 amino acids, including the glycine loop region and the invariant lysine . We conclude that the Cak1p nucleotide-binding pocket differs significantly from those of most other protein kinases and therefore might provide a specific target for an inhibitory drug.

J Biol Chem, 1999 Jan 22, 274(4), 1928 - 33
Heterochromatin organization of a natural yeast telomere . Recruitment of Sir3p through interaction with histone H4 N terminus is required for the establishment of repressive structures; Venditti S et al.; The chromatin organization of eukaryotic telomeres is essential for telomeric function and is currently receiving great attention . In yeast, the structural organization of telomeres involves a complex interplay of telomeric proteins that results in the formation of heterochromatin . This telomeric heterochromatin involves homotypic and heterotypic protein interactions that have been summarized in a general model . Recent analyses have focused on the study of the structural complexity at yeast telomeres to the level of specific nucleosomes and of the distribution of protein complexes in a natural telomeric region (LIII) . In this report, we further analyze the structural complexity of LIII and the implication of this structure on telomeric silencing . It is shown that the establishment of repressive heterochromatin structures at LIII requires the recruitment of Sir3p through interaction with the N terminus of histone H4 . The establishment of such structures does not require acetylation of any of four lysines located in the H4 N terminus (lysines 5, 8, 12, and 16).

Biochemistry, 1999 Jan 5, 38(1), 487 - 95
Effect of pH on formation of a nativelike intermediate on the unfolding pathway of a Lys 73 --> His variant of yeast iso-1-cytochrome c; Godbole S et al.; Previous work on a Lys 73 --> His (H73) variant of iso-1-cytochrome c at pH 7.5 {Godbole et al . (1997) Biochemistry 36, 119-126} showed that this variant unfolds through a nativelike intermediate that has properties consistent with replacement of the Met 80 heme ligand by His 73 . Here, the pH dependence of the equilibrium unfolding of the wild type (WT) and H73 proteins have been investigated, since a characteristic pH dependence is expected for the stability of an intermediate stabilized by histidine-heme ligation . Stability has been evaluated using guanidine hydrochloride and pH denaturation methods . Above pH 5, the m-values from guanidine hydrochloride denaturation of the WT and H73 variants remain significantly different, consistent with continued population of this intermediate . At pH 4.5 the m-values for the two proteins are within error the same . To assess stability at lower pH, acid denaturation was carried out . The midpoint is about 3.3 for both proteins but the transition is broader for the H73 protein, suggestive of intermediates again being populated during the unfolding of the H73 protein at this lower pH . Heme ligation by Met 80 was monitored (695 nm absorbance) during gdnHCl (pH 4.5 and 5.0) and acid denaturation, confirming, respectively, the absence and presence of intermediates . A thermodynamic analysis demonstrates that this complex pH dependence for the presence of histidine ligation induced intermediates is expected and implicates a titratable group with a pKa of approximately 6.6 . The analysis also demonstrates when the pH dependences of global stability and stability of an intermediate differ significantly, population of folding intermediates as a function of pH will show novel behavior.

Biochemistry, 1999 Jan 5, 38(1), 98 - 104
Calcium binding induces interaction between the N- and C-terminal domains of yeast calmodulin and modulates its overall conformation; Nakashima K et al.; Calmodulin from the yeast Saccharomyces cerevisiae binds 3 mol of Ca2+ cooperatively . We report here lines of evidence supporting the intramolecular interaction between the N- and C-terminal domains which modulates the Ca2+ binding properties of yeast calmodulin . First, the sum of the Ca2+ binding curves of the N-terminal and the C-terminal half-molecule did not yield the Ca2+ binding curve of yeast calmodulin . Second, the mean residue CD of yeast calmodulin at 222 nm (-Delta epsilon222) decreased with increases in the concentration of Ca2+, whereas those of each half-molecule increased . Finally, the C2 proton of His107 in the C-terminal domain of yeast calmodulin showed three resonance peaks with increases in the concentration of Ca2+, each corresponding to the apo, the intermediate, and the Ca2+-saturated state . The intermediate peak could not be observed in the C-terminal half-molecule of yeast calmodulin . Computer simulation considering the macroscopic Ca2+ binding constants assigned this intermediate to a species consisting of the apo C-terminal domain and the N-terminal domain with at least one of the two sites occupied by Ca2+ . Peptide segments spanning the defective fourth Ca2+ binding site may be involved in the interdomain interaction and the yeast-specific function of calmodulin.

Nucleic Acids Res, 1999 Feb 1, 27(3), 888 - 94
Genomic detection of new yeast pre-mRNA 3'-end-processing signals; Graber JH et al.; To investigate Saccharomyces cerevisiae 3'-end-processing signals, a set of 1352 unique pre-mRNA 3'-end-processing sites, corresponding to 861 different genes, was identified by alignment of expressed sequence tag sequences with the complete yeast genome . Nucleotide word frequencies in the vicinity of the cleavage sites were analyzed to reveal the signal element features . In addition to previously recognized processing signals, two previously uncharacterized components of the 3'-end-processing signal sequence were discovered, specifically a predominance of U-rich sequences located on either side of the cleavage site . One of these, the downstream U-rich signal, provides a further link between the 3'-end-processing mechanisms of yeast and higher eukaryotes . Analysis of the complete set of 3'-end-processing sites by means of a discrimination function supports a 'contextual' model in which the sum total effectiveness of the signals in all four elements determines whether or not processing occurs.

Nucleic Acids Res, 1999 Feb 1, 27(3), 736 - 42
Functional analysis of human MutSalpha and MutSbeta complexes in yeast; Clark AB et al.; Mismatch repair (MMR) is initiated when a heterodimer of hMSH2*hMSH6 or hMSH2*hMSH3 binds to mismatches . Here we perform functional analyses of these human protein complexes in yeast . We use a sensitive genetic system wherein the rate of single-base deletions in a homopolymeric run in the LYS2 gene is 10 000-fold higher in an msh2 mutant than in a wild-type strain . Expression of the human proteins alone or in combination does not reduce the mutation rate of the msh2 strain, and expression of the individual human proteins does not increase the low mutation rate of a wild-type strain . However, co-expression of hMSH2 and hMSH6 in wild-type yeast increases the mutation rate 4000-fold, while co-expression of hMSH2 and hMSH3 elevates the rate 5-fold . Analysis of cell extracts indicates that the proteins are expressed and bind to mismatched DNA . The results suggest that hMutSalpha and hMutSbeta complexes form, bind to and prevent correction of replication slippage errors in yeast . Expression of hMSH6 with hMSH2 containing a proline substituted for a conserved Arg524 eliminates the mutator effect and reduces mismatch binding . The analogous mutation in humans is associated with microsatellite instability, defective MMR and cancer, illustrating the utility of the yeast system for studying human disease alleles.

J Clin Microbiol, 1999 Feb, 37(2), 422 - 6
Simple, reliable, and cost-effective yeast identification scheme for the clinical laboratory; Koehler AP et al.; The appearance of colonies on the chromogenic medium CHROMagar Candida combined with observation of morphology on corn meal-Tween 80 agar was used for the identification of 353 clinical yeast isolates . The results were compared with those obtained with API yeast identification kits . The accuracy of identification and the turnaround time were equivalent for each method, and our cultural method was less expensive.

EMBO J, 1999 Jan 15, 18(2), 320 - 9
G1 cyclins block the Ime1 pathway to make mitosis and meiosis incompatible in budding yeast; Colomina N et al.; Diploid yeast cells switch from mitosis to meiosis when starved of essential nutrients . While G1 cyclins play a key role in initiating the mitotic cell cycle, entry into meiosis depends on Ime1, a transcriptional activator regulated by both nutritional and cell-type signals . We show here that G1 cyclins downregulate IME1 transcription and prevent the accumulation of the Ime1 protein within the nucleus, which results in repression of early-meiotic gene expression . As G1-cyclin deficient cells do not require nutrient starvation to undergo meiosis, G1 cyclin would exert its role by transmitting essential nutritional signals to Ime1 function . The existence of a negative cross-talk mechanism between mitosis and meiosis may help explain why these two developmental options are incompatible in budding yeast.

Genomics, 1999 Jan 1, 55(1), 106 - 12
A yeast artificial chromosome-based physical map of the juvenile amyotrophic lateral sclerosis (ALS2) critical region on human chromosome 2q33-q34; Hadano S et al.; The autosomal recessive form of juvenile amyotrophic lateral sclerosis (ALS2; RFALS Type 3) has previously been mapped to the 8-cM interval flanked by D2S115 and D2S155 on human chromosome 2q33-q34 . We have established a yeast artificial chromosome (YAC) contig spanning an approximately 8-Mb region of the ALS2 candidate region and mapped 52 transcribed DNA sequences including 13 known genes and 39 expressed sequenced tags within this YAC contig . The establishment of a YAC contig and transcript map that spans the region containing the ALS2 mutation is an essential step in the identification of the ALS2 gene .

Am J Trop Med Hyg, 1998 Dec, 59(6), 991 - 7
Passive transfer of growth-inhibitory antibodies raised against yeast-expressed recombinant Plasmodium falciparum merozoite surface protein-1(19); Gozalo A et al.; Purified rabbit immunoglobulin raised against yeast-expressed recombinant FVO or 3D7 Plasmodium falciparum merozoite surface protein-1 (MSP-1) 19k-D C terminal fragment (MSP-1(19)) was transfused into malaria-naive Aotus nancymai monkeys that were immediately challenged with FVO asexual stage malaria parasites . Control monkeys received rabbit immunoglobulin raised against the sexual stage antigen Pfs25 or Aotus hyperimmune serum obtained from monkeys immunized by P . falciparum infection and drug cure . Passive transfer of rabbit anti-MSP-1(19) failed to protect against homologous or heterologous challenge and, when compared with negative controls, there were no differences in prepatent periods or time to treatment . Interestingly, rabbit anti-MSP-1(19), but not anti-Pfs25, immunoglobulin, and immune monkey serum prevented the development of antibodies directed against MSP-1(19) fragment by infected monkeys, indicating that the antibodies were reactive with native MSP-1(19) antigen in vivo . The prepatent period and time to treatment was greatly delayed in the two monkeys that received Aotus immune serum, both of which developed a chronic intermittent low level infection . In vitro parasite growth inhibition assays (GIAs) confirmed the presence of inhibitory activity (40% maximum inhibition) in concentrated anti-MSP-1(19) immunoglobulin (4.8 mg/ml), but the peak concentrations we achieved in vivo (1 mg/ml) were not inhibitory in vitro . Subinhibitory levels of anti-MSP-1(19) antibodies achieved by passive transfer were not protective against P . falciparum challenge.

Yeast, 1998 Dec, 14(16), 1453 - 69
Expanding yeast knowledge online; Dolinski K et al.; The completion of the Saccharomyces cerevisiae genome sequencing project and the continued development of improved technology for large-scale genome analysis have led to tremendous growth in the amount of new yeast genetics and molecular biology data . Efficient organization, presentation, and dissemination of this information are essential if researchers are to exploit this knowledge . In addition, the development of tools that provide efficient analysis of this information and link it with pertinent information from other systems is becoming increasingly important at a time when the complete genome sequences of other organisms are becoming available . The aim of this review is to familiarize biologists with the type of data resources currently available on the World Wide Web (WWW).

J Cell Biol, 1999 Jan 11, 144(1), 83 - 98
Coronin promotes the rapid assembly and cross-linking of actin filaments and may link the actin and microtubule cytoskeletons in yeast; Goode BL et al.; Coronin is a highly conserved actin-associated protein that until now has had unknown biochemical activities . Using microtubule affinity chromatography, we coisolated actin and a homologue of coronin, Crn1p, from Saccharomyces cerevisiae cell extracts . Crn1p is an abundant component of the cortical actin cytoskeleton and binds to F-actin with high affinity (Kd 6 x 10(-9) M) . Crn1p promotes the rapid barbed-end assembly of actin filaments and cross-links filaments into bundles and more complex networks, but does not stabilize them . Genetic analyses with a crn1Delta deletion mutation also are consistent with Crn1p regulating filament assembly rather than stability . Filament cross-linking depends on the coiled coil domain of Crn1p, suggesting a requirement for Crn1p dimerization . Assembly-promoting activity is independent of cross-linking and could be due to nucleation and/or accelerated polymerization . Crn1p also binds to microtubules in vitro, and microtubule binding is enhanced by the presence of actin filaments . Microtubule binding is mediated by a region of Crn1p that contains sequences (not found in other coronins) homologous to the microtubule binding region of MAP1B . These activities, considered with microtubule defects observed in crn1Delta cells and in cells overexpressing Crn1p, suggest that Crn1p may provide a functional link between the actin and microtubule cytoskeletons in yeast.

J Cell Biol, 1999 Jan 11, 144(1), 71 - 82
Regulation of the actin cytoskeleton organization in yeast by a novel serine/threonine kinase Prk1p; Zeng G et al.; Normal actin cytoskeleton organization in budding yeast requires the function of the Pan1p/ End3p complex . Mutations in PAN1 and END3 cause defects in the organization of actin cytoskeleton and endocytosis . By screening for mutations that can suppress the temperature sensitivity of a pan1 mutant (pan1-4), a novel serine/threonine kinase Prk1p is now identified as a new factor regulating the actin cytoskeleton organization in yeast . The suppression of pan1-4 by prk1 requires the presence of mutant Pan1p . Although viable, the prk1 mutant is unable to maintain an asymmetric distribution of the actin cytoskeleton at 37 degreesC . Consistent with its role in the regulation of actin cytoskeleton, Prk1p localizes to the regions of cell growth and coincides with the polarized actin patches . Overexpression of the PRK1 gene in wild-type cells leads to lethality and actin cytoskeleton abnormalities similar to those exhibited by the pan1 and end3 mutants . In vitro phosphorylation assays demonstrate that Prk1p is able to phosphorylate regions of Pan1p containing the LxxQxTG repeats, including the region responsible for binding to End3p . Based on these findings, we propose that the Prk1 protein kinase regulates the actin cytoskeleton organization by modulating the activities of some actin cytoskeleton-related proteins such as Pan1p/End3p.

Glycobiology, 1999 Jan, 9(1), 93 - 100
A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme; Pauly M et al.; A full-length c-DNA encoding a xyloglucan-specific endo -beta-1, 4-glucanase (XEG) has been isolated from the filamentous fungus Aspergillus aculeatus by expression cloning in yeast . The colonies expressing functional XEG were identified on agar plates containing azurine-dyed cross-linked xyloglucan . The cDNA encoding XEG was isolated, sequenced, cloned into an Aspergillus expression vector, and transformed into Aspergillus oryzae for heterologous expression . The recombinant enzyme was purified to apparent homogeneity by anion-exchange and gel permeation chromatography . The recombinant XEG has a molecular mass of 23,600, an isoelectric point of 3.4, and is optimally stable at a pH of 3.4 and temperature below 30 degreesC . The enzyme hydrolyzes structurally diverse xyloglucans from various sources, but hydrolyzes no other cell wall component and can therefore be considered a xyloglucan-specific endo -beta-1, 4-glucanohydrolase . XEG hydrolyzes its substrates with retention of the anomeric configuration . The Kmof the recombinant enzyme is 3.6 mg/ml, and its specific activity is 260 micromol/min per mg protein . The enzyme was tested for its ability to solubilize xyloglucan oligosaccharides from plant cell walls . It was shown that treatment of plant cell walls with XEG yields only xyloglucan oligosaccharides, indicating that this enzyme can be a powerful tool in the structural elucidation of xyloglucans.

Microbiology, 1998 Dec, 144 ( Pt 12), 3463 - 74
Cell integrity and morphogenesis in a budding yeast septin mutant; Cid VJ et al.; The non-sporulating diploid strain V327 of Saccharomyces cerevisiae was previously isolated in a search for thermosensitive autolytic mutants . This strain is very efficient at releasing intracellular proteins into the medium when incubated at high temperatures . The expression of this lytic phenotype depends on a morphogenetic defect, consisting of the appearance of elongated chains of cells . Transmission electron microscopy revealed a mislocalization of septa at semi-permissive temperatures and a total lack of septation together with abnormal cell wall architecture at a non-permissive temperature . The septin-encoding CDC10 gene was cloned by complementation of the pleiotropic phenotype of the V327 mutant . Rescue and sequencing of CDC10 alleles from V327 revealed a point mutation that created a single amino acid change in a region which is well conserved among septins . This new allele was named cdc10-11 . The construction of a cdc10-11 haploid strain by substituting the CDC10 gene with the rescued allele permitted further genetic analyses of the mutation and allowed the construction of new homozygous cdc10-11 diploid strains that showed a reduced ability to sporulate . Fusing both the wild-type and the cdc10-11 alleles to green fluorescent protein (GFP) demonstrated that the mutation does not affect the localization of this septin to the bud neck at the standard growth temperature of 24 degrees C, although the morphogenetic phenotype at 37 degrees C parallels the disappearance of Cdc10-GFP at the ring encircling the septum area.

J Pharm Biomed Anal, 1998 Sep 1, 17(6-7), 1037 - 45
Analytical methods and stability assessment of liquid yeast derived sucrase; McIntosh KA et al.; Two independent analytical methods for determining the activity and stability profile of liquid yeast derived sucrase (YS) were established and validated in order to conduct preliminary stability studies as a function of temperature . The methods included a hexokinase-based (HK) enzymatic assay for determining the formation of glucose upon hydrolysis of sucrose by YS, and a direct polarimetric procedure to quantitate YS hydrolysis of sucrose . Both assays were validated with respect to YS dilution, incubation time, sucrose or glucose concentration, linearity of response and within- and between-day variability . A preliminary stability study was conducted over a 24 week period with liquid YS samples stored at -20, 4, 30, 40 and 50 degrees C . Enzymatic activity was monitored as a function of time using both the HK and polarimetric assays . Liquid YS samples stored at -20, 4 and 30 degrees C retained 100% activity after 24 weeks storage, while the samples stored at 40 degrees C lost approximately 70% activity over the same storage period and samples stored at 50 degrees C lost approximately 95% activity after 12 weeks storage . The two methods of analysis gave consistent results over the course of the study.

FEBS Lett, 1998 Dec 18, 441(2), 266 - 70
Identification of a human mitochondrial ABC transporter, the functional orthologue of yeast Atm1p; Csere P et al.; We have sequenced the entire coding region of the human ABC transporter ABC7 . The protein represents a 'half-transporter' and displays high sequence similarity to the mouse ABC7 protein and to the mitochondrial ABC transporter Atm1p of Saccharomyces cerevisiae . As shown by immunostaining using a specific antibody, the human ABC7 protein (hABC7) is a constituent of mitochondria . The N-terminus of hABC7 contains the information for targeting and import into the organelles . When synthesised in yeast cells defective in Atm1p (strain delta atm1/hABC7), hABC7 protein can revert the strong growth defect observed for delta atm1 cells to near wild-type behaviour . The known phenotypical consequences of inactivation of the ATM1 gene are almost fully amended by expression of hABC7 protein . delta atm1/hABC7 cells harbour wild-type levels of cytochromes and extra-mitochondrial heme-containing proteins, they contain normal levels of mitochondrial iron, and the cellular content of glutathione is substantially reduced relative to the high levels detected in delta atm1 cells . Our results suggest that hABC7 is a mitochondrial protein, and represents the functional orthologue of yeast Atm1p.

FEBS Lett, 1998 Dec 18, 441(2), 177 - 80
Glycan engineering of proteins with whole living yeast cells expressing rat liver alpha2,3-sialytransferase in the porous cell wall; Sievi E et al.; The N-glycans of recombinant proteins produced via the secretory pathway of cultured mammalian cells are often undersialylated, and insect cells lack sialytransferases . Undersialylated glycoproteins are rapidly cleared from the circulation, compromising the effect of pharmaceuticals . We show that incubation with living Saccharomyces cerevisiae cells expressing the catalytic ectodomain of rat liver alpha2,3-sialyltransferase (ST3Ne) in the porous cell wall resulted in sialylation of glycoproteins . The Km values of the yeast enzyme for several substrates were similar to those of recombinant ST3Ne from insect cells and of authentic ST3N . The yeast strain provides an inexpensive self-perpetuating source of ST3N activity for glycan engineering of recombinant proteins.

Dev Biol, 1998 Dec 15, 204(2), 451 - 63
Partial rescue of GATA-3 by yeast artificial chromosome transgenes; Lakshmanan G et al.; GATA-3 is essential for murine embryonic development, but elucidating the genetic controls over the complex temporal and tissue-specific transcriptional regulatory pattern of this transcription factor gene has been problematic . Here we report the isolation and characterization of two yeast artificial chromosomes (YACs) bearing the murine GATA-3 gene . Ordered deletions of both YACs show that they define a 1-megabase pair contig spanning the GATA-3 locus . We found that a 120-kb YAC transgene, including 35 kb of 5' as well as 60 kb of 3' flanking sequence, confers normal GATA-3 expression at sites not revealed previously through analysis of plasmid transgenic lines . However, even this 120-kb YAC does not contain sufficient information to recapitulate the complete GATA-3 expression program during embryogenesis . While not complete in its regulatory capacity, the YAC transgene is nonetheless able to complement several homozygous GATA-3 mutant phenotypes and thereby prolong embryonic life .

Biol Trace Elem Res, 1998 Nov, 65(2), 143 - 51
Bioavailability of enteral yeast-selenium in preterm infants; Bogye G et al.; There is no data or literature on the effects of supplementing infants with yeast selenium, although its intestinal absorption and bioavailability are higher in adults compared with other selenium compounds . The aim of the present investigation was to study the impact of selenium enriched yeast on the serum selenium concentration of preterm infants living in a low selenium area (Hungary) . Twenty-eight preterm infants with mean+/-SD birth weight of 962+/-129 g and gestational age 27+/-1 wk were randomized into two groups at birth with respect to selenium supplementation . In the supplemented group (n=14) infants received 4.8 mg yeast selenium containing 5 microg selenium daily via nasogastric drip during the first 14 postnatal days . The nonsupplemented infants were used as a reference group . In the supplemented group, the serum selenium concentration increased from 32.1+/-8.5 microg/L to 41.5+/-6.5 microg/L and in the nonsupplemented group it decreased from 25.9+/-6.8 microg/L to 18.2+/-6.4 microg/L from birth in two weeks time . Compared with previous studies, our results suggest that the bioavailability of selenium in the form of yeast selenium is higher than that of other selenium compounds used for preterm infants . We did not observe any complications or side-effects owing to enteral yeast selenium supplementation . We conclude that selenium enriched yeast is a safe and an effective form of short-term enteral selenium supplementation for infants.

Plant J, 1998 Nov, 16(4), 453 - 64
The plant PTS1 receptor: similarities and differences to its human and yeast counterparts; Wimmer C et al.; Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix . We have cloned and sequenced the watermelon (Citrullus vulgaris) cDNA homologue to the PTS1 receptor gene (PEX5) . Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X2-(E/S)-(Y/F/Q) in its N-terminal half . According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes . Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes . These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature . The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes . However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.

J Biol Chem, 1999 Jan 15, 274(3), 1835 - 41
A syntaxin homolog encoded by VAM3 mediates down-regulation of a yeast G protein-coupled receptor; Stefan CJ et al.; G protein-coupled receptors that transduce signals for many hormones, neurotransmitters, and inflammatory mediators are internalized and subsequently recycled to the plasma membrane, or down-regulated by targeting to lysosomes for degradation . Here we have characterized yeast alpha-factor receptors tagged with green fluorescent protein (Ste2-GFP) and used them to obtain mutants defective in receptor down-regulation . In wild type cells, Ste2-GFP was functional and localized to the plasma membrane and endocytic compartments . Although GFP was fused to the cytoplasmic tail of the receptor, GFP also accumulated in the lumen of the vacuole, suggesting that the receptor's extracellular and cytoplasmic domains are degraded within the vacuole lumen . Transposon mutagenesis and a visual screen were used to identify mutants displaying aberrant localization of Ste2-GFP . Mutants that accumulated Ste2-GFP in numerous intracellular vesicles carried disruptions of the VAM3/PTH1 gene, which encodes a syntaxin homolog (t-SNARE) required for homotypic vacuole membrane fusion, autophagy and fusion of biosynthetic transport vesicles with the vacuole . We provide evidence that Vam3 is required for the delivery of alpha-factor receptor-ligand complexes to the vacuole . Vam3 homologs in mammalian cells may mediate late steps in the down-regulation and lysosomal degradation pathways of various G protein-coupled receptors.

J Biol Chem, 1999 Jan 15, 274(3), 1199 - 202
Rad26, the yeast homolog of the cockayne syndrome B gene product, counteracts inhibition of DNA repair due to RNA polymerase II transcription; Tijsterman M et al.; Transcription-coupled DNA repair (TCR) is responsible for the preferential removal of DNA lesions from the transcribed strands of RNA polymerase II transcribed genes . Saccharomyces cerevisiae rad26 mutants and cells from patients suffering from the hereditary disease Cockayne syndrome display a TCR defective phenotype . Whether this lack of preferential repair has to be explained by a defect in repair or in general transcription is unclear at present . To discriminate between both possibilities, we analyzed repair of UV-induced cyclobutane pyrimidine dimers at single base resolution in yeast cells lacking RAD26, the homolog of the Cockayne syndrome B gene . Disrupting RAD26 affects nucleotide excision repair of transcribed DNA irrespective of the chromatin context, resulting in similar rates of removal for individual cyclobutane pyrimidine dimers throughout the transcribed strand . Notably, repair of transcribed sequences in between core nucleosomal regions is less efficient compared with nontranscribed DNA at these positions, pointing to a nucleotide excision repair impediment caused by blocked RNA polymerase . Our in vivo data demonstrate that the TCR defect in rad26 mutant cells is not due to a general transcription deficiency but results from the inability to release the transcription complex trapped at sites of base damage.

Mol Biol Cell, 1999 Jan, 10(1), 63 - 75
Structural and functional analysis of a novel coiled-coil protein involved in Ypt6 GTPase-regulated protein transport in yeast; Tsukada M et al.; The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y . We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations . SYS3 encodes a 105-kDa protein with a predicted high alpha-helical content . It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi . Like Uso1p, Sys3p is predominatly cytosolic . According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes . Its loss of function results in partial missorting of carboxypeptidase Y . Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40- to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in alpha-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment . The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicle-docking stage) between an endosomal compartment and the most distal Golgi compartment.

J Enzyme Inhib, 1998 Feb, 13(1), 57 - 68
Influence of Tris(hydroxymethyl)aminomethane on kinetic mechanism of yeast alcohol dehydrogenase; Trivic S et al.; Acetaldehyde, propionaldehyde, glyceraldehyde-3-P and 4-dimethylaminocinnamaldehyde form Schiff bases in Tris . HCl buffers; the rates of formation and dissociation of Schiff bases, and equilibrium constants for their formation are very similar for the first three aldehydes . The steady-state kinetic constants for the yeast alcohol dehydrogenase-catalyzed reaction, propan-1-ol + NAD+ reversible propionaldehyde + NADH + H+, have been determined in several Tris . HCl buffers of increasing concentration at pH 8.1 . In the forward direction, oxidation of alcohol, most kinetic constants are increased by increasing concentrations of Tris . In the reverse direction, reduction of aldehyde, substrate, NADH, Tris and Schiff base were equilibrated before enzyme reaction was started . It was found that Schiff base, rather than Tris, binds to free enzyme competitively with respect to NADH . Tris and Schiff base do not influence the binding of aldehyde to enzyme in any way.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 373 - 83
The contribution of cell wall proteins to the organization of the yeast cell wall; Kapteyn JC et al.; Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms . Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs . Both define a characteristic supramolecular complex or structural unit . The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains . The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains . The molecular architecture of the cell wall is not fixed . The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH . When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage . Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 309 - 22
Asparagine-linked glycosylation in the yeast Golgi; Dean N; The Golgi complex is the site where the terminal carbohydrate modification of proteins and lipids occurs . These carbohydrates play a variety of biological roles, ranging from the stabilization of glycoprotein structure to the provision of ligands for cell-cell interactions to the regulation of cell surface properties . Progress in our understanding of the biosynthesis and regulation of glycoconjugates has been accelerating at a rapid pace . Recent advances in the field of yeast glycobiology have been particularly impressive . This review focuses on glycosylation of proteins in the Golgi of the yeast Saccharomyces cerevisiae, with emphasis on the candidate mannosyltransferases that participate in the synthesis of N-linked oligosaccharides . Current views on how these enzymes may be regulated and how glycosylation relates on other cellular processes are also discussed.

Biochim Biophys Acta, 1999 Jan 6, 1426(2), 259 - 73
The oligosaccharyltransferase complex from yeast; Knauer R et al.; N-Glycosylation of eukaryotic secretory and membrane-bound proteins is an essential and highly conserved protein modification . The key step of this pathway is the en bloc transfer of the high mannose core oligosaccharide Glc3Man9GlcNAc2 from the lipid carrier dolichyl phosphate to selected Asn-X-Ser/Thr sequences of nascent polypeptide chains during their translocation across the endoplasmic reticulum membrane . The reaction is catalysed by the enzyme oligosaccharyltransferase (OST) . Recent biochemical and molecular genetic studies in yeast have yielded novel insights into this enzyme with multiple tasks . Nine proteins have been shown to be OST components . These are assembled into a heterooligomeric membrane-bound complex and are required for optimal expression of OST activity in vivo in wild type cells . In accord with the evolutionary conservation of core N-glycosylation, there are significant homologies between the protein sequences of OST subunits from yeast and higher eukaryotes, and OST complexes from different sources show a similar organisation as well.

Biochim Biophys Acta, 1999 Jan 4, 1426(1), 119 - 25
Effect of astaxanthin rich red yeast (Phaffia rhodozyma) on oxidative stress in rainbow trout; Nakano T et al.; The antioxidative biological effect of dietary red yeast, Phaffia rhodozyma, which is rich in astaxanthin, on rainbow trout, Oncorhynchus mykiss, was examined . The levels of serum transaminase (glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase) activities and of lipid peroxides (LPO) of fish fed oxidized oil were significantly higher than those of the control fish fed non-oxidized oil . However, the supply of red yeast considerably decreased both enzyme activities and LPO level . Furthermore, the serum lipid (triglycerides, total cholesterol and phospholipids) concentrations were also significantly decreased . Especially, the serum triglyceride level of fish fed the red yeast was as low as that of the control . It was also observed that there were no significant differences in muscle LPO levels between the fish fed red yeast and the control . The present results suggest for the first time that dietary red yeast may effectively suppress the LPO generation of tissue and normalize liver function as well as improving muscle pigmentation of trout . Thus, red yeast should have a reducing effect on oxidized oil-induced oxidative stress in fish.

Biochim Biophys Acta, 1999 Jan 5, 1409(3), 113 - 24
Expression of the bovine heart mitochondrial ADP/ATP carrier in yeast mitochondria: significantly enhanced expression by replacement of the N-terminal region of the bovine carrier by the corresponding regions of the yeast carriers; Hashimoto M et al.; To characterize the transport mechanism mediated by the mammalian mitochondrial ADP/ATP carrier (AAC), we tried to express bovine heart mitochondrial AAC (bhAAC) in Saccharomyces cerevisiae . The open reading frame of the bhAAC was introduced into the haploid strain WB-12, in which intrinsic AAC genes were disrupted . Growth of the transformant was very low in glycerol medium, and a little amount of bhAAC was detected in the mitochondrial membrane . For improvement of bhAAC expression in WB-12, we introduced DNA fragments encoding chimeric bhAACs, in which the N-terminal region of the bhAAC extending into the cytosol was replaced by the corresponding regions of the type 1 and type 2 yeast AAC isoforms (yAAC1 and yAAC2) . These transformants grew well, and the amounts of the chimeric bhAACs in their mitochondria were as high as that of yAAC2 . The carriers expressed showed essentially the same ADP transport activities as that of AAC in bovine heart mitochondria.

EMBO J, 1999 Jan 4, 18(1), 229 - 40
Mutations in both the structured domain and N-terminus of histone H2B bypass the requirement for Swi-Snf in yeast; Recht J et al.; The chromatin elements targeted by the ATPdependent, Swi-Snf nucleosome-remodeling complex are unknown . To address this question, we generated mutations in yeast histone H2B that suppress phenotypes associated with the absence of Swi-Snf . Sin- (Swi-Snf-independent) mutations occur in residues involved in H2A-H2B dimer formation, dimer- tetramer association, and in the H2B N-terminus . The strongest and most pleiotropic Sin- mutation removed 20 amino acid residues from the H2B N-terminus . This mutation allowed active chromatin to be formed at the SUC2 locus in a snf5Delta mutant and resulted in hyperactivated levels of SUC2 mRNA under inducing conditions . Thus, the H2B N-terminus may be an important target of Swi-Snf in vivo . The GCN5 gene product, the catalytic subunit of several nuclear histone acetytransferase complexes that modify histone N-termini, was also found to act in conjunction with Swi-Snf . The phenotypes of double gcn5Deltasnf5Delta mutants suggest that histone acetylation may play both positive and negative roles in the activity of the Swi-Snf-remodeling factor.

Biophys J, 1999 Jan, 76(1 Pt 1), 50 - 64
Molecular dynamics simulations of solvated yeast tRNA(Asp); Auffinger P et al.; Transfer RNA molecules are involved in a variety of biological processes, implying complex recognition events with proteins and other RNAs . From a structural point of view, tRNAs constitute a reference system for studying RNA folding and architecture . A deeper understanding of their structural and functional properties will derive from our ability to model accurately their dynamical behavior . We present the first dynamical model of a fully neutralized and solvated tRNA molecule over a 500-ps time scale . Starting from the crystallographic structure of yeast tRNA(Asp), the 75-nucleotide molecule was modeled with 8055 water molecules and 74 NH4+ counterions, using the AMBER4.1 program and the particle mesh Ewald (PME) method for the treatment of long-range electrostatic interactions . The calculations led to a dynamically stable model of the tRNA molecule . During the simulation, all secondary and tertiary base pairs are maintained while a certain lability of base triples in the tRNA core is observed . This lability was interpreted as resulting from intrinsic factors associated with the "weaker" hydrogen bonding patterns seen in these base triples and from an altered ionic environment of the tRNA molecule . Calculated thermal factors are used to compare the dynamics of the tRNA in solution and in the crystal . The present molecular dynamics simulation of a complex and highly charged nucleic acid molecule attests to the fact that simulation methods are now able to investigate not only the dynamics of proteins, but also that of large RNA molecules . Thus they also provide a basis for further investigations on the structural and functional effects of chemical and posttranscriptionally modified nucleotides as well as on ionic environmental effects.

Mutat Res, 1998 Dec 14, 409(3), 181 - 8
Defective Kin28, a subunit of yeast TFIIH, impairs transcription-coupled but not global genome nucleotide excision repair; Tijsterman M et al.; The essential Saccharomyces cerevisiae KIN28 gene encodes a subunit of general transcription factor TFIIH, a multiprotein complex required for RNA polymerase II transcription initiation and nucleotide excision repair (NER) . Kin28 is implicated in the transition from transcription initiation to transcription elongation by phosphorylation of the carboxy-terminal domain (CTD) of the largest subunit of the RNA polymerase II complex . Here, we explore the possibility that Kin28 like the other subunits of TFIIH is involved in NER in vivo, using yeast cells carrying either a wildtype or a thermosensitive KIN28 allele . The removal of UV induced cyclobutane pyrimidine dimers (CPDs) was monitored at base resolution from both strands of the RNA polymerase II transcribed genes RPB2 and URA3 . Cells carrying the thermosensitive KIN28 allele display a transcription-coupled repair (TCR) defect at the non-permissive temperature, which was most pronounced directly downstream of transcription initiation, probably as an indirect result of a general decrease in the level of RNA polymerase II transcription . The fact that CPD removal in non-transcribed DNA is completely unaffected in these cells indicates that Kin28 is not essential for general NER in vivo, providing the first example of a TFIIH subunit that is required for TCR but not for NER in general.

Proc Natl Acad Sci U S A, 1999 Jan 5, 96(1), 121 - 6
Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores; Csutora P et al.; Depletion of endoplasmic reticulum Ca2+ stores leads to the entry of extracellular Ca2+ into the cytoplasm, a process termed capacitative or store-operated Ca2+ entry . Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca2+ stores were depleted by chemical and genetic means, respectively . After microinjection into Xenopus laevis oocytes, the extracts elicited a wave of increased cytoplasmic free Ca2+ ({Ca2+}i) that spread from the point of injection across the oocyte . Extracts from cells with replete organellar Ca2+ stores were inactive . The increases depended on extracellular Ca2+, were unaffected by the inositol 1,4,5-trisphosphate (IP3) inhibitor heparin or an anti-IP3 receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation . Confocal microscopy revealed that {Ca2+}i increases were most pronounced at the periphery of the oocyte . The patterns of {Ca2+}i increases were replicated by computer simulations based on a diffusible messenger of about 700 Da that directly activates Ca2+ influx . In addition, ICRAC, a Ca2+ release-activated Ca2+ current monitored in Jurkat cells by whole-cell patch clamp recordings, was more rapidly activated when active extracts were included in the patch pipette than by the inclusion of a Ca2+ chelator or IP3 . These data support the existence in yeast and mammalian cells depleted of Ca2+ stores of a functionally conserved diffusible calcium influx factor that directly activates Ca2+ influx.

Eur J Biochem, 1998 Dec 1, 258(2), 430 - 6
Sporulation-specific expression of the yeast DIT1/DIT2 promoter is controlled by a newly identified repressor element and the short form of Rim101p; Bogengruber E et al.; Expression of the yeast genes DIT1 and DIT2 is confined to mid/late sporulation . Transcription of these two divergently arranged genes is controlled by a common 900-bp intergenic region . Random mutagenesis of this promoter and tests with appropriate reporter constructs identified an 11-bp cis-acting palindromic sequence, DIT repressor element (DRE), as a major negative regulatory site during vegetative growth . Repression is exerted by DRE in conjunction with a mid-sporulation element (MSE)-like sequence situated 26 bp away . These sequence elements are both contained within the 76-bp negative regulatory element (NRE) defined previously {Friesen H., Hepworth, S . R . & Segall, J . (1997) Mol . Cell . Biol . 17, 123-134} . The activated form of Rim101p, a transcriptional inducer of the early meiotic gene IME1, enhances expression from the DIT1 promoter both in vegetative and sporulating cells . Activation by Rim101p does not seem to involve binding of Rim101p at either of the two cis-acting sites described here, since reporter constructs with both elements or most of the NRE deleted could still be activated by the short form of Rim101p.

Bioorg Med Chem Lett, 1998 Aug 18, 8(16), 2269 - 72
Alkyl side-chain derivatives of sordaricin as potent antifungal agents against yeast; Tse B et al.; Sordarin (1) was converted to 5 and 6, which showed potent antifungal activity against yeast . A series of C1-C9 alkyl side-chain derivatives was prepared, from which it was found that the optimal activity occurred with C5 . A comparison of side chains with different unsaturation showed that the cis-alkene was the most active . This result suggested that the folding of the side chains might be crucial for the optimal activity.

Genome Res, 1998 Dec, 8(12), 1259 - 72
Homologs of the yeast longevity gene LAG1 in Caenorhabditis elegans and human; Jiang JC et al.; LAG1 is a longevity gene, the first such gene to be identified and cloned from the yeast Saccharomyces cerevisiae . A close homolog of this gene, which we call LAC1, has been found in the yeast genome . We have cloned the human homolog of LAG1 with the ultimate goal of examining its possible function in human aging . In the process, we have also cloned a homolog from the nematode worm Caenorhabditis elegans . Both of these homologs, LAG1Hs and LAG1Ce-1, functionally complemented the lethality of a lag1delta lac1delta double deletion, despite low overall sequence similarity to the yeast proteins . The proteins shared a short sequence, the Lag1 motif, and a similar transmembrane domain profile . Another, more distant human homolog, TRAM, which lacks this motif, did not complement . LAG1Hs also restored the life span of the double deletion, demonstrating that it functions in establishing the longevity phenotype in yeast . LAG1Hs mapped to 19p12, and it was expressed in only three tissues: brain, skeletal muscle, and testis . This gene possesses a trinucleotide (CTG) repeat within exon 1 . This and its expression profile raise the possibility that it may be involved in neurodegenerative disease . This possibility suggests at least one way in which LAG1Hs might be involved in human aging.

Appl Environ Microbiol, 1999 Jan, 65(1), 110 - 6
Threonine overproduction in yeast strains carrying the HOM3-R2 mutant allele under the control of different inducible promoters; Farfan MJ et al.; The HOM3 gene of Saccharomyces cerevisiae codes for aspartate kinase, which plays a crucial role in the regulation of the metabolic flux that leads to threonine biosynthesis . With the aim of obtaining yeast strains able to overproduce threonine in a controlled way, we have placed the HOM3-R2 mutant allele, which causes expression of a feedback-insensitive enzyme, under the control of four distinctive regulatable yeast promoters, namely, PGAL1, PCHA1, PCYC1-HSE2, and PGPH1 . The amino acid contents of strains bearing the different constructs were analyzed both under repression and induction conditions . Although some differences in overall threonine production were found, a maximum of around 400 nmol/mg (dry weight) was observed . Other factors, such as excretion to the medium and activity of the catabolic threonine/serine deaminase, also affect threonine accumulation . Thus, improvement of threonine productivity by yeast cells would probably require manipulation of these and other factors.

Curr Genet, 1998 Dec, 34(5), 360 - 7
Genetic analysis of the yeast NUD1 endo-exonuclease: a role in the repair of DNA double-strand breaks; Asefa B et al.; The deoxyribonucleases (DNases) have been shown genetically to be important in the vital processes of DNA repair and recombination . The NUD1 gene, which codes for an endo-exonuclease of Saccharomyces cerevisiae, was analyzed for its role in the DNA double-strand break (DSB) repair processes . While the nud1 strain is only slightly sensitive to ionizing radiation, expression of the HO-endonuclease to introduce a DSB at the MAT locus in that strain results in cell death . Cell survival is inversely proportional to the duration of HO-endonuclease expression . Analysis of the surviving colonies from the nud1 strain indicated that many of the survivors are sterile and that the proportion of these sterile survivors increases with the time of HO-endonuclease expression . On the other hand, the surviving colonies from the isogenic NUD1 strain are mating-proficient . Interestingly, double mutants of nud1 rad52 are more resistant to ionizing irradiation than the rad52 strain and have a cell-survival fraction of 32% for rad52-1 nud1 and 9% for rad52::URA3 nud1 following prolonged HO-endonuclease expression, indicating that nud1 has a suppressor effect on the DSB-induced lethality in rad52 . Polymerase chain reaction analysis showed that many of the nud1 survivors contained small alterations within theMAT locus, suggesting that the survivors arose through the process of non-homologous end-joining . These results suggest that the endo-exonuclease acts at a DSB to promote DNA repair via the homologous recombination pathway.

Cell Biochem Biophys, 1998, 29(3), 263 - 79
Ruptured fission yeast walls . Structural discontinuities related to the cell cycle; Piombo S et al.; Distributions of rupture sites of fission yeast cells ruptured by glass beads have been related to a new morphometric analysis . As shown previously (Johnson et al., Cell Biophysics, 1995), ruptures were not randomly distributed nor was their distribution dictated by geometry, rather, ruptures at the extensile end were related to cell length just as the rate of extension is related to cell length . The extension patterns of early log, mid-log, late log, and stationary phase cells from suspension cultures were found to approximate the linear growth patterns of Kubitschek and Clay (1986) . The median length of cells was found to decline through the log phase in an unbalanced manner.

Trends Biochem Sci, 1998 Dec, 23(12), 481 - 5
Structural organization of MAP-kinase signaling modules by scaffold proteins in yeast and mammals; Whitmarsh AJ et al.; MAP-kinase signaling pathways are activated by multiple extracellular stimuli . The specificity of activation and function of MAP-kinase signaling modules is determined, in part, by scaffold proteins that create multienzyme complexes . In Saccharomyces cerevisiae, two MAP-kinase-scaffold proteins have been identified . Recent studies of mammalian cells have also led to the identification of putative scaffold proteins . These scaffold proteins appear to facilitate MAP-kinase activation, in response to specific physiological stimuli, and to insulate the bound MAP-kinase module against activation by irrelevant stimuli . Scaffold proteins are therefore critical components of MAP-kinase modules and ensure signaling specificity.

J Biol Chem, 1999 Jan 1, 274(1), 543 - 8
Isolation of supernumerary yeast ATP synthase subunits e and i . Characterization of subunit i and disruption of its structural gene ATP18; Vaillier J et al.; Two subunits of the yeast ATP synthase have been isolated . Subunit e was found loosely associated to the complex . Triton X-100 at a 1% concentration removed this subunit from the ATP synthase . The N-terminal sequencing of subunit i has been performed . The data are in agreement with the sequence of the predicted product of a DNA fragment of Saccharomyces cerevisiae chromosome XIII . The ATP18 gene encodes subunit i, which is 59 amino acids long and corresponds to a calculated mass of 6687 Da . Its pI is 9.73 . It is an amphiphilic protein having a hydrophobic N-terminal part and a hydrophilic C-terminal part . It is not apparently related to any subunit described in other ATP synthases . The null mutant showed low growth on nonfermentable medium . Mutant mitochondria display a low ADP/O ratio and a decrease with time in proton pumping after ATP addition . Subunit i is associated with the complex; it is not a structural component of the enzyme but rather is involved in the oxidative phosphorylations . Similar amounts of ATP synthase were measured for wild-type and null mutant mitochondria . Because 2-fold less specific ATPase activity was measured for the null mutant than for the wild-type mitochondria, we make the hypothesis that the observed decrease in the turnover of the mutant enzyme could be linked to a proton translocation defect through F0.

J Biol Chem, 1999 Jan 1, 274(1), 354 - 9
In vivo characterization of chimeric phytochromes in yeast; Eichenberg K et al.; Phytochromes are plant photoreceptors that play a major role in photomorphogenesis . Two members of the phytochrome family have been characterized in some detail . Phytochrome A, which controls very low fluence and high irradiance responses, is rapidly degraded in the light, forms sequestered areas of phytochrome (SAPs), and does not exhibit dark reversion in monocotyledonous seedlings . Phytochrome B mediates red/far-red reversible responses, is stable in the light, and does not form SAPs . We report on the behavior in yeast of the phytochrome apoproteins of rice PHYA, tobacco PHYB, and chimeric PHYAB and PHYBA and on the behavior of the respective holoprotein adducts after assembly with phycocyanobilin chromophore (PHY*) . SAP-like formation in yeast was not observed for PHYB, but was detectable for PHYA, PHYAB, and PHYBA . Rice PHYA* did not undergo dark reversion in yeast . Surprisingly, all other tested phytochrome constructs did exhibit dark reversion, including chimeric phytochromes with a short N-terminal part of tobacco PHYB or parsley PHYA fused to rice PHYA . Furthermore, the proportion of phytochrome undergoing dark reversion and the rate of reversion were increased for both the N terminus-swapped constructs and PHYBA* . These results are discussed with respect to structure/function analysis of phytochromes A and B.

Anal Biochem, 1998 Dec 1, 265(1), 123 - 8
The yeast mitochondrial intermembrane space: purification and analysis of two distinct fractions; Martin H et al.; We have developed a protocol for the sequential release of the intermembrane space (IMS) content of Saccharomyces cerevisiae mitochondria . Two distinct fractions were obtained: a soluble IMS with cytochrome b2 as key marker and a salt-extractable IMS with cytochrome c as key marker . The identity of several proteins was determined by amino-terminal amino acid sequencing . The IMS fractions were devoid of contaminations from cytosol and mitochondrial outer and inner membranes . By subtraction analysis, the protein profiles of soluble and salt-extractable IMS fractions were depleted of contaminating bands derived from matrix proteins . The fractionation method will provide the basis for the further analysis of IMS proteins and characterization of their functions in bioenergetics, mitochondrial biogenesis, and regulatory processes.

Appl Microbiol Biotechnol, 1998 Nov, 50(5), 583 - 8
The influence of carbon source on the level and composition of ceramides of the Candida lipolytica yeast; Rupcic J et al.; Candida lipolytica yeast was grown batchwise on two different carbon sources, glucose and n-hexadecane . Free ceramides were quantitatively isolated from sphingolipid fractions of total lipids by a combination of column chromatography and preparative thin-layer chromatography . Their composition, after acid methanolysis, was analysed by gas-liquid chromatography . The ceramide content accounted for 2.6% of the total cell lipids in hexadecane-grown cells, which was 1.5 times higher than in glucose-grown cells . The fatty acid composition of ceramides was characterized by the predominance of fatty acids shorter than 20 carbon atoms and by high concentrations of fatty acids with 16 carbon atoms after growth on both carbon sources . The dominant fatty acid was hydroxylated 16:0 in the glucose-grown cells and 16:0 in the hexadecane-grown cells . The striking finding was the low degree of fatty acid hydroxylation and relatively high proportion of odd-numbered fatty acids in ceramide of the n-hexadecane-grown cells . The ceramides contained an unusual long-chain base composition . In hexadecane-grown cells more than 60% of the long-chain bases were C19 phytosphingosine . In glucose-grown cells more than one-half of the total long-chain bases were tetrahydroxy bases, 4,5-dihydroxysphinganine and 4,5-dihydroxyeicosasphinganine.

Biochemistry (Mosc), 1998 Nov, 63(11), 1307 - 11
Inactivation and conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions; Yang Y et al.; Conformational changes of yeast alcohol dehydrogenase in trifluoroethanol solutions have been followed by fluorescence emission and circular dichroism spectroscopy . At low concentration (less than 5%), trifluoroethanol shows a reversible inhibition competitive to ethanol and noncompetitive to NAD+ . The inhibition constants for native and structural-zinc-removed yeast alcohol dehydrogenase were 5.8 and 1.1 mM, respectively, suggesting that the active site becomes more flexible after the structural zinc is removed . At higher trifluoroethanol concentrations the enzyme was irreversibly inactivated . Comparison of inactivation and conformational changes of yeast alcohol dehydrogenase denatured in trifluoroethanol solutions shows that the extent of inactivation is larger than the extent of conformational changes at the same trifluoroethanol concentration . The results obtained from circular dichroism spectra show that the presence of trifluoroethanol can induce the formation of secondary structure of the enzyme.

J Cell Biol, 1998 Dec 28, 143(7), 1931 - 45
Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast; Pruyne DW et al.; The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth . Tropomyosin localizes specifically to actin cables and not cortical patches . Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle-associated Rab GTPase Sec4p depolarize rapidly . Bud growth ceases and the mother cell grows isotropically . When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays . Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p- dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence . With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables . Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth.

J Cell Biol, 1998 Dec 28, 143(7), 1775 - 87
Fission yeast bub1 is a mitotic centromere protein essential for the spindle checkpoint and the preservation of correct ploidy through mitosis; Bernard P et al.; The spindle checkpoint ensures proper chromosome segregation by delaying anaphase until all chromosomes are correctly attached to the mitotic spindle . We investigated the role of the fission yeast bub1 gene in spindle checkpoint function and in unperturbed mitoses . We find that bub1(+) is essential for the fission yeast spindle checkpoint response to spindle damage and to defects in centromere function . Activation of the checkpoint results in the recruitment of Bub1 to centromeres and a delay in the completion of mitosis . We show that Bub1 also has a crucial role in normal, unperturbed mitoses . Loss of bub1 function causes chromosomes to lag on the anaphase spindle and an increased frequency of chromosome loss . Such genomic instability is even more dramatic in Deltabub1 diploids, leading to massive chromosome missegregation events and loss of the diploid state, demonstrating that bub1(+ )function is essential to maintain correct ploidy through mitosis . As in larger eukaryotes, Bub1 is recruited to kinetochores during the early stages of mitosis . However, unlike its vertebrate counterpart, a pool of Bub1 remains centromere-associated at metaphase and even until telophase . We discuss the possibility of a role for the Bub1 kinase after the metaphase-anaphase transition.

Mol Gen Genet, 1998 Nov, 260(2-3), 295 - 9
Mouse homolog of the yeast origin recognition complex subunit ORC1 and chromosomal localization of the cognate mouse gene Orc1; Zisimopoulou P et al.; ORC1 encodes a subunit of the eukaryotic origin recognition complex in yeast, which has been shown to play a crucial role in chromosomal DNA replication and transcriptional silencing . We have identified a mouse protein closely related to Saccharomyces cerevisiae ORC1 as well as to the human, Xenopus and Drosophila homologs . MmORC1 has putative cyclin-dependent phosphorylation sites and a nucleotide triphosphate-binding motif . We have mapped the gene locus of the murine Orc1 gene to chromosome 4, band D . Conservation of structures among members of the ORC1-related proteins suggests that these proteins play a key role in the intiation of DNA replication in all eukaryotes.

Biochem Mol Biol Int, 1998 Dec, 46(5), 887 - 94
The effect of heparin and pentosan polysulfate on the thermal stability of yeast alcohol dehydrogenase; Paulikova H et al.; Heparin and pentosan polysulfate as organic polyanions inhibit yeast alcohol dehydrogenase (YADH) . The aim of this study was to determine the effect of heparin and pentosan polysulfate on the thermostability of alcohol dehydrogenase . Spectral and kinetic analyses showed that these compounds increase the thermal stability of the enzyme and eliminate entirely thermal aggregation . The thermostabilizing effect of unfractionated heparin and pentosan polysulfate was accelerated in the presence of NAD+ . The addition of NAD+ (11 microM) to the incubation medium decreased the inhibition of the YADH activity in the presence of pentosan polysulfate (1.32 microM) . Moreover, 38% of the residual activity of YADH was found after a 5-min incubation at 70 degrees C . These findings indicate that heparinoids not only modulate the enzyme activity but also can prevent the protein's thermal denaturation.

Cytogenet Cell Genet, 1998, 82(3-4), 147 - 52
Identification and characterization of STK12/Aik2: a human gene related to aurora of Drosophila and yeast IPL1; Kimura M et al.; Mutations in aurora of Drosophila and related Saccharomyces cerevisiae IPL1 protein kinases are known to cause abnormal chromosome segregation . We earlier isolated a cDNA encoding a novel human protein kinase Aik which shares high amino acid identity with the Aurora/Ipl1 protein kinase family . In the present study, a second human cDNA highly homologous to aurora/IPL1 (Aik2) was identified and the nucleotide sequence was determined (gene symbol STK12) . The C-terminal kinase domain of the STK12 encoded protein shares high amino acid sequence identity with those of mouse STK-1 (90%), rat AIM-1 (90%), human Aik (69%), mouse IAK1/Ayk1 (69%), Xenopus pEg2 (68%), Drosophila Aurora (62%), and yeast Ipl1 (45%), whereas the N-terminal domain of the STK12 protein shares little homology with those of Aurora/Ipl1 family members except for AIM-1 and STK-1 . Northern blotting analyses revealed that STK12 expression was high in thymus, while low level expression was detected in small intestine, testis, colon, spleen, and brain . The STK12 protein content in HeLa cells is low in S phase, but it accumulates during M phase . STK12 was mapped to human chromosome 17p13.1 by fluorescence in situ hybridization . The chromosome location of STK12 was further defined using a radiation hybrid panel (Stanford G3), that showed a linkage with marker WI-7901 (LOD Score 7.83) located between D17S938 and D17S786.

Mol Cell Biol, 1999 Jan, 19(1), 934 - 40
RNA polymerase II transcription suppresses nucleosomal modulation of UV-induced (6-4) photoproduct and cyclobutane pyrimidine dimer repair in yeast; Tijsterman M et al.; The nucleotide excision repair (NER) pathway is able to remove a wide variety of structurally unrelated lesions from DNA . NER operates throughout the genome, but the efficiencies of lesion removal are not the same for different genomic regions . Even within a single gene or DNA strand repair rates vary, and this intragenic heterogeneity is of considerable interest with respect to the mutagenic potential of carcinogens . In this study, we have analyzed the removal of the two major types of genotoxic DNA adducts induced by UV light, i.e., the pyrimidine (6-4)-pyrimidone photoproduct (6-4PP) and the cyclobutane pyrimidine dimer (CPD), from the Saccharomyces cerevisiae URA3 gene at nucleotide resolution . In contrast to the fast and uniform removal of CPDs from the transcribed strand, removal of lesions from the nontranscribed strand is generally less efficient and is modulated by the chromatin environment of the damage . Removal of 6-4PPs from nontranscribed sequences is also profoundly influenced by positioned nucleosomes, but this type of lesion is repaired at a much higher rate . Still, the transcribed strand is repaired preferentially, indicating that, as in the removal of CPDs, transcription-coupled repair predominates in the removal of 6-4PPs from transcribed DNA . The hypothesis that transcription machinery operates as the rate-determining damage recognition entity in transcription-coupled repair is supported by the observation that this pathway removes both types of UV photoproducts at equal rates without being profoundly influenced by the sequence or chromatin context.

Mol Cell Biol, 1999 Jan, 19(1), 585 - 93
The yeast a1 and alpha2 homeodomain proteins do not contribute equally to heterodimeric DNA binding; Jin Y et al.; In diploid cells of the yeast Saccharomyces cerevisiae, the alpha2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression . Although both proteins bind to the DNA, in the alpha2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro . This result brings up the question of the contribution of each protein in the heterodimer complex to the DNA-binding affinity and specificity . To determine the requirements for the a1-alpha2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-alpha2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro . Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the alpha2 or a1 protein . Interestingly, an alpha2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein . These results suggest that the specificity of alpha2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts . We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex . In contrast to the lack of a defective phenotype produced by mutations in the alpha2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-alpha2-mediated repression and DNA binding . This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex.

Mol Cell Biol, 1999 Jan, 19(1), 484 - 94
cDNA of the yeast retrotransposon Ty5 preferentially recombines with substrates in silent chromatin; Ke N et al.; The yeast retrotransposon Ty5 preferentially integrates into regions of silent chromatin . Ty5 cDNA also recombines with homologous sequences, generating tandem elements or elements that have exchanged markers between cDNA and substrate . In this study, we demonstrate that Ty5 integration depends upon the conserved DD(35)E domain of integrase and cis-acting sequences at the end of the long terminal repeat (LTR) implicated in integrase binding . cDNA recombination requires Rad52p, which is responsible for homologous recombination . Interestingly, Ty5 cDNA recombines at least three times more frequently with substrates in silent chromatin than with a control substrate at an internal chromosomal locus . This preference depends upon the Ty5 targeting domain that is responsible for integration specificity, suggesting that localization of cDNA to silent chromatin results in the enhanced recombination . Recombination with a telomeric substrate occasionally generates highly reiterated Ty5 arrays, and mechanisms for tandem element formation were explored by using a plasmid-based recombination assay . Point mutations were introduced into plasmid targets, and recombination products were characterized to determine recombination initiation sites . Despite our previous observation of the importance of the LTR in forming tandem elements, recombination cannot simply be explained by crossover events between the LTRs of substrate and cDNA . We propose an alternative model based on single-strand annealing, where single-stranded cDNA initiates tandem element formation and the LTR is required for strand displacement to form a looped intermediate . Retrotransposons are increasingly found associated with chromosome ends, and amplification of Ty5 by both integration and recombination exemplifies how retroelements can contribute to telomere dynamics.

Mol Cell Biol, 1999 Jan, 19(1), 384 - 91
Ribosomal protein L3 mutants alter translational fidelity and promote rapid loss of the yeast killer virus; Peltz SW et al.; Programmed -1 ribosomal frameshifting is utilized by a number of RNA viruses as a means of ensuring the correct ratio of viral structural to enzymatic proteins available for viral particle assembly . Altering frameshifting efficiencies upsets this ratio, interfering with virus propagation . We have previously demonstrated that compounds that alter the kinetics of the peptidyl-transfer reaction affect programmed -1 ribosomal frameshift efficiencies and interfere with viral propagation in yeast . Here, the use of a genetic approach lends further support to the hypothesis that alterations affecting the ribosome's peptidyltransferase activity lead to changes in frameshifting efficiency and virus loss . Mutations in the RPL3 gene, which encodes a ribosomal protein located at the peptidyltransferase center, promote approximately three- to fourfold increases in programmed -1 ribosomal frameshift efficiencies and loss of the M1 killer virus of yeast . The mak8-1 allele of RPL3 contains two adjacent missense mutations which are predicted to structurally alter the Mak8-1p . Furthermore, a second allele that encodes the N-terminal 100 amino acids of L3 (called L3Delta) exerts a trans-dominant effect on programmed -1 ribosomal frameshifting and killer virus maintenance . Taken together, these results support the hypothesis that alterations in the peptidyltransferase center affect programmed -1 ribosomal frameshifting.

Mol Cell Biol, 1999 Jan, 19(1), 86 - 98
Functional organization of the yeast SAGA complex: distinct components involved in structural integrity, nucleosome acetylation, and TATA-binding protein interaction; Sterner DE et al.; SAGA, a recently described protein complex in Saccharomyces cerevisiae, is important for transcription in vivo and possesses histone acetylation function . Here we report both biochemical and genetic analyses of members of three classes of transcription regulatory factors contained within the SAGA complex . We demonstrate a correlation between the phenotypic severity of SAGA mutants and SAGA structural integrity . Specifically, null mutations in the Gcn5/Ada2/Ada3 or Spt3/Spt8 classes cause moderate phenotypes and subtle structural alterations, while mutations in a third subgroup, Spt7/Spt20, as well as Ada1, disrupt the complex and cause severe phenotypes . Interestingly, double mutants (gcn5Delta spt3Delta and gcn5Delta spt8Delta) causing loss of a member of each of the moderate classes have severe phenotypes, similar to spt7Delta, spt20Delta, or ada1Delta mutants . In addition, we have investigated biochemical functions suggested by the moderate phenotypic classes and find that first, normal nucleosomal acetylation by SAGA requires a specific domain of Gcn5, termed the bromodomain . Deletion of this domain also causes specific transcriptional defects at the HIS3 promoter in vivo . Second, SAGA interacts with TBP, the TATA-binding protein, and this interaction requires Spt8 in vitro . Overall, our data demonstrate that SAGA harbors multiple, distinct transcription-related functions, including direct TBP interaction and nucleosomal histone acetylation . Loss of either of these causes slight impairment in vivo, but loss of both is highly detrimental to growth and transcription.

Mol Cell Biol, 1999 Jan, 19(1), 31 - 45
The yeast telomere length counting machinery is sensitive to sequences at the telomere-nontelomere junction; Ray A et al.; Saccharomyces cerevisiae telomeres consist of a continuous 325 +/- 75-bp tract of the heterogeneous repeat TG1-3 which contains irregularly spaced, high-affinity sites for the protein Rap1p . Yeast cells monitor or count the number of telomeric Rap1p molecules in a negative feedback mechanism which modulates telomere length . To investigate the mechanism by which Rap1p molecules are counted, the continuous telomeric TG1-3 sequences were divided into internal TG1-3 sequences and a terminal tract separated by nontelomeric spacers of different lengths . While all of the internal sequences were counted as part of the terminal tract across a 38-bp spacer, a 138-bp disruption completely prevented the internal TG1-3 sequences from being considered part of the telomere and defined the terminal tract as a discrete entity separate from the subtelomeric sequences . We also used regularly spaced arrays of six Rap1p sites internal to the terminal TG1-3 repeats to show that each Rap1p molecule was counted as about 19 bp of TG1-3 in vivo and that cells could count Rap1p molecules with different spacings between tandem sites . As previous in vitro experiments had shown that telomeric Rap1p sites occur about once every 18 bp, all Rap1p molecules at the junction of telomeric and nontelomeric chromatin (the telomere-nontelomere junction) must participate in telomere length measurement . The conserved arrangement of these six Rap1p molecules at the telomere-nontelomere junction in independent transformants also caused the elongated TG1-3 tracts to be maintained at nearly identical lengths, showing that sequences at the telomere-nontelomere junction had an effect on length regulation . These results can be explained by a model in which telomeres beyond a threshold length form a folded structure that links the chromosome terminus to the telomere-nontelomere junction and prevents telomere elongation.

Zentralbl Veterinarmed A, 1998 Nov, 45(9), 551 - 7
Selenite and selenium yeast as feed supplements to growing fattening pigs; Ortman K et al.; Twenty-four cross-bred fattening pigs weighing, on average, 24 kg were divided into three equal groups and fed a basic diet containing 0.1 mg selenium/kg . The diet of group A was supplemented with 0.3 mg selenium/kg derived from selenium yeast, group B with 0.1 mg selenium/kg from the same source, and group C with 0.3 mg/kg from sodium selenite . The pigs were slaughtered after 103 days . There was no significant difference between the mean whole blood selenium concentration of the pigs supplemented with selenium yeast neither after five nor after nine weeks of supplementation (group A, 201 and 213 micrograms/litre; group B 192 and 201 micrograms/litre, respectively), but the group supplemented with selenite had significantly lower concentrations both after five and nine weeks (158 and 159 micrograms/litre, respectively) . There were no significant differences between the mean activity of glutathione peroxidase (GSH-Px; in the whole blood of the three groups (group A, 348 and 327 mukat*/litre, group B, 342 and 302 mukat/litre; group C, 332 and 300 mukat/litre, after five and nine weeks, respectively) . The concentration of selenium in the liver of the pigs supplemented with organic selenium was significantly higher than in the pigs supplemented with selenite (group A, 0.54 mg/kg wet weight; group B, 0.54 mg/kg; group C, 0.40 mg/kg) . No deposits of lipofuscin or ceroid pigments were observed in the histological preparations of liver and heart from any of the pigs.

EMBO J, 1998 Dec 15, 17(24), 7454 - 68
Control of cleavage site selection during mRNA 3' end formation by a yeast hnRNP; Minvielle-Sebastia L et al.; Endonucleolytic cleavage of pre-mRNAs is the first step during eukaryotic mRNA 3' end formation . It has been proposed that cleavage factors CF IA, CF IB and CF II are required for pre-mRNA 3' end cleavage in yeast . CF IB is composed of a single polypeptide, Nab4p/Hrp1p, which is related to the A/B group of metazoan heterogeneous nuclear ribonucleoproteins (hnRNPs) that function as antagonistic regulators of 5' splice site selection . Here, we provide evidence that Nab4p/Hrp1p is not required for pre-mRNA 3' end endonucleolytic cleavage . We show that CF IA and CF II devoid of Nab4p/Hrp1p are sufficient to cleave a variety of RNA substrates but that cleavage occurs at multiple sites . Addition of Nab4p/Hrp1p prevents these alternative cleavages in a concentration-dependent manner, suggesting an essential and conserved role for some hnRNPs in pre-mRNA cleavage site selection.

EMBO J, 1998 Dec 15, 17(24), 7442 - 53
A role for the yeast La protein in U6 snRNP assembly: evidence that the La protein is a molecular chaperone for RNA polymerase III transcripts; Pannone BK et al.; The first protein that binds to all newly synthesized RNA polymerase III transcripts is a highly conserved phosphoprotein known as the La autoantigen . Although binding by the yeast La protein Lhp1p to pre-tRNAs is required for the normal pathway of tRNA maturation, the role of the La protein in the biogenesis of other polymerase III transcripts has been unclear . We identified a mutation in a novel component of the U6 snRNP that causes yeast cells to require Lhp1p for growth . This protein, Lsm8p, is a member of a family of proteins, known as Sm-like proteins, that shares two conserved motifs with the core Sm proteins of the U1, U2, U4 and U5 snRNPs . The lsm8-1 cells have drastically reduced levels of the mature U6 snRNP, consistent with a defect in U6 snRNP assembly . In these cells, Lhp1p stabilizes newly synthesized U6 RNA, thus facilitating assembly of the RNA into the U6 snRNP . These results provide evidence that Lhp1p is a molecular chaperone for polymerase III-transcribed RNAs and implicate Lsm8p as a key component in the very early steps of U6 snRNP assembly.

EMBO J, 1998 Dec 15, 17(24), 7416 - 29
Crm1p mediates regulated nuclear export of a yeast AP-1-like transcription factor; Yan C et al.; The yeast AP-1-like transcription factor, Yap1p, activates genes required for the response to oxidative stress . Yap1p is normally cytoplasmic and inactive, but will activate by nuclear translocation if cells are placed in an oxidative environment . Here we show that Yap1p is a target of the beta-karyopherin-like nuclear exporter, Crm1p . Yap1p is constitutively nuclear in a crm1 mutant, and Crm1p binds to a nuclear export sequence (NES)-like sequence in Yap1p in the presence of RanGTP . Recognition of Yap1p by Crm1p is inhibited by oxidation, and this inhibition requires at least one of the three cysteine residues flanking the NES . These results suggest that Yap1p localization is largely regulated at the level of nuclear export, and that the oxidation state affects the accessibility of the Yap1p NES to Crm1p directly . We also show that a mutation in RanGAP (rna1-1) is synthetically lethal with crm1 mutants . Yap1p export is inhibited in both rna1-1 and prp20 (RanGNRF) mutant strains, but Yap1p rapidly accumulates at the nuclear periphery after shifting rna1-1, but not other mutant cells to the non-permissive temperature . Thus, disassembly of export complexes in response to RanGTP hydrolysis may be required for release of substrate from a terminal binding site at the nuclear pore complex (NPC).

EMBO J, 1998 Dec 15, 17(24), 7190 - 8
Mutations in the pore regions of the yeast K+ channel YKC1 affect gating by extracellular K+; Vergani P et al.; The product of the Saccharomyces cerevisiae K+-channel gene YKC1 includes two pore-loop sequences that are thought to form the hydrophilic lining of the pore . Gating of the channel is promoted by membrane depolarization and is regulated by extracellular K+ concentration ({K+}o) both in the yeast and when expressed in Xenopus oocytes . Analysis of the wild-type current now shows that: (i) {K+}o suppresses a very slowly relaxing component, accelerating activation; (ii) {K+}o slows deactivation in a dose-dependent fashion; and (iii) Rb+, Cs+ and, to a lesser extent, Na+ substitute for K+ in its action on gating . We have identified single residues, L293 and A428, at equivalent positions within the two pore loops that affect the {K+}o sensitivity . Substitution of these residues gave channels with reduced sensitivity to {K+}o in macroscopic current kinetics and voltage dependence, but had only minor effects on selectivity among alkali cations in gating and on single-channel conductance . In some mutants, activation was slowed sufficiently to confer a sigmoidicity to current rise at low {K+}o . The results indicate that these residues are involved in {K+}o sensing . Their situation close to the permeation pathway points to an interaction between gating and permeation.

EMBO J, 1998 Dec 15, 17(24), 7170 - 8
Yeast mitochondrial F1F0-ATP synthase exists as a dimer: identification of three dimer-specific subunits; Arnold I et al.; Using the technique of blue native gel electrophoresis, the oligomeric state of the yeast mitochondrial F1F0-ATP synthase was analysed . Solubilization of mitochondrial membranes with low detergent to protein ratios led to the identification of the dimeric state of the ATP synthase . Analysis of the subunit composition of the dimer, in comparison with the monomer, revealed the presence of three additional small proteins . These dimer-specific subunits of the ATP synthase were identified as the recently described subunit e/Tim11 (Su e/Tim11), the putative subunit g homolog (Su g) and a new component termed subunit k (Su k) . Although, as shown here, these three proteins are not required for the formation of enzymatically active ATP synthase, Su e/Tim11 and Su g are essential for the formation of the dimeric state . Su e/Tim11 appears to play a central role in this dimerization process . The dimer-specific subunits are associated with the membrane bound F0-sector . The F0-sector may thereby be involved in the dimerization of two monomeric F1F0-ATP synthase complexes . We speculate that the F1F0-ATP synthase of yeast, like the other complexes of oxidative phosphorylation, form supracomplexes to optimize transduction of energy and to enhance the stability of the complex in the membrane.

Biochem J, 1999 Jan 1, 337 ( Pt 1), 29 - 35
A conserved motif in the yeast nucleolar protein Nop2p contains an essential cysteine residue; King M et al.; Nop2p is an essential nucleolar protein in Saccharomyces cerevisiae that is involved in large ribosomal subunit assembly . It has substantial homology with human p120, the proliferation-associated nucleolar antigen that is overexpressed in many human cancers . A motif containing an invariant Pro-Cys dipeptide is found in Nop2p, p120 and the bacterial Fmu proteins . A total of nine conserved residues, including Pro423 and Cys424, were individually altered in Nop2p by site-directed mutagenesis . Nop2p function was abolished by conversion of Cys424 into either alanine or serine . All of the other Nop2p mutations tested sustained yeast viability, including glycine replacement of Pro423 and the conversion of a second conserved cysteine into alanine . The crucial role of Cys424 in Nop2p is intriguing, due to the critical roles that cysteine residues adjacent to a proline have in a number of nucleotide-modifying enzymes.

Biochem J, 1999 Jan 1, 337 ( Pt 1), 13 - 7
Groucho/transducin-like enhancer of split (TLE) family members interact with the yeast transcriptional co-repressor SSN6 and mammalian SSN6-related proteins: implications for evolutionary conservation of transcription repression mechanisms; Grbavec D et al.; The yeast proteins TUP1 and SSN6 form a transcription repressor complex that is recruited to different promoters via pathway-specific DNA-binding proteins and regulates the expression of a variety of genes . TUP1 is functionally related to invertebrate and vertebrate transcriptional repressors of the Groucho/transducin-like Enhancer of split (TLE) family . The aim was to examine whether similar mechanisms underlie the transcription repression functions of TUP1 and Groucho/TLEs by determining whether TLE family members can interact with yeast SSN6 and mammalian SSN6-like proteins . It is shown in the present work that SSN6 binds to TLE1 and mediates transcriptional repression when expressed in mammalian cells . Moreover, TLE1 and TLE2 interact with two mammalian proteins related to SSN6, designated as the products of the ubiquitously transcribed tetratricopeptide-repeat genes on the Y (or X) chromosomes (UTY/X) . These findings suggest that mammalian TLE and UTY/X proteins may mediate repression mechanisms similar to those performed by TUP1-SSN6 in yeast.

Nat Biotechnol, 1998 Dec, 16(13), 1334 - 7
Identification of surrogate agonists for the human FPRL-1 receptor by autocrine selection in yeast; Klein C et al.; We describe a procedure for isolating agonists for mammalian G protein-coupled receptors of unknown function . Human formyl peptide receptor like-1 (FPRL-1) receptor, originally identified as an orphan G protein-coupled receptor related to the formyl peptide receptor (FPR1), was expressed in Saccharomyces cells designed to couple receptor activation to histidine prototrophy . Selection for histidine prototrophs among transformants obtained with a plasmid-based library encoding random peptides identified six different agonists, each of whose production yielded autocrine stimulation of the receptor expressed in yeast . A synthetic version of each peptide promoted activation of FPRL-1 expressed in human embryonic kidney (HEK293) cells, and five of the peptides exhibited significant selectivity for activation of FPRL-1 relative to FPR1 . One selective peptide was tested and found to mobilize calcium in isolated human neutrophils . This demonstrates that stimulation of FPRL-1 results in neutrophil activation and suggests that the receptor functions as a component of the inflammatory response . This autocrine selection protocol may be a generally applicable method for providing pharmacological tools to evaluate the physiological roles of the growing number of mammalian orphan G protein-coupled receptors.

J Protein Chem, 1998 Oct, 17(7), 591 - 8
Structural dissection of the DNA-binding domain of the yeast transcriptional activator GAL4 reveals an alpha-helical region responsible for dimerization; Gadhavi PL; Limited proteolysis of the DNA-binding domain (residues 1-147) of the yeast transcriptional activator GAL4 has been used to define more precisely the subdomain structure required for DNA binding and dimerization . Two regions of the protein were found to be resistant to proteolysis: the cysteine-rich, zinc-binding region (residues 6-43) and a hydrophobic sequence between residues 52 and 97 . Carboxy-terminal deletion fragments of the DNA-binding domain were generated and assayed by DNase 1 footprinting . This showed that the affinity of DNA binding depends on the sequence between residues 65 and 94 . Structural comparisons by UV circular dichroism (CD) were made and the difference CD spectra indicate that strong alpha-helical content is found specifically in the region between residues 65 and 94, which previous studies have shown to enable dimerization and in this study the formation of a stable protein-DNA complex.

J Biol Chem, 1998 Dec 18, 273(51), 34611 - 5
BMCP1, a novel mitochondrial carrier with high expression in the central nervous system of humans and rodents, and respiration uncoupling activity in recombinant yeast; Sanchis D et al.; We report here the cloning and functional analysis of a novel homologue of the mitochondrial carriers predominantly expressed in the central nervous system and referred to as BMCP1 (brain mitochondrial carrier protein-1) . The predicted amino acid sequence of this novel mitochondrial carrier indicates a level of identity of 39, 31, or 30%, toward the mitochondrial oxoglutarate carrier, phosphate carrier, or adenine nucleotide translocator, respectively, and a level of identity of 34, 38, or 39% with the mitochondrial uncoupling proteins UCP1, UCP2, or UCP3, respectively . Northern analysis of mouse, rat, or human tissues demonstrated that mRNA of this novel gene is mainly expressed in brain, although it is 10-30-fold less expressed in other tissues . In situ hybridization analysis of brain showed it is particularly abundant in cortex, hippocampus, thalamus, amygdala, and hypothalamus . Chromosomal mapping indicates that BMCP1 is located on chromosome X of mice and at Xq24 in man . Expression of the protein in yeast strongly impaired growth rate . Analysis of respiration of total recombinant yeast or yeast spheroplasts and in particular of the relationship between respiratory rate and membrane potential of yeast spheroplasts revealed a marked uncoupling activity of respiration, suggesting that although BMCP1 sequence is more distant from the uncoupling proteins (UCPs), this protein could be a fourth member of the UCP family.

J Biol Chem, 1998 Dec 18, 273(51), 34328 - 34
Evidence for a salt bridge between transmembrane segments 5 and 6 of the yeast plasma-membrane H+-ATPase; Gupta SS et al.; The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell . This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase . In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase . Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum . Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M . B., Ambesi, A., and Slayman, C . W . (1998) J . Biol . Chem . 273, 17411-17417) . To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated . Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping . These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide.

J Biol Chem, 1998 Dec 18, 273(51), 34240 - 6
Mutations in the AF-2/hormone-binding domain of the chimeric activator GAL4.estrogen receptor.VP16 inhibit hormone-dependent transcriptional activation and chromatin remodeling in yeast; Stafford GA et al.; GAL4.estrogen receptor.VP16 (GAL4.ER.VP16), which contains the GAL4 DNA-binding domain, the human ER hormone binding (AF-2) domain, and the VP16 activation domain, functions as a hormone-dependent transcriptional activator in yeast (Louvion, J.-F., Havaux-Copf, B., and Picard, D . (1993) Gene (Amst.) 131, 129-134) . Previously, we showed that this activator can remodel chromatin in yeast in a hormone-dependent manner . In this work, we show that a weakened VP16 activation domain in GAL4.ER.VP16 still allows hormone-dependent chromatin remodeling, but mutations in the AF-2 domain that abolish activity in the native ER also eliminate the ability of GAL4.ER.VP16 to activate transcription and to remodel chromatin . These findings suggest that an important role of the AF-2 domain in the native ER is to mask the activation potential of the AF-1 activation domain in the unliganded state; upon ligand activation, a conformational change releases AF-2-mediated repression and transcriptional activation ensues . We also show that the AF-2 domain, although inactive at simple promoters on its own in yeast, can enhance transcription by the MCM1 activator in hormone-dependent manner, consistent with its having a role in activation as well as repression in the native ER.

J Biol Chem, 1998 Dec 18, 273(51), 34180 - 9
Affinity purification and partial characterization of a yeast multiprotein complex for nucleotide excision repair using histidine-tagged Rad14 protein; Rodriguez K et al.; The nucleotide excision repair (NER) pathway of eukaryotes involves approximately 30 polypeptides . Reconstitution of this pathway with purified components is consistent with the sequential assembly of NER proteins at the DNA lesion . However, recent studies have suggested that NER proteins may be pre-assembled in a high molecular weight complex in the absence of DNA damage . To examine this model further, we have constructed a histidine-tagged version of the yeast DNA damage recognition protein Rad14 . Affinity purification of this protein from yeast nuclear extracts resulted in the co-purification of Rad1, Rad7, Rad10, Rad16, Rad23, RPA, RPB1, and TFIIH proteins, whereas none of these proteins bound to the affinity resin in the absence of recombinant Rad14 . Furthermore, many of the co-purifying proteins were present in approximately equimolar amounts . Co-elution of these proteins was also observed when the nuclear extract was fractionated by gel filtration, indicating that the NER proteins were associated in a complex with a molecular mass of >1000 kDa prior to affinity chromatography . The affinity purified NER complex catalyzed the incision of UV-irradiated DNA in an ATP-dependent reaction . We conclude that active high molecular weight complexes of NER proteins exist in undamaged yeast cells.

J Biol Chem, 1998 Dec 18, 273(51), 33889 - 92
A new protein conjugation system in human . The counterpart of the yeast Apg12p conjugation system essential for autophagy; Mizushima N et al.; Autophagy is an intracellular process for bulk degradation of cytoplasmic components . We recently found a protein conjugation system essential for autophagy in the yeast, Saccharomyces cerevisiae . The C-terminal glycine of a novel modifier protein, Apg12p, is conjugated to a lysine residue of Apg5p via an isopeptide bond . This conjugation reaction is mediated by Apg7p, a ubiquitin activating enzyme (E1)-like enzyme, and Apg10p, suggesting that it is a ubiquitination-like system (Mizushima, N., Noda, T., Yoshimori, T., Tanaka, Y., Ishii, T., George, M . D., Klionsky, D . J., Ohsumi, M . , and Ohsumi, Y . (1998) Nature 395, 395-398) . Although autophagy is a ubiquitous process in eukaryotic cells, no molecule involved in autophagy has yet been identified in higher eukaryotes . We reasoned that this conjugation system could be conserved . Here we report cloning and characterization of the human homologue of Apg12 (hApg12) . It is a 140-amino acid protein and possesses 27% identity and 48% similarity with the yeast Apg12p, but no apparent homology to ubiquitin . Northern blot analysis showed that its expression was ubiquitous in human tissues . We found that it was covalently attached to another protein . This target protein was identified to be the human Apg5 homologue (hApg5) . Mutagenic analyses suggested that this conjugation was formed via an isopeptide bond between the C-terminal glycine of hApg12 and Lys-130 of hApg5 . These findings indicate that the Apg12 system is well conserved and may function in autophagy also in human cells.

Science, 1998 Dec 11, 282(5396), 2022 - 8
Comparison of the complete protein sets of worm and yeast: orthology and divergence; Chervitz SA et al.; Comparative analysis of predicted protein sequences encoded by the genomes of Caenorhabditis elegans and Saccharomyces cerevisiae suggests that most of the core biological functions are carried out by orthologous proteins (proteins of different species that can be traced back to a common ancestor) that occur in comparable numbers . The specialized processes of signal transduction and regulatory control that are unique to the multicellular worm appear to use novel proteins, many of which re-use conserved domains . Major expansion of the number of some of these domains seen in the worm may have contributed to the advent of multicellularity . The proteins conserved in yeast and worm are likely to have orthologs throughout eukaryotes; in contrast, the proteins unique to the worm may well define metazoans.

Exp Cell Res, 1998 Dec 15, 245(2), 379 - 88
Heat stress-induced life span extension in yeast; Shama S et al.; The yeast Saccharomyces cerevisiae has a limited life span that can be measured by the number of times individual cells divide . Several genetic manipulations have been shown to prolong the yeast life span . However, environmental effects that extend longevity have been largely ignored . We have found that mild, nonlethal heat stress extended yeast life span when it was administered transiently early in life . The increased longevity was due to a reduction in the mortality rate that persisted over many cell divisions (generations) but was not permanent . The genes RAS1 and RAS2 were necessary to observe this effect of heat stress . The RAS2 gene is consistently required for maintenance of life span when heat stress is chronic or in its extension when heat stress is transient or absent altogether . RAS1, on the other hand, appears to have a role in signaling life extension induced by transient, mild heat stress, which is distinct from its life-span-curtailing effect in the absence of stress and its lack of involvement in the response to chronic heat stress . This distinction between the RAS genes may be partially related to their different effects on growth-promoting genes and stress-responsive genes . The ras2 mutation clearly hindered resumption of growth and recovery from stress, while the ras1 mutation did not . The HSP104 gene, which is largely responsible for induced thermotolerance in yeast, was necessary for life extension induced by transient heat stress . An interaction between mitochondrial petite mutations and heat stress was found, suggesting that mitochondria may be necessary for life extension by transient heat stress . The results raise the possibility that the RAS genes and mitochondria may play a role in the epigenetic inheritance of reduced mortality rate afforded by transient, mild heat stress .

Exp Cell Res, 1998 Dec 15, 245(2), 368 - 78
Role of RAS2 in recovery from chronic stress: effect on yeast life span; Shama S et al.; The replicative life span of Saccharomyces cerevisiae was previously shown to be modulated by the homologous signal transducers Ras1p and Ras2p in a reciprocal manner . We have used thermal stress as a life span modulator in order to uncover functional differences between the RAS genes that may contribute to their divergent effects on life span . Chronic exposure of cells throughout life to recurring heat shocks at sublethal temperatures decreased their replicative life span . ras2 mutants, however, suffered the largest decrease compared to wild-type and ras1 mutant cells . The decrease was correlated with a substantial delay in resumption of budding upon recovery from these heat shocks, indicating an impaired renewal of cell cycling . Detailed analysis of gene expression showed that, during recovery, ras2 mutants were selectively impaired in down-regulation of stress-responsive genes and up-regulation of growth-promoting genes . Our results suggest that one of the functions of RAS2 in maintaining life span, for which RAS1 does not substitute, is to ensure renewal of growth and cell division after bouts of stress that cells encounter during their life . This activity of RAS2 is effected by the cyclic AMP pathway . Overexpression of RAS2, but not RAS2(ser42) which is deficient in the activation of adenylate cyclase, completely reversed the effect of chronic stress on life span . Thus, RAS2 is limiting for longevity in the face of chronic stress . Since RAS2 is known to down-regulate stress responses, this demonstrates that for longevity the ability to recover from stress is at least as important as the ability to mount a stress response .

Eur J Biochem, 1998 Nov 15, 258(1), 19 - 28
Under respiratory growth conditions, Bcl-x(L) and Bcl-2 are unable to overcome yeast cell death triggered by a mutant Bax protein lacking the membrane anchor; Clow A et al.; We have reported earlier that cytosolic expression of the full-length human apoptosis inducer Bax-alpha (Bax) in the yeast Saccharomyces cerevisiae suppresses growth and induces mortality in cells containing functional mitochondria . Human Bcl-x(L) overcomes this toxicity . Here we describe that a mutant Bax protein, with a missing membrane anchor region (Bax delta), also inhibits growth and causes cell death in yeast . However, the death inhibitory proteins Bcl-x(L) and Bcl-2 fail to rescue Bax delta-mediated growth inhibition under conditions promoting respiration, although they bind Bax delta in the cell . Results in Jurkat T-cells corroborate that Bcl-x(L) is much less efficient at rescuing mammalian cells from the effect of Bax delta than from full length Bax . We have also inquired if the respiration-dependent toxicity of Bax and Bax delta in yeast is nullified by Bcl-x(L)delta and Bcl-2delta, molecules which lack membrane anchors but bind Bax in the yeast two-hybrid system . It appears that, under conditions which facilitate respiration in yeast, Bcl-x(L)delta and Bcl-2delta are incapable of rescuing both Bax-containing and Bax delta-containing cells . Our results open up the interesting possibility that there might exist proteins, unrelated to the Bcl-2 family, which could negate death induced by a membrane anchor-free form of Bax.

FEMS Microbiol Lett, 1998 Dec 1, 169(1), 191 - 7
Stress tolerance in a yeast sterol auxotroph: role of ergosterol, heat shock proteins and trehalose; Swan TM et al.; The role of ergosterol in yeast stress tolerance, together with heat shock proteins (hsps) and trehalose, was examined in a sterol auxotrophic mutant of Saccharomyces cerevisiae . Ergosterol levels paralleled viability data, with cells containing higher levels of the sterol exhibiting greater tolerances to heat and ethanol . Although the mutant synthesised hsps and accumulated trehalose upon heat shock to the same levels as the wild-type cells, these parameters did not relate to stress tolerance . These results indicate that the role of ergosterol in stress tolerance is independent of hsps or trehalose.

RNA, 1998 Dec, 4(12), 1610 - 22
The structure of the ITS2-proximal stem is required for pre-rRNA processing in yeast; Peculis BA et al.; Accurate and efficient processing of pre-rRNA is critical to the accumulation of mature functional ribosomal subunits for maintenance of cell growth . Processing requires numerous factors which act in trans as well as RNA sequence/ structural elements which function in cis . To examine the latter, we have used directed mutagenesis and expression of mutated pre-rRNAs in yeast . Specifically, we tested requirements for formation of an ITS2-proximal stem on processing, a structure formed by an interaction between sequences corresponding to the 3' end of 5.8S rRNA and the 5' end of 25S . Pre-rRNA processing is inhibited in templates encoding mutations that prevent the formation of the ITS2-proximal stem . Compensatory, double mutations, which alter the sequence of this region but restore the structure of the stem, also restore processing, although at lower efficiency . This reduction in efficiency is reflected in decreased levels of mature 5.8S and 25S rRNA and increased levels of 35S pre-rRNA and certain processing intermediates . This phenotype is reminiscent of the biochemical depletion of U8 snoRNA in vertebrates for which the ITS2-proximal stem has been proposed as a potential site for interaction with U8 RNP . Thus, formation of the ITS2-proximal stem may be a requirement common to yeast and vertebrate pre-rRNA processing.

Nucleic Acids Res, 1999 Jan 1, 27(1), 69 - 73
The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data; Hodges PE et al.; The Yeast Proteome Database (YPD) is a model for the organization and presentation of comprehensive protein information . Based on the detailed curation of the scientific literature for the yeast Saccharomyces cerevisiae, YPD contains more than 50 000 annotations lines derived from the review of 8500 research publications . The information concerning each of the approximately 6100 yeast proteins is structured around a convenient one-page format, the Yeast Protein Report, with additional information provided as pop-up windows . Protein classification schema have been revised this year, defining each protein's cellular role, function and pathway, and adding a Functional to the Yeast Protein Report . These changes provide the user with a succinct summary of the protein's function and its place in the biology of the cell, and they enhance the power of YPD Search functions . Precalculated sequence alignments have been added, to provide a crossover point for comparative genomics . The first transcript profiling data has been integrated into the YPD Protein Reports, providing the framework for the presentation of genome-wide functional data . The Yeast Proteome Database can be accessed on the Web at http://www.proteome.com/YPDhome.html

Hokkaido Igaku Zasshi, 1998 Sep, 73(5), 505 - 17
{Evaluation of yeast functional assay to detect p53 gene mutation in minute endoscopic samples of colonic mucosal neoplasia}; Nakamura Y; Mutation in the p53 tumor suppressor gene is known to play a critical role in the carcinogenesis of many types of human cancers, especially in colon carcinogenesis . To investigate the early stage of neoplastic processes, a simple and reliable method is needed to evaluate the state of p53 mutation . For this purpose, we tested the availability of the recently developed yeast functional assay to minute endoscopic samples . The assay consisted of 3 steps: 1) extraction of mRNA from the minute samples, 2) reverse transcriptional polymerase chain reaction (RT-PCR) amplification of the mRNA, and 3) transformation of yeasts by means of crude PCR products . This was an effective way to analyze many samples within a short period of time (2 days) . The results were visible either as white colonies (wild type) or red colonies (mutant type) . At first, we tested whether the quantity of mutants was measurable or not by analyzing a calibrated mixture of the cells known to harbor mutant and wild-type . The percentage of the red colonies that showed mutant p53 alleles was in proportion to the initial input percentage of the mutant p53 cells . Secondly, in clinical cases, it was found that minute samples (10-20 mg) were sufficient both for the analysis of yeast functional assays and pathologic examinations . The samples taken from normal mucosa and adenomas revealed fewer red colonies than the background value of this assay (10%) . All the samples from adenocarcinomas yielded red colonies over 20% . The percentage of red colonies was well consistent with the ratio of cancer cell numbers to total cells . However, the percentage was not always correlated with the number of immunohistochemically positive cells stained with monoclonal antibody to detect abnormal p53 . These results indicate that this assay combined with histological examination may provide useful information on determining carcinogenesis in endoscopic samples.

Mol Cell, 1998 Nov, 2(5), 675 - 82
The histone H3-like TAF is broadly required for transcription in yeast; Moqtaderi Z et al.; In yeast cells, independent depletion of TAFs (130, 67, 40, and 19) found specifically in TFIID results in selective effects on transcription, including a common effect on his3 core promoter function . In contrast, depletion of TAF17, which is also present in the SAGA histone acetylase complex, causes a decrease in transcription of most genes . However, TAF17-depleted cells maintain Ace1-dependent activation, and they induce de novo activation by heat shock factor in a manner predominantly associated with the activator, not the core promoter . Thus, TAF17 is broadly, but not universally, required for transcription in yeast, TAF17 depletion and TAF130 depletion each disrupt TFIID integrity yet cause different transcriptional consequences, suggesting that the widespread influence of TAF17 might not be due solely to its function in TFIID.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14886 - 90
Transgenic knockout mice exclusively expressing human hemoglobin S after transfer of a 240-kb betas-globin yeast artificial chromosome: A mouse model of sickle cell anemia; Chang JC et al.; Sickle cell anemia (SCA) and thalassemia are among the most common genetic diseases worldwide . Current approaches to the development of murine models of SCA involve the elimination of functional murine alpha- and beta-globin genes and substitution with human alpha and betas transgenes . Recently, two groups have produced mice that exclusively express human HbS . The transgenic lines used in these studies were produced by coinjection of human alpha-, gamma-, and beta-globin constructs . Thus, all of the transgenes are integrated at a single chromosomal site . Studies in transgenic mice have demonstrated that the normal gene order and spatial organization of the members of the human beta-globin gene family are required for appropriate developmental and stage-restricted expression of the genes . As the cis-acting sequences that participate in activation and silencing of the gamma- and beta-globin genes are not fully defined, murine models that preserve the normal structure of the locus are likely to have significant advantages for validating future therapies for SCA . To produce a model of SCA that recapitulates not only the phenotype, but also the genotype of patients with SCA, we have generated mice that exclusively express HbS after transfer of a 240-kb betas yeast artificial chromosome . These mice have hemolytic anemia, 10% irreversibly sickled cells in their peripheral blood, reticulocytosis, and other phenotypic features of SCA.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14799 - 804
The terminal tail region of a yeast myosin-V mediates its attachment to vacuole membranes and sites of polarized growth; Catlett NL et al.; The Saccharomyces cerevisiae myosin-V, Myo2p, has been implicated in the polarized movement of several organelles and is essential for yeast viability . We have shown previously that Myo2p is required for the movement of a portion of the lysosome (vacuole) into the bud and consequently for proper inheritance of this organelle during cell division . Class V myosins have a globular carboxyl terminal tail domain that is proposed to mediate localization of the myosin, possibly through interaction with organelle-specific receptors . Here we describe a myo2 allele whose phenotypes support this hypothesis . vac15-1/myo2-2 has a single mutation in this globular tail domain, causing defects in vacuole movement and inheritance . Although a portion of wild-type Myo2p fractionates with the vacuole, the myo2-2 gene product does not . In addition, the mutant protein does not concentrate at sites of active growth, the predominant location of wild-type Myo2p . Although deletion of the tail domain is lethal, the myo2-2 gene product retains the essential functions of Myo2p . Moreover, myo2-2 does not cause the growth defects and lethal genetic interactions seen in myo2-66, a mutant defective in the actin-binding domain . These observations suggest that the myo2-2 mutation specifically disrupts interactions with selected myosin receptors, namely those on the vacuole membrane and those at sites of polarized growth.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14640 - 5
Yeast chorismate mutase in the R state: simulations of the active site; Ma J et al.; The isomerization of chorismate to prephenate by chorismate mutase in the biosynthetic pathway that forms Tyr and Phe involves C5---O (ether) bond cleavage and C1---C9 bond formation in a Claisen rearrangement . Development of negative charge on the ether oxygen, stabilized by Lys-168 and Glu-246, is inferred from the structure of a complex with a transition state analogue (TSA) and from the pH-rate profile of the enzyme and the E246Q mutant . These studies imply a protonated Glu-246 well above pH 7 . Here, several 500-ps molecular dynamics simulations test the stability of enzyme-TSA complexes by using a solvated system with stochastic boundary conditions . The simulated systems are (i) protonated Glu-246 (stable), (ii) deprotonated Glu-246 (unstable), (iii) deprotonated Glu-246 plus one H2O between Glu-246 and the ether oxygen (unstable), (iv) the E246Q mutant (stable), and (v) addition of OH- between protonated Glu-246 and the ether oxygen . In (v), a local conformational change of Lys-168 displaced the OH- into the solvent region, suggesting a possible rate-determining step that precedes the catalytic step . In a 500-ps simulation of the enzyme complexed with the reactant chorismate or the product prephenate, no water molecule remained near the oxygen of the ligand . Calculations using the linearized Poisson-Boltzmann equation show that the effective pKa of Glu-246 is shifted from 5.8 to 8.1 as the negative charge on the ether oxygen of the TSA is changed from -0.56 electron to -0.9 electron . Altogether, these results support retention of a proton on Glu-246 to high pH and the absence of a water molecule in the catalytic steps.

Proc Natl Acad Sci U S A, 1998 Dec 8, 95(25), 14614 - 21
Comparative analyses of the secondary structures of synthetic and intracellular yeast MFA2 mRNAs; Doktycz MJ et al.; The overall folded (global) structure of mRNA may be critical to translation and turnover control mechanisms, but it has received little experimental attention . Presented here is a comparative analysis of the basic features of the global secondary structure of a synthetic mRNA and the same intracellular eukaryotic mRNA by dimethyl sulfate (DMS) structure probing . Synthetic MFA2 mRNA of Saccharomyces cerevisiae first was examined by using both enzymes and chemical reagents to determine single-stranded and hybridized regions; RNAs with and without a poly(A) tail were compared . A folding pattern was obtained with the aid of the MFOLD program package that identified the model that best satisfied the probing data . A long-range structural interaction involving the 5' and 3' untranslated regions and causing a juxtaposition of the ends of the RNA, was examined further by a useful technique involving oligo(dT)-cellulose chromatography and antisense oligonucleotides . DMS chemical probing of A and C nucleotides of intracellular MFA2 mRNA was then done . The modification data support a very similar intracellular structure . When low reactivity of A and C residues is found in the synthetic RNA, approximately 70% of the same sites are relatively more resistant to DMS modification in vivo . A slightly higher sensitivity to DMS is found in vivo for some of the A and C nucleotides predicted to be hybridized from the synthetic structural model . With this small mRNA, the translation process and mRNA-binding proteins do not block DMS modifications, and all A and C nucleotides are modified the same or more strongly than with the synthetic RNA.

Curr Biol, 1998 Dec 3, 8(24), 1310 - 21
Interaction of yeast Rvs167 and Pho85 cyclin-dependent kinase complexes may link the cell cycle to the actin cytoskeleton; Lee J et al.; Background: . PHO85 encodes the catalytic subunit of a cyclin-dependent kinase (Cdk) in budding yeast and functions in phosphate and glycogen metabolism . Pho85 associated with the G1 cyclins Pcl1 and Pcl2 is also required for cell cycle progression in the absence of the Cdc28 cyclins Cln1 and Cln2 . Loss of Pcl1, Pcl2 and related Pho85 cyclins results in budding defects, suggesting that Pcl-Pho85 complexes function in cell morphogenesis early in the cell cycle; their precise role is not clear, however . Results: . To identify targets for Pcl-Pho85 kinases, we performed yeast two-hybrid interaction screens using Pcl2 and the related cyclin Pcl9 . We identified RVS167, a gene involved in endocytosis, organization of the actin cytoskeleton, and cell survival after starvation . Like rvs167Delta mutants, pho85 mutants or strains deleted for the Pcl1,2-type Pho85 cyclins showed abnormal cell morphology on starvation, sensitivity to salt, random budding in diploids, and defects in endocytosis and in the actin cytoskeleton . Overexpression of Rvs167 in wild-type cells caused morphological abnormalities and growth arrest at high temperatures; these phenotypes were exacerbated by deleting PHO85 . Rvs167 has a Src homology 3 (SH3) domain and five potential Pho85 phosphorylation sites; recombinant Rvs167 was phosphorylated by the Pcl2-Pho85 kinase in vitro . Maximal phosphorylation of Rvs167 in vivo required Pho85 and the Pcl1,2-type cyclins . Conclusions: . Rvs167 interacts with Pho85 cyclins and is implicated as a target of Pho85 kinases in vivo . Our results identify a connection between Cdks and the actin cytoskeleton; interaction of Rvs167 and Pcl-Pho85 Cdks might contribute to actin cytoskeleton regulation in response to stresses such as starvation.

Mol Biol Cell, 1998 Dec, 9(12), 3533 - 45
Specific molecular chaperone interactions and an ATP-dependent conformational change are required during posttranslational protein translocation into the yeast ER; McClellan AJ et al.; The posttranslational translocation of proteins across the endoplasmic reticulum (ER) membrane in yeast requires ATP hydrolysis and the action of hsc70s (DnaK homologues) and DnaJ homologues in both the cytosol and ER lumen . Although the cytosolic hsc70 (Ssa1p) and the ER lumenal hsc70 (BiP) are homologous, they cannot substitute for one another, possibly because they interact with specific DnaJ homologues on each side of the ER membrane . To investigate this possibility, we purified Ssa1p, BiP, Ydj1p (a cytosolic DnaJ homologue), and a GST-63Jp fusion protein containing the lumenal DnaJ region of Sec63p . We observed that BiP, but not Ssa1p, is able to associate with GST-63Jp and that Ydj1p stimulates the ATPase activity of Ssa1p up to 10-fold but increases the ATPase activity of BiP by <2-fold . In addition, Ydj1p and ATP trigger the release of an unfolded polypeptide from Ssa1p but not from BiP . To understand further how BiP drives protein translocation, we purified four dominant lethal mutants of BiP . We discovered that each mutant is defective for ATP hydrolysis, fails to undergo an ATP-dependent conformational change, and cannot interact with GST-63Jp . Measurements of protein translocation into reconstituted proteoliposomes indicate that the mutants inhibit translocation even in the presence of wild-type BiP . We conclude that a conformation- and ATP-dependent interaction of BiP with the J domain of Sec63p is essential for protein translocation and that the specificity of hsc70 action is dictated by their DnaJ partners.

Mol Biol Cell, 1998 Dec, 9(12), 3383 - 97
The syntaxin Tlg1p mediates trafficking of chitin synthase III to polarized growth sites in yeast; Holthuis JC et al.; Tlg1p and Tlg2p, members of the syntaxin family of SNAREs in yeast, have been implicated in both endocytosis and the retention of late Golgi markers . We have investigated the functions of these and the other endocytic syntaxins Pep12p and Vam3p . Remarkably, growth is possible in the absence of all four proteins . In the absence of the others, Pep12p and Tlg1p can each create endosomes accessible to the endocytic tracer dye FM4-64 . However, although Pep12p is required for the ligand-induced internalization of the alpha factor receptor and its passage via Pep12p-containing membranes to the vacuole, Tlg1p is not . In contrast, Tlg1p is required for the efficient localization of the catalytic subunit of chitin synthase III (Chs3p) to the bud neck, a process that involves endocytosis and polarized delivery of Chs3p . In wild-type cells, internalized Chs3p cofractionates with Tlg1p and Tlg2p, and in a strain lacking the other endocytic syntaxins, either Tlg1p or Tlg2p is sufficient for correct localization of the enzyme . Pep12p is neither necessary nor sufficient for this process . We conclude that there are two endocytic routes in yeast that can operate independently and that Tlg1p is located at the junction of one of these with the polarized exocytic pathway.

Biochemistry, 1998 Nov 24, 37(47), 16591 - 600
Relationship between conserved consensus site residues and the productive conformation for the TPQ cofactor in a copper-containing amine oxidase from yeast; Schwartz B et al.; A highly conserved asparagine residue is contained in the consensus site sequences of all known copper-containing amine oxidases (CAOs) . On the basis of published crystallographic structures, the asparagine is found to reside proximal to the active site redox cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ) . In this study, the conserved asparagine was changed to an alanine in a CAO from Hansenula polymorpha expressed in Saccharomyces cerevisiae, and the mutant's catalytic properties were characterized using steady-state kinetics and resonance Raman spectroscopy . Several lines of evidence point to TPQ exisiting in an nonproductive orientation in the mutant, including reductions in several steady-state parameters and an accumulation of an inactive product Schiff base complex when the enzyme is incubated with methylamine as the substrate . This product Schiff base complex was previously found to form following mutation of another conserved consensus site residue, a glutamate (or aspartate) at the C + 1 position from TPQ {Cai, D., Dove, J., Nakamura, N., Sanders-Loehr, J., and Klinman, J . P . (1997) Biochemistry 36, 11472-11478} . The results suggest that these two residues are crucial in maintaining the balance of cofactor mobility versus rigidity expected to be necessary during the dual processes of biogenesis and catalysis, respectively, that all CAOs must accomplish . In addition, a previously unidentified structural linkage between these two highly conserved residues is proposed which spans both subunits of the dimeric CAOs, and may have implications for intersubunit communication.

J Immunol Methods, 1998 Nov 1, 220(1-2), 179 - 88
A yeast surface display system for the discovery of ligands that trigger cell activation; Cho BK et al.; Opposing cells often communicate signalling events using multivalent interactions between receptors present on their cell surface . For example, T cells are typically activated when the T cell receptor (TCR) and its associated costimulatory molecules are multivalently engaged by the appropriate ligands present on an antigen presenting cell . In this report, yeast expressing high cell-surface levels of a TCR ligand (a recombinant antibody to the TCR Vbeta domain) were shown to act as 'pseudo' antigen presenting cells and induce T cell activation as monitored by increased levels of CD25 and CD69 and by downregulation of cell surface TCR . Similar levels of T cell activation could occur even when a 30-fold excess of irrelevant yeast was present, suggesting that such a yeast display system, by virtue of its ability to present ligands multivalently, may be used in highly sensitive procedures to identify novel polypeptides that interact multivalently with cell surface receptors and thereby trigger specific cellular responses.

Plant J, 1998 Oct, 16(2), 163 - 72
Expression of a yeast-derived invertase in developing cotyledons of Vicia narbonensis alters the carbohydrate state and affects storage functions; Weber H et al.; In plants the carbohydrate state provides signals to adjust metabolism to specific physiological conditions . Storage-active sink organs like seeds often contain high levels of sucrose . In order to change the sugar status during seed development a yeast-derived invertase gene was expressed in Vicia narbonensis under control of the LeguminB4 promoter . A signal sequence targeted the invertase to the apoplast in maturing embryos . In the cotyledons, sucrose was decreased whereas hexoses strongly accumulated . There was a major reduction of starch whereas proteins were less affected . Vacuoles of cotyledon cells were enlarged and dry seeds wrinkled . Transcripts and enzyme activity of sucrose synthase, the small and large subunit of ADP-glucose pyrophosphorylase as well as vicilin were downregulated . Sucrose phosphate synthase and legumin-mRNAs were not affected . Analysing single seeds with different sucrose levels revealed a positive correlation of sucrose concentration to mRNA levels of sucrose synthase and most pronounced to ADP-glucose pyrophosphorylase-mRNA levels as well as to starch content . Glucose on the other hand did not show any correlation . After feeding 14C-sucrose in vitro, the invertase-expressing cotyledons partitioned less carbon into starch compared to the wild-type . In the transgenic cotyledons, a relatively higher amount was directed into proteins compared to starch . We conclude that starch accumulation in developing cotyledons could be a function of sucrose concentration . Our results are consistent with a possible sucrose-mediated induction of storage-associated differentiation indicated by upregulation of specific genes of the starch synthesis pathway.

Acta Microbiol Pol, 1998, 47(2), 131 - 40
Genetical and biochemical characterization of cycle Krebs mutant in yeast; Boniewska-Bernacka E et al.; We present the results of genetical and biochemical investigations of yeast mutants which presumably are deficient in different steps of the Krebs cycle . The investigation of the activities of the Krebs cycle enzymes suggests that we have isolated two types of mutants: structural and regulatory.

Biochim Biophys Acta, 1998 Nov 26, 1443(1-2), 139 - 48
Yeast ribosomal proteins L4, L17, L20, and L25 exhibit different binding characteristics for the yeast 35S precursor rRNA; Yeh LC et al.; In vitro synthesized radioactive yeast 35S precursor rRNA (35S pre-rRNA) molecules were used to determine the binding characteristics of 13 proteins from the yeast 60S ribosome subunit . L4, L17, L20 and L25 were found to bind the 35S pre-rRNA molecule in vitro in the absence of any other cellular components as determined by a modified membrane filtration assay and an agarose gel mobility shift assay . In all cases, RNA-protein complex formation was proportional to the amount of protein added to the binding reaction mixture . Binding to the pre-rRNA could be saturated yielding a molar RNA/protein ratio approaching one . Non-radioactive 35S pre-rRNA transcript competed for the binding in a dosage-dependent manner . Presence of 18S rRNA species and poly(A) did not affect their binding to the 35S RNA . However, in the presence of the 25S rRNA species, the four proteins exhibited distinct binding characteristics for the pre-rRNA molecule . L4 did not bind the 25S rRNA but interacted specifically with the 35S pre-rRNA molecule with a binding constant of 4.4x10(6)/M . L17 bound the pre-rRNA molecule preferentially (Ka=17x10(6)/M) but also bound the mature 25S rRNA species (Ka=10x10(6)/M) . L20 bound both the pre-rRNA molecule and the 25S rRNA species equally well (Ka=11-12x10(6)/M) . L25 also bound both the 35S pre-rRNA and the mature 25S rRNA with slightly different affinities, with Ka=3.1 vs . 2.5x10(6)M, respectively . We speculate that L4, L17, and L25 are among the early assembled ribosomal proteins but L4 may be one of the first ribosomal proteins that bind to the 35S pre-rRNA molecule during ribosome biogenesis.

J Biol Chem, 1998 Dec 11, 273(50), 33795 - 802
Reconstitution of yeast RNA polymerase I transcription in vitro from purified components . TATA-binding protein is not required for basal transcription; Keener J et al.; Five purified protein components, RNA polymerase I, Rrn3p, core factor, TBP (TATA-binding protein), and upstream activation factor, are sufficient for high level transcription in vitro from the Saccharomyces cerevisiae rDNA promoter . Rrn3p and pol I form a complex in solution that is active in specific initiation . Three protein components, pol I, Rrn3p, and core factor, and promoter sequence to -38, suffice for basal transcription . Unlike pol II and pol III, yeast pol I basal transcription does not require TBP . Instead, TBP, upstream activation factor, and the upstream element of the promoter together stimulate pol I basal transcription to a fully activated level . The role of TBP in pol I transcription is fundamentally different from its role in pol II or pol III transcription.

J Biol Chem, 1998 Dec 11, 273(50), 33360 - 6
Y'-Help1, a DNA helicase encoded by the yeast subtelomeric Y' element, is induced in survivors defective for telomerase; Yamada M et al.; The yeast Y' element is a highly polymorphic repetitive sequence present in the subtelomeric regions of many yeast telomeres . The Y' element is classed as either Y'-L or Y'-S, depending on its length . It has been reported that survivors arising from telomerase-deficient yeast mutants compensate for telomere loss by the amplification of Y' elements . The total Saccharomyces cerevisiae genome DNA data base was searched for Y' elements, and 11 Y'-Ls and eight Y'-Ss were identified . As reported previously, many of the sequences were found to contain long open reading frames which potentially encode helicase . We examined the expression of the Y' elements in telomerase-deficient Deltatlc1 survivors, in which the TLC1 gene encoding the yeast telomerase template RNA had been disrupted, and found that the Y' element is highly expressed in the survivors, but not in the wild-type cells . Moreover, we demonstrated that the survivors produce a Y'-encoded protein designated as Y'-Help1 (Y'-helicase protein 1), and that this protein possesses helicase activity . Therefore, we suggest that the Y' element has a novel and potentially important role in trans, in addition to the well characterized role in cis, in telomerase-independent telomere maintenance in yeast.

J Biol Chem, 1998 Dec 11, 273(50), 33273 - 8
Targeting to the endoplasmic reticulum in yeast cells by determinants present in transmembrane domains; Letourneur F et al.; The transmembrane domains (TMDs) of many type I integral membrane proteins contain determinants that cause localization in the endoplasmic reticulum (ER) in mammalian cells by an unknown mechanism . Here we show that the yeast ER localization machinery recognizes determinants in TMDs that are very similar to those identified previously in mammalian cells . These determinants are recognized in post-ER compartments and recycled back to the ER, thus acting as ER retrieval signals . Moreover determinants in TMDs are inefficiently sorted in several previously characterized yeast mutants with defects in the ER retrieval machinery . Similar ER retrieval signals are also recognized in the TMDs of polytopic integral membrane proteins, apparently by the same sorting machinery . The isolation of new mutants defective in sorting of membrane determinants might provide a better understanding of the molecular mechanisms involved in this process.

J Biol Chem, 1998 Dec 11, 273(50), 33111 - 4
Preferential binding of yeast Rad4.Rad23 complex to damaged DNA; Jansen LE et al.; The yeast Rad4 and Rad23 proteins form a complex that is involved in nucleotide excision repair (NER) . Their function in this process is not known yet, but genetic data suggest that they act in an early step in NER . We have purified an epitope-tagged Rad4.Rad23 (tRad4 . Rad23) complex from yeast cells, using a clone overproducing Rad4 with a hemagglutinin-tag at its C terminus . tRad4.Rad23 complex purified by both conventional and immuno-affinity chromatography complements the in vitro repair defect of rad4 and rad23 mutant extracts, demonstrating that these proteins are functional in NER . Using electrophoretic mobility shift assays, we show preferential binding of the tRad4.Rad23 complex to damaged DNA in vitro . UV-irradiated, as well as N-acetoxy-2-(acetylamino)fluorene-treated DNA, is efficiently bound by the protein complex . These data suggest that Rad4.Rad23 interacts with DNA damage during NER and may play a role in recognition of the damage.

J Mol Biol, 1998 Dec 11, 284(4), 975 - 88
Yeast RNase III as a key processing enzyme in small nucleolar RNAs metabolism; Chanfreau G et al.; The variety of biogenesis pathways for small nucleolar RNAs (snoRNAs) reflects the diversity of their genomic organization . We have searched for yeast snoRNAs which are affected by the depletion of the yeast ortholog of bacterial RNase III, Rnt1 . In a yeast strain inactivated for RNT1, almost half of the snoRNAs tested are depleted with significant accumulation of monocistronic or polycistronic precursors . snoRNAs from both major families of snoRNAs (C/D and H/ACA) are affected by RNT1 disruption . In vitro, recombinant Rnt1 specifically cleaves pre-snoRNA precursors in the absence of other factors, generating intermediates which require the action of other enzymes for processing to the mature snoRNA . Most Rnt1 cleavage sites fall within potentially double-stranded regions closed by tetraloops with a novel consensus sequence AGNN . These results demonstrate that biogenesis of a large number of snoRNAs from the two major families of snoRNAs requires a common RNA endonuclease and a putative conserved structural motif .

Front Biosci, 1998 Dec 01, 3, D1241 - 52
Inhibition of internal entry site (IRES)-mediated translation by a small yeast RNA: a novel strategy to block hepatitis C virus protein synthesis; Das S et al.; The observation that poliovirus mRNA is not translated in the yeast Saccharomyces cerevisiae has led to the discovery of a small RNA (60 nt, called IRNA, inhibitor RNA) which was later shown to specifically inhibit internal ribosome entry site (IRES)-mediated translation of naturally uncapped mRNAs . Translation of cellular capped mRNAs was not significantly inhibited by IRNA . IRNA also specifically inhibited hepatitis C virus (HCV) IRES-mediated translation in vitro and in vivo . A hepatoma cell line constitutively expressing IRNA was refractory to infection by a chimeric poliovirus (PV/HCV) in which PV IRES is replaced by HCV-IRES . In contrast, a PV/EMCV chimeric virus containing the EMCV IRES was not significantly inhibited in the IRNA-hepatoma cell line compared to the control hepatoma cells . UV-crosslinking studies showed that the IRNA binds a number of cellular proteins that appear to be important for IRES-mediated translation . Interaction of these proteins with the viral IRES elements is believed to be important in recruiting ribosomes to the 5( UTR of viral RNAs . The binding of the purified La autoantigen to the HCV IRES element was efficiently and specifically competed by IRNA . These results provide a basis for development of novel drugs effective against HCV infection.

Semin Cell Dev Biol, 1998 Oct, 9(5), 527 - 33
Novel pathways, membrane coats and PI kinase regulation in yeast lysosomal trafficking; Burd CG et al.; Analysis of membrane transport in the yeast Saccharomyces cerevisiae continues to provide important insights into the molecular mechanisms that direct endocytic and lysosomal sorting pathways in eukaryotic cells . Recent findings include the identification of two novel endomembrane transport pathways, a Golgi-to-vacuole biosynthetic pathway requiring the adaptor protein-3 (AP-3) complex, and a vacuolar membrane recycling pathway regulated by PtdIns(3,5)P2 . At the molecular level, a candidate vesicle coat protein complex mediating endosome-to-Golgi recycling has been identified . In addition, protein sorting signals directing phosphorylation-dependent ubiquitination of endocytic cargoes, and a recognition motif for AP-3-dependent sorting have been characterized . Important mechanistic insights into SNARE-mediated, NSF-dependent membrane fusion reactions also have been made using yeast-based in vitro assays and the identification of the zinc-binding FYVE domain as a PtdIns(3)P-specific binding domain has linked phosphoinositide signaling to the regulation of vesicle docking/fusion, as well as other membrane transport reactions along the lysosomal sorting pathways .

J Cell Biol, 1998 Nov 30, 143(5), 1167 - 82
Involvement of long chain fatty acid elongation in the trafficking of secretory vesicles in yeast; David D et al.; Members of the synaptobrevin/VAMP family of v-SNAREs are thought to be essential for vesicle docking and exocytosis in both lower and higher eukaryotes . Here, we describe yeast mutants that appear to bypass the known v-SNARE requirement in secretion . Recessive mutations in either VBM1 or VBM2, which encode related ER-localized membrane proteins, allow yeast to grow normally and secrete in the absence of Snc v-SNAREs . These mutants show selective alterations in protein transport, resulting in the differential trafficking and secretion of certain protein cargo . Yet, processing of the vacuolar marker, carboxypeptidase Y, and the secreted protein, invertase, appear normal in these mutants indicating that general protein trafficking early in the pathway is unaffected . Interestingly, VBM1 and VBM2 are allelic to ELO3 and ELO2, two genes that have been shown recently to mediate the elongation of very long chain fatty acids and subsequent ceramide and inositol sphingolipid synthesis . Thus, the v-SNARE requirement in constitutive exocytosis is abrogated by mutations in early components of the secretory pathway that act at the level of lipid synthesis to affect the ability of secretory vesicles to sort and deliver protein cargo.

Genetics, 1998 Dec, 150(4), 1407 - 17
Mapping of a yeast G protein betagamma signaling interaction; Dowell SJ et al.; The mating pathway of Saccharomyces cerevisiae is widely used as a model system for G protein-coupled receptor-mediated signal transduction . Following receptor activation by the binding of mating pheromones, G protein betagamma subunits transmit the signal to a MAP kinase cascade, which involves interaction of Gbeta (Ste4p) with the MAP kinase scaffold protein Ste5p . Here, we identify residues in Ste4p required for the interaction with Ste5p . These residues define a new signaling interface close to the Ste20p binding site within the Gbetagamma coiled-coil . Ste4p mutants defective in the Ste5p interaction interact efficiently with Gpa1p (Galpha) and Ste18p (Ggamma) but cannot function in signal transduction because cells expressing these mutants are sterile . Ste4 L65S is temperature-sensitive for its interaction with Ste5p, and also for signaling . We have identified a Ste5p mutant (L196A) that displays a synthetic interaction defect with Ste4 L65S, providing strong evidence that Ste4p and Ste5p interact directly in vivo through an interface that involves hydrophobic residues . The correlation between disruption of the Ste4p-Ste5p interaction and sterility confirms the importance of this interaction in signal transduction . Identification of the Gbetagamma coiled-coil in Ste5p binding may set a precedent for Gbetagamma-effector interactions in more complex organisms.

Can J Microbiol, 1998 Aug, 44(8), 718 - 22
Candida ipomoeae, a new yeast species related to large-spored Metschnikowia species; Lachance MA et al.; Numerous strains of an unusual asexual yeast species were isolated from flowers of morning glory (Ipomoea spp., Convolvulaceae) and associated drosophilids and sap beetles of the genus Conotelus sampled in Hawaii and in Brazil . The nutritional profile of this yeast is similar to those of Metschnikowia hawaiiensis and Metschnikowia continentalis, which share the same habitats . The cells are large, hydrophobic, and tend to remain attached after budding, causing the colonies on agar media to have a convoluted appearance, reminiscent of popcorn . The sequences of the D1/D2 domain of large subunit rDNAs of strains from three different localities confirmed that a single species is involved, and that it is related to large-spored Metschnikowia species . The type strain is UWO(PS)91-672.1 (CBS 8466).

Mol Gen Genet, 1998 Oct, 260(1), 108 - 14
The yeast nuclear gene DSS1, which codes for a putative RNase II, is necessary for the function of the mitochondrial degradosome in processing and turnover of RNA; Dziembowski A et al.; The yeast nuclear gene DSS1 codes for a mitochondrial protein containing regions of homology to bacterial RNase II and can act as a multicopy suppressor of a deletion of the SUV3 gene, which encodes an RNA helicase . In order to establish the function of the DSS1 gene in mitochondrial biogenesis we studied RNA metabolism in yeast strains disrupted for SUV3 or DSS1 . The results indicate that in the absence of DSS1 the in vitro activity of 3'-5' exoribonuclease is abolished and mitochondrial translation is blocked . In disruption strains harboring intronless mitochondrial genomes steady-state levels of COB mRNA and 16S rRNA were very low, while in the presence of a mitochondrial genome containing the omega intron in the 21S rRNA gene the excised intron accumulates . Moreover we observed an accumulation of precursors of 21S rRNA and the VAR1 mRNA . All these phenotypes are virtually identical to those of strains in which SUV3 is disrupted . We suggest that the DSS1 gene product, like the SUV3 gene product, is a subunit of the yeast mitochondrial degradosome (mtEXO), and that this protein complex participates in intron-independent turnover and processing of mitochondrial transcripts . In addition our studies exclude any role for the NUC1 nuclease in these phenomena.

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1431 - 43
Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, five new related yeast species from flowers and associated insects; Lachance MA et al.; Five new yeast species, Wickerhamiella australiensis, Wickerhamiella cacticola, Wickerhamiella occidentalis, Candida drosophilae and Candida lipophila, are described to accommodate isolates recovered from flowers and floricolous insects of Australian Hibiscus trees, cosmopolitan morning glories (Ipomoea spp.) and Brazilian cereoid cacti . The new Wickerhamiella species are heterothallic, occur in the haploid condition and are clearly separated reproductively from one another . Although they exhibit little physiological variation, they are easily delineated from Wickerhamiella domercqiae, the only species known previously, by their resistance to cycloheximide and the production of strong extracellular lipases . C . drosophilae and C . lipophila share the latter property, but unlike the Wickerhamiella species, they fail to utilize nitrate as sole nitrogen source . PFGE indicates that these yeasts have an unusually low number of chromosomes . The large-subunit rDNA (D1/D2) sequences demonstrate a close relationship between the five species and Candida vanderwaltii and Candida azyma . Their relationship with W . domercqiae is more distant, but all share, with some other Candida species, a single monophyletic clade . The type and isotype strains are as follows: W . australiensis strains UWO(PS)95-604.3T (h+; CBS 8456T) and UWO(PS)95-631.3I (h-; CBS 8457I); W . cacticola strains UFMG96-267T (h+; CBS 8454T) and UFMG96-381I (h-; CBS 8455I); W . occidentalis strains UWO(PS)91-698.4T (h+; CBS 8452T) and UFMG96-212I (h-; CBS 8453I); C . drosophilae UWO(PS)91-716.3T (CBS 8459T); and C . lipophila UWO(PS)91-681.3T (CBS 8458T).

Int J Syst Bacteriol, 1998 Oct, 48 Pt 4, 1413 - 7
The yeast genus Starmerella gen . nov . and Starmerella bombicola sp . nov., the teleomorph of Candida bombicola (Spencer, Gorin & Tullock) Meyer & Yarrow; Rosa CA et al.; Seven strains of a heterothallic haploid yeast species were isolated from flowers of Calystegia sepium (hedge bindweed, Convolvulaceae) and associated sap beetles of the genus Conotelus . Conjugation was observed between some of the isolates and the type strain of Candida bombicola, resulting in evanescent asci with one ascospore with a convoluted surface . The sequences of the D1/D2 variable domain of the large subunit of the rDNAs of three strains differed by only one or two bases from that of the type strain . The new genus Starmerella, with the single species Starmerella bombicola, is proposed to accommodate the teleomorph of C . bombicola . The designated isotype is strain UWO(PS)97-118I (H-; CBS 8451I).

Biosens Bioelectron, 1998 Oct 1, 13(7-8), 831 - 8
Development of a green fluorescent protein reporter for a yeast genotoxicity biosensor; Billinton N et al.; A reporter system, constructed for a laboratory screen for new genes involved in DNA repair in the brewer's yeast Saccharomyces cerevisiae, has been developed for use in a genotoxicity biosensor . The strain produces green fluorescent protein (yEGFP) when DNA damage has occurred . yEGFP is codon optimised for yeasts . The reporter does not respond to chemicals which delay mitosis, and responds appropriately to the genetic regulation of DNA repair . Data is presented which demonstrate strain improvements appropriate to biosensor technology: improved signal to noise ratio, ease of data collection and uncomplicated material handling.

Arch Environ Contam Toxicol, 1999 Jan, 36(1), 7 - 12
Action of inorganic tin and organotins on a hydrocarbon-using yeast, Candida maltosa; Tobin JM et al.; Two inorganic tin compounds, as well as mono- and tri-substituted methyl-, butyl-, and phenyltins were examined for their interactions with a hydrocarbon-using strain of Candida maltosa . Neither of the inorganic tins (SnII and SnIV) inhibited growth at concentrations up to 0.8 mM while binding to the yeast cells occurred to levels of 0.3 and 0.23 mM Sn/g cells, respectively . Neither inorganic tin caused leakage of potassium from the yeast cells . Among the organotins, tributyl- and triphenyltins caused total viability loss and near maximum potassium loss at initial concentrations of 0.08 mM . For these compounds binding to the cells increased with increasing initial concentrations to maximum values of 0.51 and 0.65 mM Sn/g cells respectively . The other organotin compounds were not inhibitory and did not cause potassium leakage from the cells . Tin from them became cell associated only in the cases of monobutyl- and monophenyl tin, which were bound at markedly lower levels (<0.1 mM Sn/g cells) . These results are consistent with the conclusion that the inhibitory compounds can act on the yeast cell membrane but, although binding to the cells is a prerequisite for growth inhibition and cell leakage, no simple correlation was established between binding levels and toxicity effects.

Nucleic Acids Res, 1998 Dec 1, 26(23), 5402 - 8
Differential regulation of two closely clustered yeast genes, MAG1 and DDI1, by cell-cycle checkpoints; Zhu Y et al.; Eukaryotic DNA-damage checkpoint genes have been shown to not only arrest cells at certain stages, but are also involved in the transcriptional response to DNA damage . However, while the signal transduction for cell-cycle checkpoint is well characterized, it is not clear whether the same signal transduction pathway is responsible for the regulation of all DNA damage-inducible genes . In order to understand how different checkpoint genes are involved in gene regulation, the effects of various checkpoint mutations on the expression of a unique yeast MAG1 - DDI1 dual promoter were examined in this study . MAG1 and DDI1 are transcribed from a common promoter region and co-induced by a variety of DNA damaging agents . However, gene-specific cis -acting elements were also identified, and the two genes are indeed differentially expressed under certain conditions . We found that DDI1 induction was not affected in any of the checkpoint mutants . In contrast, MAG1 induction was completely abolished in the pol2 and rad53 mutants . However, in the mec1-1 or any of the G1/S and G2/M checkpoint mutants, including rad9, rad17 and rad24, DNA damage-induced MAG1 expression was not significantly affected, and a rad9 rad17 double mutation only slightly reduced MAG1 induction . Based on this and previous studies, we present two models for the role of checkpoint genes in transcriptional regulation in response to DNA damage.

J Mol Biol, 1998 Dec 4, 284(3), 673 - 87
Functional and structural characterization of the prp3 binding domain of the yeast prp4 splicing factor; Ayadi L et al.; Nuclear pre-mRNA splicing occurs in a large RNA-protein complex containing four small nuclear ribonucleoprotein particles (snRNPs) and additional protein factors . The yeast Prp4 (yPrp4) protein is a specific component of the U4/U6 and U4/U6-U5 snRNPs, which associates transiently with the spliceosome before the first step of splicing . In this work, we used the in vivo yeast two-hybrid system and in vitro immunoprecipitation assays to show that yPrp4 interacts with yPrp3, another U4/U6 snRNP protein . To investigate the domain of yPrp4 that directly contacts yPrp3, we introduced deletions in the N-terminal half of yPrp4 and point mutations in the C-terminal half of the molecule, and we tested the resulting prp4 mutants for cell viability and for their ability to interact with yPrp3 . We could not define any particular sequence in the first 161 amino acid residues that are specifically required for protein-protein interactions . However, deletion of a small basic-rich region of 30 amino acid residues is lethal to the cells . Analysis of the C terminus prp4 mutants obtained clearly shows that this region of yPrp4 represents the primary domain of interaction with yPrp3 . Interestingly, yPrp4 shows significant similarity in its C-terminal half to the beta-subunits of G proteins . We have generated a three-dimensional computer model of this domain, consisting of a seven-bladed beta-propeller based on the crystalline structure of beta-transducin . Several lines of evidence suggested that yPrp4 is contacting yPrp3 through a large flat surface formed by the long variable loops linking the beta-strands of the propeller . This surface could be used as a scaffold for generating an RNA-protein complex .

J Biol Chem, 1998 Nov 27, 273(48), 32254 - 64
Identification of highly conserved amino-terminal segments of dTAFII230 and yTAFII145 that are functionally interchangeable for inhibiting TBP-DNA interactions in vitro and in promoting yeast cell growth in vivo; Kotani T et al.; TFIID is a multiprotein complex composed of TBP and several TAFIIs . Small amino-terminal segments (TAF N-terminal domain (TAND)) of Drosophila TAFII230 (dTAFII230) and yeast TAFII145 (yTAFII145) bind strongly to TBP and inhibit TBP-DNA interactions . yTAFII145 TAND (yTAND) was divided into two subdomains, yTANDI10-37 and yTANDII46-71, that function cooperatively . Here, we identify dTANDII within the amino terminus of dTAFII230 at 118-143 amino acids in addition to dTANDI18-77, reported previously . dTANDII exhibits pronounced sequence similarity to yTANDII, and the two were shown to be functionally equivalent in binding to TBP and inhibiting TBP-DNA interactions in vitro . Alanine scanning mutation analysis demonstrated that Phe-57 (yTANDII) and Tyr-129 (dTANDII) are critically required for the interaction with TBP . Yeast strains containing mutant yTAFII145 lacking yTANDI or yTANDII showed a temperature-sensitive growth phenotype . The conserved core of dTANDII could substitute for the yTANDII core, and Phe-57 or Tyr-129 described above was critically required for the function of this segment in promoting normal cell growth at 37 degreesC . In these respects, the impact of yTANDII mutations on cell growth paralleled their effects on TBP binding in vitro, strongly suggesting that the yTAFII145-TBP interaction and its negative effects on TFIID binding to core promoters are physiologically important.

J Biol Chem, 1998 Nov 27, 273(48), 31648 - 51
Involvement of cell surface glycosyl-phosphatidylinositol-linked aspartyl proteases in alpha-secretase-type cleavage and ectodomain solubilization of human Alzheimer beta-amyloid precursor protein in yeast; Komano H et al.; Human beta-amyloid precursor protein (APP) introduced into yeast undergoes alpha-secretase-type cleavage, suggesting that yeast have alpha-secretase-like protease(s) . Here we report that two structurally and functionally related glycosyl-phosphatidylinositol-linked yeast aspartyl proteases, Mkc7p and Yap3p (collectively termed yapsin), are responsible for alpha-secretase-type cleavage of APP expressed in yeast, resulting in release of soluble APP into the extracellular space . Disruption of MKC7 and YAP3 in a vacuolar protease-deficient strain abolished this APP cleavage/release, and APP cleavage/release could be restored by introduction of MKC7 or YAP3 on a single copy plasmid . Purified Mkc7p cleaved an internally quenched fluorogenic APP peptide substrate at the alpha-secretase cleavage site . Measurement of proteolytic activity either in yeast homogenates or on the yeast cell surface revealed that most Mkc7p and Yap3p activities were localized at the cell surface . These results establish a molecular basis for alpha-secretase-type cleavage in yeast and support the generally held concept that alpha-secretase cleavage of APP occurs at the cell surface.

EMBO J, 1998 Nov 16, 17(22), 6541 - 50
Recognition specificity of individual EH domains of mammals and yeast; Paoluzi S et al.; The Eps homology (EH) domain is a recently described protein binding module that is found, in multiple or single copies, in several proteins in species as diverse as human and yeast . In this work, we have investigated the molecular details of recognition specificity mediated by this domain family by characterizing the peptide-binding preference of 11 different EH domains from mammal and yeast proteins . Ten of the eleven EH domains could bind at least some peptides containing an Asn-Pro-Phe (NPF) motif . By contrast, the first EH domain of End3p preferentially binds peptides containing an His-Thr/Ser-Phe (HT/SF) motif . Domains that have a low affinity for the majority of NPF peptides reveal some affinity for a third class of peptides that contains two consecutive amino acids with aromatic side chains (FW or WW) . This is the case for the third EH domain of Eps15 and for the two N-terminal domains of YBL47c . The consensus sequences derived from the peptides selected from phage-displayed peptide libraries allows for grouping of EH domains into families that are characterized by different NPF-context preference . Finally, comparison of the primary sequence of EH domains with similar or divergent specificity identifies a residue at position +3 following a conserved tryptophan, whose chemical characteristics modulate binding preference.

Science, 1998 Nov 20, 282(5393), 1511 - 6
The role of Far1p in linking the heterotrimeric G protein to polarity establishment proteins during yeast mating; Butty AC et al.; Heterotrimeric guanosine triphosphate (GTP)-binding proteins (G proteins) determine tissue and cell polarity in a variety of organisms . In yeast, cells orient polarized growth toward the mating partner along a pheromone gradient by a mechanism that requires Far1p and Cdc24p . Far1p bound Gbetagamma and interacted with polarity establishment proteins, which organize the actin cytoskeleton . Cells containing mutated Far1p unable to bind Gbetagamma or polarity establishment proteins were defective for orienting growth toward their mating partner . In response to pheromones, Far1p moves from the nucleus to the cytoplasm . Thus, Far1p functions as an adaptor that recruits polarity establishment proteins to the site of extracellular signaling marked by Gbetagamma to polarize assembly of the cytoskeleton in a morphogenetic gradient.

Folia Microbiol (Praha), 1998, 43(5), 443 - 52
Glucose repression of maltase and methanol-oxidizing enzymes in the methylotrophic yeast Hansenula polymorpha: isolation and study of regulatory mutants; Alamae T et al.; Regulation of the synthesis of maltase and methanol-oxidizing enzymes by the carbon source has been analyzed in the methylotrophic yeast Hansenula polymorpha . Maltase was shown to be responsible for the growth of H . polymorpha not only on maltose, but also on sucrose . The affinity of maltase towards maltase substrates decreased in the order: 4-nitrophenyl glucoside (PNPG) < sucrose < maltose . Mutants with glucose repression-insensitive synthesis of alcohol oxidase and maltase were obtained from H . polymorpha by mutagenesis and subsequent selection on methanol medium in the presence of 2-deoxy-D-glucose . One of the isolated mutants, L63, was studied in more detail . Mutant L63 was recessive and monogenic and it was not deficient in hexokinase . Its analysis revealed that H . polymorpha most probably has a repressor protein that in the presence of glucose can down-regulate expression of both maltase and enzymes of methanol oxidation.

Mol Cell Biol, 1998 Dec, 18(12), 7064 - 74
Reversible association between the V1 and V0 domains of yeast vacuolar H+-ATPase is an unconventional glucose-induced effect; Parra KJ et al.; The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification . The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore . Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains . To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes . Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p . Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required . A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly . Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A . We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.

Mol Cell Biol, 1998 Dec, 18(12), 7009 - 19
Requirement of PKR dimerization mediated by specific hydrophobic residues for its activation by double-stranded RNA and its antigrowth effects in yeast; Patel RC et al.; The roles of protein dimerization and double-stranded RNA (dsRNA) binding in the biochemical and cellular activities of PKR, the dsRNA-dependent protein kinase, were investigated . We have previously shown that both properties of the protein are mediated by the same domain . Here we show that dimerization is mediated by hydrophobic residues present on one side of an amphipathic alpha-helical structure within this domain . Appropriate substitution mutations of residues on that side produced mutants with increased or decreased dimerization activities . Using these mutants, we demonstrated that dimerization is not essential for dsRNA binding . However, enhancing dimerization artificially, by providing an extraneous dimerization domain, increased dsRNA binding of both wild-type and mutant proteins . In vitro, the dimerization-defective mutants could not be activated by dsRNA but were activated normally by heparin . In Saccharomyces cerevisiae, unlike wild-type PKR, these mutants could not inhibit cell growth and the dsRNA-binding domain of the dimerization-defective mutants could not prevent the antigrowth effect of wild-type PKR . These results demonstrate the biological importance of the dimerization properties of PKR.

J Cell Sci, 1998 Dec 18, 111 ( Pt 24), 3585 - 96
Localization and regulation of the cdk-activating kinase (Cak1p) from budding yeast; Kaldis P et al.; Eukaryotic cell cycles are controlled by the activities of cyclin-dependent kinases (cdks) . The major cdk in budding yeast, Saccharomyces cerevisiae, is Cdc28p . Activation of Cdc28p requires phosphorylation on threonine 169 and binding to a cyclin . Thr-169 is phosphorylated by the cdk-activating kinase (CAK), Cak1p, which was recently identified as the physiological CAK in budding yeast . Here we present our further characterization of yeast Cak1p . We have found that Cak1p is dispersed throughout the cell as shown by immunofluorescence; biochemical subcellular fractionation confirmed that most of the Cak1p is found in the cytoplasm . Cak1p is a monomeric enzyme in crude yeast lysates . Mutagenesis of potential sites of activating phosphorylation had little effect on the activity of Cak1p in vitro or in vivo . Furthermore, Cak1p contains no posttranslational modifications detectable by two-dimensional isoelectric focusing . We found that Cak1p is a stable protein during exponential growth but that its expression decreases considerably when cells enter stationary phase . In contrast, Cak1p levels oscillate dramatically during meiosis, reflecting regulation at both the transcriptional and post-translational level . The localization and regulation of Cak1p are in contrast to those of the known vertebrate CAK, p40(MO15).

Yeast, 1998 Jun 30, 14(9), 813 - 25
Quantitative imaging of TATA-binding protein in living yeast cells; Patterson GH et al.; We describe the quantitative monitoring of TATA-binding protein (TBP) localization and expression in living Saccharomyces cerevisiae cells . We replaced the endogenous TBP with a green fluorescent protein (GFP) x TBP fusion, which was imaged quantitatively by laser scanning confocal microscopy (LSCM) . When GFP x TBP expression was altered by using various promoters, the levels measured by LSCM correlated well with the levels determined by immunoblot of whole cell extract protein . These results show that GFP x TBP imaging not only offers a method of measurement equivalent to a more conventional technique but also provides real-time quantitation in living cells and subcellular localization information . Time-lapse confocal imaging of GFP x TBP in mitotic yeast cells revealed that it remains localized to the nucleus and displays an asymmetric distribution (1:0.7) between mother and daughter cells . Based on this and data from a mutant which underexpresses GFP x TBP, we suggest that intracellular levels of TBP are near rate-limiting for growth and viability.

Yeast, 1998 Jun 30, 14(9), 805 - 12
Development of a transformation system for the multinuclear yeast Dipodascus (Endomyces) magnusii; Adamikova L et al.; We have developed the first system for genetic transformation of the multinuclear yeast Dipodascus magnusii . The system is based on a dominant selectable marker and an autonomously replicating sequence . We have constructed a plasmid vector which contains a marker conferring resistance to zeocin and the segment of non-transcribed spacer of D . magnusii ribosomal DNA which supports the autonomous replication of plasmid DNA in yeast cells . Plasmid DNA has been transferred into D . magnusii cells by electroporation.

J Microencapsul, 1998 Nov-Dec, 15(6), 761 - 73
Microencapsulation in yeast cells; Bishop JR et al.; A method for encapsulating high concentrations of essential oils into bakers' yeast (Saccharomyces cerevisiae) is described . The process involves mixing an aqueous suspension of yeast and an essential oil, which allows the oil to pass freely through the cell wall and membrane and remain passively within the cell . Oil droplets sequestered within the cell were clearly visible using confocal microscopy . Transmission electron microscopy demonstrated that the cell wall and membrane remain intact during the process . Cells quickly lost viability during the process and it appeared unnecessary for the cells to be viable for the process to occur . Encapsulated oil was recovered from the cells using a water/ethanol extraction procedure and analysed by gas chromatography . No significant differences were noted between encapsulated and unencapsulated oil profiles . The rate of permeation of oil into the yeast cells was found to increase significantly at higher temperatures due to the phase transition of the lipid membrane . The rates at which different essential oils permeated the cell varied considerably due to variations in terpene chemistry . The encapsulation of straight chain hydrocarbons highlighted the effects of molecular size, shape and the presence of hydroxl groups on the process . The process occurs by passive diffusion as a result of hydrophobic flavour components partitioning into the cell membrane and intracellular lipid . This paper briefly reviews the patented literature and reports some of the initial observations of the transport mechanisms involved during the accumulation of essential oils by yeast cells.

J Cell Biol, 1998 Nov 16, 143(4), 935 - 46
The high osmolarity glycerol response (HOG) MAP kinase pathway controls localization of a yeast golgi glycosyltransferase; Reynolds TB et al.; The yeast alpha-1,3-mannosyltransferase (Mnn1p) is localized to the Golgi by independent transmembrane and lumenal domain signals . The lumenal domain is localized to the Golgi complex when expressed as a soluble form (Mnn1-s) by exchange of its transmembrane domain for a cleavable signal sequence (Graham, T . R., and V . A . Krasnov . 1995 . Mol . Biol . Cell . 6:809-824) . Mutants that failed to retain the lumenal domain in the Golgi complex, called lumenal domain retention (ldr) mutants, were isolated by screening mutagenized yeast colonies for those that secreted Mnn1-s . Two genes were identified by this screen, HOG1, a gene encoding a mitogen-activated protein kinase (MAPK) that functions in the high osmolarity glycerol (HOG) pathway, and LDR1 . We have found that basal signaling through the HOG pathway is required to localize Mnn1-s to the Golgi in standard osmotic conditions . Mutations in HOG1 and LDR1 also perturb localization of intact Mnn1p, resulting in its loss from early Golgi compartments and a concomitant increase of Mnn1p in later Golgi compartments.

J Cell Biol, 1998 Nov 16, 143(4), 921 - 33
A role for the DnaJ homologue Scj1p in protein folding in the yeast endoplasmic reticulum; Silberstein S et al.; Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ . Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER . However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants . Here, we show that the Deltascj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins . Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Deltascj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced . Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation . The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER . We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.

RNA, 1998 Nov, 4(11), 1321 - 31
Poly(A)-tail-promoted translation in yeast: implications for translational control; Preiss T et al.; The cap structure and the poly(A) tail synergistically activate mRNA translation in vivo . Recent work using Saccharomyces cerevisiae spheroplasts and a yeast cell-free translation system revealed that the poly(A) tail can function as an independent promotor for ribosome recruitment, to internal initiation sites within an mRNA . This raises the question of how regulatory upstream open reading frames and translational repressor proteins binding to the 5'UTR can function, as well as how regulated polyadenylation can support faithful activation of protein synthesis . We investigated the function of the regulatory upstream open reading frame 4 from the yeast GCN 4 gene and the effect of IRP-1 binding to an iron-responsive element introduced into the 5' UTR of reporter mRNAs . Both manipulations effectively block cap-dependent translation, whereas ribosome recruitment promoted by the poly(A) tail under non-competitive conditions can efficiently bypass both blocks . We show that the synergistic use of both, the cap structure and the poly-A tail enforced by mRNA competition reinstates the full extent of translational control by both types of 5' UTR regulatory elements . With a view towards regulated polyadenylation, we studied the function of poly(A) tails of defined length on the translation of capped mRNAs . We find that poly(A) tail elongation increases translational efficiency, particularly under competitive conditions . Our results integrate recent findings on the function of the poly(A) tail into an understanding of translational control.

J Cell Biol, 1998 Nov 2, 143(3), 737 - 49
Polymerization of purified yeast septins: evidence that organized filament arrays may not be required for septin function; Frazier JA et al.; The septins are a family of proteins required for cytokinesis in a number of eukaryotic cell types . In budding yeast, these proteins are thought to be the structural components of a filament system present at the mother-bud neck, called the neck filaments . In this study, we report the isolation of a protein complex containing the yeast septins Cdc3p, Cdc10p, Cdc11p, and Cdc12p that is capable of forming long filaments in vitro . To investigate the relationship between these filaments and the neck filaments, we purified septin complexes from cells deleted for CDC10 or CDC11 . These complexes were not capable of the polymerization exhibited by wild-type preparations, and analysis of the neck region by electron microscopy revealed that the cdc10Delta and cdc11Delta cells did not contain detectable neck filaments . These results strengthen the hypothesis that the septins are the major structural components of the neck filaments . Surprisingly, we found that septin dependent processes like cytokinesis and the localization of Bud4p to the neck still occurred in cdc10Delta cells . This suggests that the septins may be able to function in the absence of normal polymerization and the formation of a higher order filament structure.

J Cell Biol, 1998 Nov 2, 143(3), 687 - 94
Time-lapse microscopy reveals unique roles for kinesins during anaphase in budding yeast; Straight AF et al.; The mitotic spindle is a complex and dynamic structure . Genetic analysis in budding yeast has identified two sets of kinesin-like motors, Cin8p and Kip1p, and Kar3p and Kip3p, that have overlapping functions in mitosis . We have studied the role of three of these motors by video microscopy of motor mutants whose microtubules and centromeres were marked with green fluorescent protein . Despite their functional overlap, each motor mutant has a specific defect in mitosis: cin8Delta mutants lack the rapid phase of anaphase B, kip1Delta mutants show defects in the slow phase of anaphase B, and kip3Delta mutants prolong the duration of anaphase to the point at which the spindle becomes longer than the cell . The kip3Delta and kip1Delta mutants affect the duration of anaphase, but cin8Delta does not.

J Cell Biol, 1998 Nov 2, 143(3), 577 - 88
Molecular architecture of the yeast nuclear pore complex: localization of Nsp1p subcomplexes; Fahrenkrog B et al.; The nuclear pore complex (NPC), a supramolecular assembly of approximately 100 different proteins (nucleoporins), mediates bidirectional transport of molecules between the cytoplasm and the cell nucleus . Extensive structural studies have revealed the three- dimensional (3D) architecture of Xenopus NPCs, and eight of the approximately 12 cloned and characterized vertebrate nucleoporins have been localized within the NPC . Thanks to the power of yeast genetics, 30 yeast nucleoporins have recently been cloned and characterized at the molecular level . However, the localization of these nucleoporins within the 3D structure of the NPC has remain elusive, mainly due to limitations of preparing yeast cells for electron microscopy (EM) . We have developed a new protocol for preparing yeast cells for EM that yielded structurally well-preserved yeast NPCs . A direct comparison of yeast and Xenopus NPCs revealed that the NPC structure is evolutionarily conserved, although yeast NPCs are 15% smaller in their linear dimensions . With this preparation protocol and yeast strains expressing nucleoporins tagged with protein A, we have localized Nsp1p and its interacting partners Nup49p, Nup57p, Nup82p, and Nic96p by immuno-EM . Accordingly, Nsp1p resides in three distinct subcomplexes which are located at the entry and exit of the central gated channel and at the terminal ring of the nuclear basket.

J Biol Chem, 1998 Nov 20, 273(47), 31541 - 6
Affinity of yeast nucleotide excision repair factor 2, consisting of the Rad4 and Rad23 proteins, for ultraviolet damaged DNA; Guzder SN et al.; Saccharomyces cerevisiae Rad4 and Rad23 proteins are required for the nucleotide excision repair of UV light-damaged DNA . Previous studies have indicated that these two DNA repair proteins are associated in a tight complex, which we refer to as nucleotide excision repair factor 2 (NEF2) . In a reconstituted nucleotide excision repair reaction, incision of UV-damaged DNA is dependent on NEF2, indicating a role of NEF2 in an early step of the repair process . NEF2 does not, however, possess an enzymatic activity, and its function in the damage-specific incision reaction has not yet been defined . Here we use a DNA mobility shift assay to demonstrate that NEF2 binds specifically to UV-damaged DNA . Elimination of cyclobutane pyrimidine dimers from the UV-damaged DNA by enzymatic photoreactivation has little effect on the affinity of NEF2 for the DNA, suggesting that NEF2 recognizes the 6-(1, 2)-dihydro-2-oxo-4-pyrimidinyl)-5-methyl-2,4-(1H,3H)-pyrimidinedione photoproducts in the damaged DNA . These results highlight the intricacy of the DNA damage-demarcation reaction during nucleotide excision repair in eukaryotes.

J Biol Chem, 1998 Nov 20, 273(47), 30903 - 8
Interaction of Kar2p and Sls1p is required for efficient co-translational translocation of secreted proteins in the yeast Yarrowia lipolytica; Boisrame A et al.; The yeast Yarrowia lipolytica is a model organism for in vivo study of the signal recognition particle-dependent targeting pathway . In this report, we defined solubilization conditions and set up a fractionation procedure of Y . lipolytica microsomes to determine the amounts of Sec61p-containing translocation pores linked to ribosomes . In contrast to Saccharomyces cerevisiae, from 70 to 80% of Sec61p associates with ribosomes in this yeast . The chaperone protein Kar2p and the Sls1p product, a resident protein of the endoplasmic reticulum lumen, partially fractionate with this Sec61p population . Moreover, Sls1p can be co-immunoprecipitated with Kar2p, and the two polypeptides are shown to directly interact in the yeast two-hybrid system . A site-directed mutagenesis was performed on the SLS1 coding sequence that allowed us to define a functional domain in Sls1p . Indeed, co-translational translocation of a reporter protein is affected when one of these mutant proteins is expressed . Moreover, this protein has lost its capacity to interact with Kar2p, and the two lumenal polypeptides might thus cooperate to promote secretory protein co-translational translocation.

Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13783 - 7
The three yeast A kinases have specific signaling functions in pseudohyphal growth; Robertson LS et al.; The three yeast A kinase catalytic subunit isoforms are redundant for viability . We demonstrate that they have dramatically different roles in pseudohyphal development: Tpk2 is essential, whereas Tpk3 inhibits . Tpk1 has no discernible effect . Two-hybrid analysis identified the transcription factor Sfl1 as a protein that interacts specifically with Tpk2, but not Tpk1 or Tpk3 . Deletion of SFL1 enhances pseudohyphal and invasive growth . Flo11, a cell surface flocculin required for pseudohyphal development, is transcriptionally regulated by Tpk2 and Sfl1 . Genetic evidence indicates that Tpk2 acts upstream of Sfl1 in the regulation of Flo11.

Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13641 - 5
Chloride is an allosteric effector of copper assembly for the yeast multicopper oxidase Fet3p: an unexpected role for intracellular chloride channels; Davis-Kaplan SR et al.; GEF1 is a gene in Saccharomyces cerevisiae, which encodes a putative voltage-regulated chloride channel . gef1 mutants have a defect in the high-affinity iron transport system, which relies on the cell surface multicopper oxidase Fet3p . The defect is due to an inability to transfer Cu+ to apoFet3p within the secretory apparatus . We demonstrate that the insertion of Cu into apoFet3p is dependent on the presence of Cl- . Cu-loading of apoFet3p is favored at acidic pH, but in the absence of Cl- there is very little Cu-loading at any pH . Cl- has a positive allosteric effect on Cu-loading of apoFet3p . Kinetic studies suggest that Cl- may also bind to Fet3p and that Cu+ has an allosteric effect on the binding of Cl- to the enzyme . Thus, Cl- may be required for the metal loading of proteins within the secretory apparatus . These results may have implications in mammalian physiology, as mutations in human intracellular chloride channels result in disease.

J Bacteriol, 1998 Nov, 180(22), 5885 - 90
Regulation and physiological role of the DAS1 gene, encoding dihydroxyacetone synthase, in the methylotrophic yeast Candida boidinii; Sakai Y et al.; The physiological role of dihydroxyacetone synthase (DHAS) in Candida boidinii was evaluated at the molecular level . The DAS1 gene, encoding DHAS, was cloned from the host genome, and regulation of its expression by various carbon and nitrogen sources was analyzed . Western and Northern analyses revealed that DAS1 expression was regulated mainly at the mRNA level . The regulatory pattern of DHAS was similar to that of alcohol oxidase but distinct from that of two other enzymes in the formaldehyde dissimilation pathway, glutathione-dependent formaldehyde dehydrogenase and formate dehydrogenase . The DAS1 gene was disrupted in one step in the host genome (das1Delta strain), and the growth of the das1Delta strain in various carbon and nitrogen sources was compared with that of the wild-type strain . The das1Delta strain had completely lost the ability to grow on methanol, while the strain with a disruption of the formate dehydrogenase gene could survive (Y . Sakai et al., J . Bacteriol . 179:4480-4485, 1997) . These and other experiments (e.g., those to determine the expression of the gene and the growth ability of the das1Delta strain on media containing methylamine or choline as a nitrogen source) suggested that DAS1 is involved in assimilation rather than dissimilation or detoxification of formaldehyde in the cells.

Trends Biochem Sci, 1998 Oct, 23(10), 394 - 8
DNA end-joining: from yeast to man; Critchlow SE et al.; DNA non-homologous end-joining (NHEJ) is a crucial process that has been conserved highly throughout eukaryotic evolution . At its heart is a multiprotein complex containing the KU70-KU80 heterodimer . Recent work has identified additional proteins involved in this pathway, providing insights into the mechanism of NHEJ and revealing exciting links with the control of transcription, telomere length and chromatin structure.

DNA Cell Biol, 1998 Oct, 17(10), 823 - 33
Protein kinase profile of sperm and eggs: cloning and characterization of two novel testis-specific protein kinases (AIE1, AIE2) related to yeast and fly chromosome segregation regulators; Tseng TC et al.; We have analyzed the general protein kinase expression profile in mouse sperm and eggs . A total of 41 different kinases were identified . In this study, we describe two novel protein kinases, designated AIE1 (mouse) and AIE2 (human), which share high amino acid identities with the serine/threonine (S/T) kinase domain of yeast Ip11, fly aurora, and frog Eg2 . Mutations in Ip11 and aurora have been reported to cause abnormal chromosome segregation and centrosome separation . Both AIE1 and AIE2 contain a typical S/T kinase domain (251 aa) flanked by a short polypeptide at both ends . Two other AIE-related kinases (STK-1 and IAK1/Ayk1) were also identified in mature mouse oocytes . The central kinase domain of AIE1 revealed 77.6% and 66.3% identity with that of STK-1 and IAK1/Ayk1, but much less homology was found in the sequence outside the kinase domain . Northern blot analysis revealed that both AIE1 and AIE2 are specifically expressed in testis, whereas STK-1 and IAK1/Ayk1 are expressed in many tissues rich in proliferating cells . An in vitro kinase assay showed that AIE1 can phosphorylate casein, AIE1 itself, and an uncharacterized cellular protein (p16) . The kinase activity of AIE1 can be destroyed by heat inactivation . In summary, we suggest that AIE is a new member of the S/T kinase family, which may be regulated in a fashion distinct from other AIE-related kinases.

Mol Cell, 1998 Oct, 2(4), 437 - 45
Localization of ASH1 mRNA particles in living yeast; Bertrand E et al.; ASH1 mRNA localizes to the bud tip in Saccharomyces cerevisiae to establish asymmetry of HO expression, important for mating type switching . To visualize real time localization of the mRNA in living yeast cells, green fluorescent protein (GFP) was fused to the RNA-binding protein MS2 to follow a reporter mRNA containing MS2-binding sites . Formation and localization of a GFP particle in the bud required ASH1 3'UTR (untranslated region) sequences . The SHE mutants disrupt RNA and particle localization and SHE 2 and 3 mutants inhibit particle formation as well . Both She3myc and She1myc colocalized with the particle . Video microscopy demonstrated that She1p/Myo4p moved particles to the bud tip at 200-440 nm/sec . Therefore, the ASH1 3'UTR-dependent particle serves as a marker for RNA transport and localization.

Physiol Chem Phys Med NMR, 1998, 30(1), 49 - 62
Effect of glucose on the conformation of ADPMg(II) bound at the active site of yeast hexokinase PI; Maity H et al.; The conformation of ADPMg(II) bound at the active site of yeast hexokinase PI has been determined using transferred nuclear Overhauser effect spectroscopy (TRNOESY) . We have measured the time dependent NOE buildup of all the proton pairs of ADP in enzyme . ADPMg(II) and enzyme . glucose.ADPMg(II) complexes at 500 MHz and 10 degrees C . The data have been analyzed using complete relaxation matrix approach to obtain various inter-proton distances . These distances were used as restraints in the molecular dynamics and energy minimization to obtain the conformation of the bound nucleotide . The results from these calculations suggest that in both the complexes, the nucleotide binds in an anti conformation with a glycosidic torsion angle chi = 55 +/- 5 degrees and 52 +/- 5 degrees in PI.ADPMg(II) and PI.glucose.ADPMg(II) complexes, respectively . However, the phase angle of pseudorotation (P) which defines the sugar pucker in the two complexes was found to be 99 degrees, corresponding to 0T1 for PI.ADPMg(II) complex and 69 degrees corresponding to 4T0 for PI.glucose.ADPMg(II) complex . The cleft closure conformational change in the enzyme induced by glucose seems to affect the conformation of the ribosyl moiety of the bound nucleotide.

Biochem J, 1998 Nov 15, 336 ( Pt 1), 91 - 9
Structural and functional properties of a yeast xylitol dehydrogenase, a Zn2+-containing metalloenzyme similar to medium-chain sorbitol dehydrogenases; Lunzer R et al.; The NAD+-dependent xylitol dehydrogenase from the xylose-assimilating yeast Galactocandida mastotermitis has been purified in high yield (80%) and characterized . Xylitol dehydrogenase is a heteronuclear multimetal protein that forms homotetramers and contains 1 mol of Zn2+ ions and 6 mol of Mg2+ ions per mol of 37.4 kDa protomer . Treatment with chelating agents such as EDTA results in the removal of the Zn2+ ions with a concomitant loss of enzyme activity . The Mg2+ ions are not essential for activity and are removed by chelation or extensive dialysis without affecting the stability of the enzyme . Results of initial velocity studies at steady state for d-sorbitol oxidation and d-fructose reduction together with the characteristic patterns of product inhibition point to a compulsorily ordered Theorell-Chance mechanism of xylitol dehydrogenase in which coenzyme binds first and leaves last . At pH 7.5, the binding of NADH (Ki approximately 10 microM) is approx . 80-fold tighter than that of NAD+ . Polyhydroxyalcohols require at least five carbon atoms to be good substrates of xylitol dehydrogenase, and the C-2 (S), C-3 (R) and C-4 (R) configuration is preferred . Therefore xylitol dehydrogenase shares structural and functional properties with medium-chain sorbitol dehydrogenases.

Mutat Res, 1998 Sep 11, 408(3), 183 - 94
The N-degron protein degradation strategy for investigating the function of essential genes: requirement for replication protein A and proliferating cell nuclear antigen proteins for nucleotide excision repair in yeast extracts; Huang W et al.; Nucleotide excision repair (NER) of DNA in the yeast Saccharomyces cerevisiae and in human cells has been shown to be a biochemically complex process involving multiple gene products . In yeast, the involvement of the DNA replication accessory proteins, replication protein A (RPA1) and proliferating cell nuclear antigen (PCNA) in NER has not been demonstrated genetically . In this study we have generated temperature-degradable rfa1 and pcna mutants and show that these mutants are defective in NER in vitro under conditions that promote degradation of the RFA1 and PCNA gene products . We also demonstrate a physical interaction between RPA1 protein and subunits of the RNA polymerase II basal transcription factor IIH (TFIIH).

Mol Cell Endocrinol, 1998 Aug 25, 143(1-2), 133 - 42
Studies of dehydroepiandrosterone (DHEA) with the human estrogen receptor in yeast; Nephew KP et al.; Dehydroepiandrosterone (DHEA) is a C19 adrenal steroid synthesized in the human adrenal cortex and serving as a biosynthetic precursor to testosterone and 17beta-estradiol . Despite the fact that it is one of the most abundant steroid hormones in circulation, the physiological role of DHEA in humans remains unclear . The action of DHEA itself, such as its interactions with receptors and nuclear transcription factors, is not well understood, and a specific DHEA receptor has yet to be identified . Although the activity of DHEA can be due to its metabolism into androgens and estrogens, DHEA has been shown to interact with the androgen receptor and the estrogen receptor (ER) in vitro . We demonstrate in this study that DHEA (3beta-Hydroxy-5alpha-androstan-17-one) inhibits 17beta-estradiol (E2) binding to its receptor in vivo in yeast . DHEA stimulates human ER dimerization in yeast, as determined by ER fusion protein interactions, GAL4 reconstitution and subsequent measurement of increased beta-galactosidase activity . DHEA causes an increase in estrogen response element-dependent beta-galactosidase activity, demonstrating that the ER dimer induced by DHEA is transcriptionally active, but at a concentration of DHEA about 1000 times greater than E2 . Inclusion of the nuclear receptor co-activator RIP140 in the yeast enhances ER transactivation by DHEA or E2 in a ligand-dependent manner; moreover, only in the presence of RIP140 is DHEA able to stimulate beta-galactosidase activity to levels similar to those achieved by E2 . Ligand-receptor interaction for other C19-steroids was also examined . While 5-androstene-3beta, 17beta-diol (ADIOL) displayed estrogenic activity in this system, 4-androstene-17-dione (androstenedione) and 4-androstene-17beta-ol,3-one (testosterone) did not . We have investigated whether DHEA can interact with the human ER in vivo . Our findings demonstrate a mechanism by which DHEA interacts directly with estrogen signaling systems; however, because DHEA is several orders of magnitude less potent than E2 in this system, we conclude that it essentially is not an estrogen agonist.

Biochim Biophys Acta, 1998 Nov 11, 1414(1-2), 260 - 4
Evidence of a subunit 4 (subunit b) dimer in favor of the proximity of ATP synthase complexes in yeast inner mitochondrial membrane; Spannagel C et al.; Yeast mitochondria having either the D54C or E55C mutations in subunit 4 (subunit b), which is a component of the ATP synthase stator, displayed a spontaneous disulfide bridge between two subunits 4 . This dimer was not soluble upon Triton X-100 extraction either at concentrations which extract the yeast ATP synthase or at higher concentrations . Increasing detergent concentrations led to a lack of the oligomycin-sensitive ATPase activity, thus showing an uncoupling between the two sectors of the mutated enzymes due to the dissociation of the subunit 4 dimer from the mutant enzyme . There is only one subunit 4 (subunit b) per eukaryotic ATP synthase . As a consequence, the results are interpreted as the proximity of ATP synthase complexes within the inner mitochondrial membrane.

FEBS Lett, 1998 Oct 16, 437(1-2), 56 - 60
Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants; Mizoguchi T et al.; A possible MAP kinase (MAPK) cascade of Arabidopsis thaliana was identified on the basis of both yeast 2-hybrid analysis and complementation analysis of yeast mutants . Specific protein-protein interactions between ATMPK4 (a MAPK) and MEK1 (a MAPKK) and interactions between MEK1 and ATMEKK1 (a MAPKKK) were detected by using the 2-hybrid system . A growth defect of the yeast mpk1delta mutant was reversed by coexpression of ATMPK4 and MEK1 . Coexpression of the N-terminal deletion form of ATMEKK1 increased the ability of MEK1 to suppress a growth defect of the yeast pbs2delta mutant . These results suggest that ATMPK4, MEK1, and ATMEKK1 may interact with each other and constitute a specific MAPK cascade in Arabidopsis . This is the first demonstration of a possible MAPK cascade in plants.

Indian J Biochem Biophys, 1998 Jun, 35(3), 157 - 60
Modification of arginine residues at the substrate binding site of yeast glutathione reductase; Pandey A et al.; Yeast glutathione reductase (GR) was inactivated by phenylglyoxal (PG), which specifically modifies arginine residues of the enzyme . Inactivation followed psuedo-first order rate kinetics . There was no reversible complex formation prior to inactivation . Analysis of the kinetic data showed the order of reaction to be unity with respect to the modifier . Inactivation of GR was completely prevented by the presence of oxidised glutathione (GSSG), whereas NADP gave only partial protection . Stoichiometric studies showed that around four arginine residues per subunit were modified by PG in the absence of GSSG, whereas only one was modified in its presence . From these observations, it is concluded that essential arginine residues are present at the substrate binding site.

Mol Biol Cell, 1998 Nov, 9(11), 3071 - 83
Hsp90 is required for pheromone signaling in yeast; Louvion JF et al.; The heat-shock protein 90 (Hsp90) is a cytosolic molecular chaperone that is highly abundant even at normal temperature . Specific functions for Hsp90 have been proposed based on the characterization of its interactions with certain transcription factors and kinases including Raf in vertebrates and flies . We therefore decided to address the role of Hsp90 for MAP kinase pathways in the budding yeast, an organism amenable to both genetic and biochemical analyses . We found that both basal and induced activities of the pheromone-signaling pathway depend on Hsp90 . Signaling is defective in strains expressing low levels or point mutants of yeast Hsp90 (Hsp82), or human Hsp90beta instead of the wild-type protein . Ste11, a yeast equivalent of Raf, forms complexes with wild-type Hsp90 and depends on Hsp90 function for accumulation . For budding yeast, Ste11 represents the first identified endogenous "substrate" of Hsp90 . Moreover, Hsp90 functions in steroid receptor and pheromone signaling can be genetically separated as the Hsp82 point mutant T525I and the human Hsp90beta are specifically defective for the former and the latter, respectively . These findings further corroborate the view that molecular chaperones must also be considered as transient or stable components of signal transduction pathways.

Microbiology, 1998 Oct, 144 ( Pt 10), 2739 - 48
Pleiotropic effects of the opi1 regulatory mutation of yeast: its effects on growth and on phospholipid and inositol metabolism; Jiranek V et al.; Key factors which impact on the biosynthesis and subsequent fate of the phospholipid precursor inositol were studied as a function of growth phase in the yeast Saccharomyces cerevisiae . Both wild-type and strains disrupted for the OPI1 gene, the principal negative regulator of the phospholipid biosynthetic genes, were examined . Overexpression of the INO1 gene and overproduction of both inositol and the major inositol-containing phospholipid, phosphatidylinositol, varied as a function of growth phase . In opi1 cells, INO1 expression was constitutive at a high level throughout growth, although the level of transcript was reduced at stationary phase when the cells were grown in defined medium . In the wild-type strain, INO1 expression was limited to a peak in the exponential phase of growth in cells grown in the absence of inositol . Interestingly, the pattern of OPI1 expression in the wild-type strain resembled that of its putative target, INO1 . Intracellular inositol contents of the opi1 strain were higher than those of the wild-type strain, with peak levels occurring in the stationary phase . Membrane phosphatidylinositol content paralleled intracellular inositol content, with opi1 strains having a higher phosphatidylinositol content in stationary phase . The proportion of the predominant phospholipid, phosphatidylcholine, exhibited a profile that was the inverse of the phosphatidylinositol content: phosphatidylcholine content was lowest in opi1 cells in stationary phase . The opi1 mutation was also found to have effects beyond phospholipid biosynthesis . opi1 cells were smaller, and opi1 cultures achieved a cell density twice as high as comparable wild-type cultures . opi1 cells were also more salt tolerant than wild-type cells: they were partly resistant to shrinking, more rapidly resumed growth, and attained a higher culture density after upshift to medium supplemented with 8% NaCl.

Biochem Mol Biol Int, 1998 Oct, 46(2), 277 - 86
HIV-1 Nef protein causes death in stressed yeast cells due to determinants near the N-terminus and elsewhere in Nef; Macreadie I et al.; The Human Immunodeficiency Virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis . In order to determine more about the effects of Nef on basic cellular functions Nef was produced in yeast under a variety of conditions and in multiple cell types . Production of Nef caused cell death in acutely copper- or heat-stressed diploid cells . The N-terminal melittin-like region of Nef was involved in toxicity since a Trp5-->Ala change within Nef change caused increased toxicity . However, another determinant was also involved in toxicity since production of Nef20-206 was also still toxic . In each of these Nef-producing cells there was coincident membrane permeabilisation . These results suggest the possibility of a novel yeast bioassay for Nef inhibitors and that cells producing high levels of Nef may be selectively killed by stress.

Genomics, 1998 Nov 1, 53(3), 406 - 9
Cloning of STK13, a third human protein kinase related to Drosophila aurora and budding yeast Ipl1 that maps on chromosome 19q13.3-ter; Bernard M et al.; This report describes the identification of a cDNA encoding STK13, a third human protein kinase related to the Drosophila Aurora and the budding yeast Ipl1 kinases . After screening of a human placental cDNA library with a Xenopus laevis cDNA encoding the pEg2 protein kinase and 5' RACE on testis mRNA, a full-length cDNA was isolated . The chromosomal localization of STK13 on 19q13.3-ter between the markers D19S210 and D19S218 was established by a combination of somatic cell and radiation hybrid panel PCR screening . The localization of STK13 on human chromosome 19 was confirmed by fluorescence in situ hybridization (FISH) using a genomic clone containing STK13 as a probe .

EMBO J, 1998 Nov 2, 17(21), 6412 - 25
Distinct roles of two separable in vitro activities of yeast Mre11 in mitotic and meiotic recombination; Furuse M et al.; In Saccharomyces cerevisiae, Mre11 protein is involved in both double-strand DNA break (DSB) repair and meiotic DSB formation . Here, we report the correlation of nuclease and DNA-binding activities of Mre11 with its functions in DNA repair and meiotic DSB formation . Purified Mre11 bound to DNA efficiently and was shown to have Mn2+-dependent nuclease activities . A point mutation in the N-terminal phosphoesterase motif (Mre11D16A) resulted in the abolition of nuclease activities but had no significant effect on DNA binding . The wild-type level of nuclease activity was detected in a C-terminal truncated protein (Mre11DeltaC49), although it had reduced DNA-binding activity . Phenotypes of the corresponding mutations were also analyzed . The mre11D16A mutation conferred methyl methanesulfonate-sensitivity to mitotic cells and caused the accumulation of unprocessed meiotic DSBs . The mre11DeltaC49 mutant exhibited almost wild-type phenotypes in mitosis . However, in meiosis, no DSB formation could be detected and an aberrant chromatin configuration was observed at DSB sites in the mre11DeltaC49 mutant . These results indicate that Mre11 has two separable functional domains: the N-terminal nuclease domain required for DSB repair, and the C-terminal dsDNA-binding domain essential to its meiotic functions such as chromatin modification and DSB formation . Keywords: DNA binding/double-strand break repair/DSB formation/Mre11/nuclease

EMBO J, 1998 Nov 2, 17(21), 6348 - 58
The yeast U2A'/U2B complex is required for pre-spliceosome formation; Caspary F et al.; Human U2 snRNP contains two specific proteins, U2A' and U2B", that interact with U2 snRNA stem-loop IV . In Saccharomyces cerevisiae, only the counterpart of human U2B", Yib9p, has been identified . Database searches revealed a gene potentially coding for a protein with striking similarities to human U2A', henceforth called LEA1 (looks exceptionally like U2A') . We demonstrate that Lea1p is a specific component of the yeast U2 snRNP . In addition, we show that Lea1p interacts directly with Yib9p . In vivo association of Lea1p with U2 snRNA requires Yib9p . Reciprocally, Yib9p binds to the U2 snRNA only in the presence of Lea1p in vivo, even though it has been previously shown to associate on its own with the U2 snRNA stem-loop IV in vitro . Strains lacking LEA1 and/or YIB9 grow slowly, are temperature sensitive and contain reduced levels of U2 snRNA . Pre-mRNA splicing is strongly impaired in these cells . In vitro studies demonstrate that spliceosome assembly is blocked prior to addition of U2 snRNP . This phenotype can be rescued partially, but specifically, by addition of the corresponding recombinant protein(s) . This demonstrates a specific role for the yeast U2 snRNP specific proteins during formation of the pre-spliceosome.

EMBO J, 1998 Nov 2, 17(21), 6327 - 36
Multiple transcriptional activation complexes tether the yeast activator Met4 to DNA; Blaiseau PL et al.; The transcriptional regulation of the sulfur amino acid pathway in Saccharomyces cerevisiae depends on a single activator, Met4p, whose function requires different combinations of the auxiliary factors Cbf1p, Met28p, Met31p and Met32p . The first description of how these factors cooperate to activate transcription was provided by the identification of the Cbf1-Met4-Met28 complex which is assembled on the regulatory region of the MET16 gene . In this paper, we demonstrate that other pathways are used to recruit Met4p on the 5' upstream region of the two genes, MET3 and MET28 . In these cases, Met4p is tethered to DNA through two alternative complexes associating Met4p with Met28p and either Met31p or Met32p . These complexes are formed over the AAACTGTG sequence, a cis-acting element found upstream of several MET genes . The identification of a domain within Met4p that mediates its interaction with Met31p and Met32p allowed in vivo analysis of the specificity of the Met4p-containing complexes . The results therefore demonstrate that the co-regulation of a single gene network may be gained through different molecular mechanisms . In addition the sulfur system exacerbates the structural variety of the nucleoprotein complexes in which a single bZIP factor can be engaged.

EMBO J, 1998 Nov 2, 17(21), 6144 - 54
Subcellular distribution of proteasomes implicates a major location of protein degradation in the nuclear envelope-ER network in yeast; Enenkel C et al.; 26S proteasomes are the key enzyme complexes responsible for selective turnover of short-lived and misfolded proteins . Based on the assumption that they are dispersed over the nucleoplasm and cytoplasm in all eukaryotic cells, we wanted to determine the subcellular distribution of 26S proteasomes in living yeast cells . For this purpose, we generated yeast strains that express functional green fluorescent protein (GFP) fusions of proteasomal subunits . An alpha subunit of the proteolytically active 20S core complex of the 26S proteasome, Pre6/YOL038w, as well as an ATPase-type subunit of the regulatory 19S cap complex, Cim5/YOL145w, were tagged with GFP . Both chimeras were shown to be incorporated completely into active 26S proteasomes . Microscopic analysis revealed that GFP-labelled 20S as well as 19S subunits are accumulated mainly in the nuclear envelope (NE)-endoplasmic reticulum (ER) network in yeast . These findings were supported by the co-localization and co-enrichment of 26S proteasomes with NE-ER marker proteins . A major location of proteasomal peptide cleavage activity was visualized in the NE-ER network, indicating that proteasomal degradation takes place mainly in this subcellular compartment in yeast.

Genes Cells, 1998 Aug, 3(8), 521 - 32
Crm1 (XpoI) dependent nuclear export of the budding yeast transcription factor yAP-1 is sensitive to oxidative stress; Kuge S et al.; BACKGROUND: The yAP-1 transcription factor is crucial for the oxidative stress response of the budding yeast Saccharomyces cerevisiae; its activity is induced in response to oxidative stress, and as a consequence the expression of a number of target genes is enhanced . We have shown previously that yAP-1 is mainly found in the cytoplasm, but that upon the imposition of oxidative stress it localizes to the nucleus . In this study, we addressed the mechanism through which yAP-1 nuclear localization is regulated . RESULTS: Here we show that yAP-1 localization is mediated by active export from the nucleus, resulting from the activity of Crm1 (XpoI), a conserved protein that functions as an export receptor which recognizes the nuclear export signal (NES) . When Crm1 expression was repressed, yAP-1 was localized in the nucleus and induced the expression of a yAP-1 dependent target gene . Our results also suggest that the cysteine rich domain (CRD), at the C-terminus of yAP-1, functions as an export recognition sequence . yAP-1 and Crm1 interact in vivo and this interaction is reduced in response to oxidative stress . CONCLUSIONS: These results suggest a novel regulatory mechanism of nucleocytoplasmic transport which is dependent upon a redox sensitive nuclear export pathway.

Genes Cells, 1998 Aug, 3(8), 485 - 98
Stress-activated signalling pathways in yeast; Toone WM et al.; Eukaryotic cells have developed response mechanisms to combat the harmful effects of a variety of stress conditions . In the majority of cases, such responses involve changes in the gene expression pattern of the cell, leading to increased levels and activities of proteins that have stress-protective functions . Over the last few years, considerable progress has been made in understanding how stress-dependent transcriptional changes are brought about, and it transpires that the underlying mechanisms are highly conserved, being similar in organisms ranging from yeast to man . Many of the stress signals derive from the extracellular environment and accordingly these signals require transduction from the cell surface to the nucleus . This is accomplished through stress-activated signalling pathways, key amongst which are the highly conserved stress-activated MAP kinase pathways . Stimulation of these pathways leads to the increased activity of specific transcription factors and consequently the increased expression of certain stress-related genes . In this review, we focus on the progress that has been made in understanding these stress responses in yeast.

Vaccine, 1998 Dec, 16(20), 1933 - 5
Long-term persistence of anti-HBs after vaccination with a recombinant DNA yeast-derived hepatitis B vaccine: 8-year results; Van Herck K et al.; The aim of this study was to evaluate the persistence of antibodies 7 years after hepatitis B booster administration in healthy adult volunteers who were vaccinated in 1986 . In October 1986, 188 seronegative, healthy adult volunteers (117 men and 71 women) were vaccinated with a 20 micrograms dose recombinant DNA yeast-derived hepatitis B vaccine . Mean age of the study group was 23.3 years (+/- 0.28) . Immunisation was carried out according to a 0-1-2 month vaccination schedule, with a booster dose at 12 months . Of the 159 subjects who received the full vaccination course, 63 (40%) had a blood sample taken 8 years after the first vaccination . Of these 63 subjects, five were excluded from the analysis due to an irregular vaccination schedule and four subjects did not complete the accompanying questionnaire on possible booster administration . So, 54 subjects remained available for further analysis . Fourteen individuals had received an additional booster of hepatitis B vaccine sometime between 1989 and 1994 . The geometric mean titre (GMT) at month 13 for these 14 individuals was 1494 mIU ml-1, compared with 3103 mIU ml-1 for those who did not receive an interim booster . Forty subjects, who received no additional booster dose besides that of month 12, met the inclusion criteria of the follow-up study . Of these, all subjects except one were seropositive for anti-HBs at month 96 (GMT: 215.9 mIU ml-1) . All subjects were still anti-HBc negative at that time . Distribution of individual antibody titres revealed that overall 92.5% of subjects retained protective antibody levels (> or = 10 mIU ml-1); 72.5% of vaccinees retained high levels of anti-HBs (> or = 100 mIU ml-1) as compared to 99.2 and 97.0% at month 13, respectively . A positive correlation was found between the subjects' titres at month 13 and month 96 . A 0-1-2 dose vaccination course with a booster dose administered at month 12, induces a protective immune response which lasts at least until 7 years after the full vaccination course of the subjects . A positive correlation was found between the anti-HBs antibody titres at month 13 and month 96.

Curr Opin Genet Dev, 1998 Oct, 8(5), 560 - 4
Regulation of glucose utilization in yeast; Carlson M; Our understanding of the regulatory mechanisms controlling the glucose response in yeast has advanced in the past year . Two transporter-like proteins have been shown to function as glucose sensors in a pathway for glucose induction of transporter genes . Our understanding of the glucose repression mechanism has been augmented by studies of the Snf1 protein kinase, the Mig1 repressor, and the role of Snf1 in inhibiting Mig1 and regulating activators . DNA microarrays have been used to assess glucose-regulated gene expression.

Biochem J, 1998 Nov 1, 335 ( Pt 3), 647 - 52
Clustering of the YNA1 gene encoding a Zn(II)2Cys6 transcriptional factor in the yeast Hansenula polymorpha with the nitrate assimilation genes YNT1, YNI1 and YNR1, and its involvement in their transcriptional activation; Avila J et al.; The genes encoding the nitrate transporter (YNT1), nitrite reductase (YNI1) and nitrate reductase (YNR1) are clustered in the yeast Hansenula polymorpha . In addition, DNA sequencing of the region containing these genes demonstrated that a new open reading frame called YNA1 (yeast nitrate assimilation) was located between YNR1 and YNI1 . The YNA1 gene encodes a protein of 529 residues belonging to the family of Zn(II)2Cys6 fungal transcriptional factors, and has the highest similarity to the transcriptional factors encoded by nirA, and to a smaller extent to nit-4, involved in the nitrate induction of the gene involved in the assimilation of this compound in filamentous fungi . Northern blot analysis showed the presence of the YNA1 transcript in cells incubated in nitrate, nitrate plus ammonium, ammonium, and nitrogen-free media, with a decrease in its levels in those cells incubated in ammonium . In nitrate the strain Deltayna1::URA3, with a disrupted YNA1 gene, neither grew nor expressed the genes YNT1, YNI1 and YNR1 . In the gene cluster YNT1-YNI1-YNA1-YNR1, the four genes were transcribed independently in the YNT1-->YNR1 direction and the transcription start sites were determined by primer extension.

Tsitologiia, 1998, 40(7), 661 - 76
{Stereospecific anomalies in the structure of five completely sequenced yeast chromosomes}; Soidla TR et al.; Using an original computer program we analysed complete nucleotide sequences of chromosomes I, II, III, VI and IX in yeast cells . As a general rule, we found large stereospecific anomalies near genes with a presumed high expression level (a full catalogue of such anomalies for 5 genes with highest CAI in each chromosome is presented) . As a rule, they are also present at mobile genetic elements . Many large stereospecific anomalies are situated next to the sites of specific anomalies of general nucleotide composition-regions devoid of specific dinucleotides . We have noticed many "trains" (lines) of different stereospecific anomalies, possibly showing areas of cooperative binding of different regulatory and structural proteins to DNA . In several, but not all, analysed chromosomes we found a new class of especially large stereospecific anomalies related to repetitive DNA of small length (less than or around 100 nucleotides).

J Biochem (Tokyo), 1998 Nov, 124(5), 886 - 91
The carboxyl-terminal region of the yeast ATPase inhibitor is indispensable for the stability of the protein in mitochondria; Ichikawa N et al.; The role of the carboxyl-terminal region of the yeast mitochondrial ATPase inhibitor was investigated . Three progressive C-terminal deletion mutants of the inhibitor were constructed: (i) Ile58-->end; (ii) Ile51-->end; and (iii) Gln43-->end . The truncated inhibitor was detected in extracts of Ile58-->end mutant yeast cells . For the Ile51-->end mutant, the truncated inhibitor was only detected when the cells were grown on medium containing the membrane-permeable metal chelator, o-phenanthroline, which inhibits mitochondrial proteases . The most greatly truncated inhibitor protein, Gln43-->end, was never detected even in the cells grown in the presence of the metal chelator . The rates of ATP synthesis and hydrolysis in the mutant mitochondria containing the Ile51-->end inhibitor were similar to those in wild type control cells, while the Ile51-->end inhibitor protein was degraded in the cells unless they were incubated in the presence of the chelator . These results indicate that the carboxyl-terminal region of the ATPase inhibitor is not involved in the its inhibitory action on the F1Fo-ATPase, but is required for the stable conformation of the protein which is protected against degradation by proteases.

Biochem Biophys Res Commun, 1998 Oct 20, 251(2), 520 - 6
Regulation of protein phosphatase 2A catalytic activity by alpha4 protein and its yeast homolog Tap42; Nanahoshi M et al.; Recent studies have revealed that the alpha4 protein, a mammalian homolog of yeast Tap42, is associated with the protein phosphatase 2A catalytic subunit (PP2A-C), however, effects of the association of alpha4 with PP2A-C on its phosphatase activity have not been examined, especially using physiologically relevant substrates in the signaling pathway of mTOR (the mammalian target of rapamycin) protein . Here, we report how this association affects the enzymatic activity of PP2A-C using the recombinant eIF-4E binding protein (4E-BP1) phosphorylated by immunoprecipitated mTOR as a substrate . PP2A-C dephosphorylated 4E-BP1 in vitro . The association of alpha4 and Tap42 with PP2A-C inhibited the phosphatase activity toward 4E-BP1 . Rapamycin treatment, however, neither induced restoration of the phosphatase activity of PP2A-C nor caused dissociation of alpha4 and Tap42 from PP2A-C . Our study is the first report to reveal a potential regulatory role of alpha4 and Tap42 to inibit the phosphatase activity of PP2A-C toward the physiologically relevant substrate in the mTOR signaling .

Yeast, 1998 Sep 30, 14(13), 1175 - 87
Regulation of peroxisomal proteins and organelle proliferation by multiple carbon sources in the methylotrophic yeast, Candida boidinii; Sakai Y et al.; A methylotrophic yeast, Candida boidinii, was grown on various combinations of peroxisome-inducing carbon source(s) (PIC(s)), i.e . methanol, oleate and D-alanine, and the regulation of peroxisomal proteins (both matrix and membrane ones) and organelle proliferation were studied . This regulation was followed (1) at the protein or enzyme level by means of the peroxisomal enzyme activity and Western analysis; (2) at the mRNA level by Northern analysis; and (3) at the organelle level by direct observation of peroxisomes under a fluorescent microscope . Peroxisomal proliferation was followed in vivo by using a C . boidinii strain producing a green fluorescent protein having peroxisomal targeting signal 1 . When multiple PICs were used for cell growth, C . boidinii induced specific peroxisomal proteins characteristic of all PIC(s) present in the medium, responding to all PIC(s) simultaneously . Thus, these PICs were considered to induce peroxisomal proliferation independently and not to repress peroxisomes induced by other PICs . Next, the sensitivity of the peroxisomal induction to glucose repression was studied . While the peroxisomal induction by methanol or oleate was completely repressed by glucose, the D-alanine-induced activities of D-amino acid oxidase and catalase, Pmp47, and the organelle proliferation were not . These results indicate that peroxisomal proliferation in yeasts is not necessarily sensitive to glucose repression . Lastly, this regulation was shown to occur at the mRNA level.

J Biol Chem, 1998 Nov 6, 273(45), 29481 - 8
The yeast RAD7 and RAD16 genes are required for postincision events during nucleotide excision repair . In vitro and in vivo studies with rad7 and rad16 mutants and purification of a Rad7/Rad16-containing protein complex; Reed SH et al.; In eukaryotes, nucleotide excision repair (NER) is a complex reaction requiring multiple proteins . In the yeast Saccharomyces cerevisiae, two of these proteins, Rad7 and Rad16, are specifically involved in the removal of lesions from transcriptionally silent regions of the genome in vivo . Extracts prepared from rad7 or rad16 mutant cells are deficient, but not totally defective, in both oligonucleotide excision and repair synthesis of damaged plasmid DNA . We show that these extracts are, however, fully proficient in the incision step of the NER reaction in vitro . Furthermore, using a cdc9 mutant to trap incision intermediates, we demonstrate that rad7 and rad16 mutants are proficient in NER-dependent DNA incision in vivo . A purified protein complex containing both Rad7 and Rad16 proteins complements the oligonucleotide excision and repair synthesis defects in rad7 and rad16 mutant extracts . We conclude that the products of the RAD7 and RAD16 genes are involved in a postincision event(s) during NER in yeast.

J Biol Chem, 1998 Nov 6, 273(45), 29283 - 6
delta-N-methylarginine is a novel posttranslational modification of arginine residues in yeast proteins; Zobel-Thropp P et al.; We have found a novel modification of protein arginine residues in the yeast Saccharomyces cerevisiae . Intact yeast cells lacking RMT1, the gene encoding the protein omega-NG-arginine methyltransferase, were labeled with the methyl donor S-adenosyl-L-{methyl-3H}methionine . The protein fraction was acid-hydrolyzed to free amino acids, which were then fractionated on a high resolution sulfonated polystyrene cation exchange column at pH 5.27 and 55 degreesC . In the absence of the omega-NG, NG-{3H}dimethylarginine product of the RMT1 methyltransferase, we were able to detect a previously obscured 3H-methylated species that migrated in the region of methylated arginine derivatives . The {3H}methyl group(s) of this unknown species were not volatilized by treatment with 2 M NaOH at 55 degreesC for up to 48 h, suggesting that they were not modifications of the terminal omega-guanidino nitrogen atoms . However, this base treatment did result in the formation of a new 3H-methylated derivative that co-chromatographed with delta-N-methylornithine on high resolution cation exchange chromatography, on reverse phase high pressure liquid chromatography, and on thin layer chromatography . From these data, we suggest that the identity of the original unknown methylated residue is delta-N-monomethylarginine . The presence of this methylated residue in yeast cells defines a novel type of protein modification reaction in eukaryotes.

Biochem Biophys Res Commun, 1998 Oct 29, 251(3), 903 - 6
The use of the yeast two hybrid system to evaluate ErbB-3 interactions with SH2 domain containing proteins; Yoo JY et al.; Several mutations in the tyrosine kinase domain of ErbB-3 have been postulated to render this enzyme catalytically inactive . To test which amino acid mutations in ErbB-3 might be critical for kinase inactivation, we used a yeast two hybrid assay of protein-protein interaction . We monitored restoration of ErbB-3 kinase activity by investigating the ability of wild type or mutant ErbB-3 to associate with the SH2 containing proteins Syp and Phosphatidyl-inositol-3-kinase (PI3K) . Our results demonstrate that changing individual amino acids to tyrosine kinase consensus sequences did not increase the interaction of ErbB-3 with Syp or PI3K . Mutation of the consensus Asp832 of rat ErbB-3 to Asn observed in human and bovine ErbB-3 significantly increased the interaction of ErbB-3 and Syp and PI3K 11 or 26 fold respectively . A double mutant (Asp832Asn, Asp757 His) exhibited a 96 or 350 fold increase in the ability to bind PI3K and Syp .

Biochem Biophys Res Commun, 1998 Oct 29, 251(3), 818 - 23
Growth-dependent change of the 26S proteasome in budding yeast; Fujimuro M et al.; The 26S proteasome is assembled from the 20S proteasome and the regulatory subunit complex in an ATP-dependent manner . In the present study, we found that the ATP-dependent activity and the protein amount of the 26S proteasome change during growth of the budding yeast Saccharomyces cerevisiae . Both levels in the stationary phase are higher than those in the exponentially growing phase . On the other hand, the levels of the 20S proteasome appear to remain unchanged during growth . These results suggest that the 26S proteasome undergoes a growth-dependent change and that the 26S proteasome plays a role in the survival of yeast cells under starvation conditions .

Biochem Biophys Res Commun, 1998 Oct 9, 251(1), 296 - 300
Isoform-specific phosphorylation of fission yeast type 2C protein phosphatase; Kobayashi T et al.; Protein phosphatase 2C (PP2C) is one of the four major protein serine/threonine phosphatases of eukaryotes and is implicated in the regulation of various cellular functions . With the goal of elucidating the mechanism responsible for regulating PP2C functions, we investigated the significance of phosphorylation of fission yeast Ptc1, Ptc2, and Ptc3, the yeast orthologs of mammalian PP2C . Both Ptc2 and Ptc3 but not Ptc1 were phosphorylated stoichiometrically by casein kinase II on serine residues at their carboxy-terminal regions . Mutational analysis of Ptc2 and Ptc3 revealed that serine residues of the conserved sequence (Ser-X-Ser-X-X-Glu/Asp) of these proteins were the phosphorylation sites . Interestingly, the activities of Ptc2 and Ptc3 were decreased 25 +/- 7.5% and increased 55 +/- 3.7%, respectively, by phosphorylation . In addition, the same site(s) of Ptc2 was phosphorylated when the protein was expressed in fission yeast cells . These results suggest that phosphorylation of PP2C plays important physiological roles in fission yeast cells .

J Mol Biol, 1998 Nov 6, 283(4), 727 - 30
Second-site reversion analysis is not a reliable method to determine distances in membrane proteins: an assessment using mutations in yeast cytochrome c oxidase subunits I and II; Meunier B et al.; We have examined deficiency mutations and reversions in subunits I and II of yeast cytochrome c oxidase in order to test the reliability of second-site reversion analysis in prediction of tertiary structure of a membrane protein complex . It appears that the method can not provide information on distance between residues, since reversions can be up to 30 A from the primary mutations . However, the reversions are not randomly located in the structure but reveal regions essential for assembly or functional units .

Biochemistry, 1998 Oct 27, 37(43), 15238 - 46
pH dependence of the reaction catalyzed by yeast Mg-enolase; Vinarov DA et al.; The pH dependence of the chemical shifts of the 31P resonances of enzyme-bound substrates 2-phosphoglycerate (PGA) and phosphoenolpyruvate (PEP) were measured to obtain further insight into the catalytic mechanism of yeast enolase . The 31P resonances of PGA and PEP bound to the enolase-Mg complex are individually observed by NMR . The Keq,internal = 1.5 favoring PEP was measured . A pH dependence of the 31P chemical shifts gives pKa values of 5.82 and 6.16 for bound PGA and PEP, respectively, indicating that both ligands bind predominantly with their phosphate groups as the dianionic species and their ionization has been altered . The phosphoryl group of PGA has been suggested as playing a role in catalysis {Nowak, T., Mildvan, A . S., and Kenyon, G . L . (1973) Biochemistry 12, 1690-1701} . The pH dependence of the kinetic parameters for Mg-enolase shows a single break in the plot of pKm, PGA vs pH at pH 6.27 with a pH independence above pH 7 . This is consistent with the trianion of PGA preferably binding to the enzyme . The kcat profile gives pKA values of 5.94 and 8.35, and kcat/Km profiles give pKA values of 5.85, 6.25, and 8.39 . Activation studies with Mg2+ show a pH independence for the activator constant (Ka), but a pH-dependent inhibition at higher concentrations of Mg2+ . The log kcat and kcat/Ka profiles from Mg2+ activation give pKA values of about 5.9 and 8.4 . These results confirm the importance of residues with pKA values of about 5.9 and 8.4 (His and Lys residues?) but do not support a function for the phosphoryl group of the substrate . The pH dependence of the Ki,Mg2+ gives pKA fits of 5 . 95, 7.13, and 8.35 . Data from cation inhibition suggest that the phosphate of the substrate and a His residue on enolase may bind the inhibitory Mg2+.

Mol Gen Genet, 1998 Sep, 259(4), 429 - 36
Oligomers of the Cdc7/Dbf4 protein kinase exist in the yeast cell; Shellman YG et al.; Cdc7/Dbf4 protein kinase is required for the initiation of DNA replication in Saccharomyces cerevisiae . Cdc7/Dbf4 protein kinase is not a cyclin-dependent kinase (CDK), but is regulated in a similar fashion in that the Cdc7 kinase subunit is inactive in the absence of the regulatory subunit Dbf4 . In contrast to what is known about CDKs, Cdc7/Dbf4 protein kinase is shown to be an oligomer in the cell in this report . Genetic data that support this claim include interallelic complementation between several cdc7ts alleles and the cdc7T281A allele and also the results of experiments using the two-hybrid system with Cdc7 in both DNA-binding and transactivation domain plasmids . A molecular interaction between two different Cdc7 molecules was shown by using a HA-tagged Cdc7 protein that differs in size from the wild-type Cdc7 protein: an anti-HA antibody immunoprecipitates both proteins in approximately equal stoichiometry . Analysis of the native molecular weight of Cdc7/Dbf4 protein kinase is consistent with oligomerization of the Cdc7 protein in that complexes of about 180 and 300 kDa were found . Oligomers of Cdc7 protein may exist for the purpose of allosteric regulation or to allow phosphorylation of multiple substrate protein molecules.

Mol Gen Genet, 1998 Sep, 259(4), 388 - 92
Flank matrix attachment regions (MARs) from chicken, bean, yeast or tobacco do not prevent homology-dependent trans-silencing in transgenic tobacco plants; Vaucheret H et al.; The effect of flanking matrix attachment regions (MARs) on homology-dependent trans-silencing was tested using two strong trans-silencing loci . The transgenic tobacco line 271 carries at a single locus a p35S-RiN-tNos transgene which is able to silence, in trans and at the transcriptional level, the expression of any p35S-driven transgene irrespective of its position . The transgenic tobacco line 6b8 carries at a single locus a p35S-uidA-tRbcS transgene which is able to silence in trans, at the post-transcriptional level, the expression of any uidA-expressing transgene irrespective of its position . Various transgenic tobacco lines carrying a target p35S-uidA-tNos transgene, flanked on each side by MARs from chicken, bean, yeast or tobacco, were crossed with lines carrying the 271 and 6b8 loci . Expression of the target transgene was silenced in all hybrids, irrespective of the presence or absence of MAR sequences . These results therefore demonstrate that MARs are not able to protect transgene expression from strong silencing loci that act in trans.

Mol Gen Genet, 1998 Sep, 259(4), 372 - 8
Yeast Skn7p activity is modulated by the Sln1p-Ypd1p osmosensor and contributes to regulation of the HOG pathway; Ketela T et al.; Activation and control of the yeast HOG (High Osmolarity Glycerol) MAP kinase cascade is accomplished, in part, by a two-component sensory-response circuit comprised of the osmosensing histidine protein kinase Sln1p, the phospho-relay protein Ypd1p, and the response regulator protein Ssk1p . We found that deletion of SLN1 and/or YPD1 reduces reporter gene transcription driven by a second two-component response regulator -- Skn7p . The effect of sln1delta and ypd1delta mutations upon Skn7p activity is dependent on a functional two-component phosphorylation site (D427) in Skn7p, suggesting that Sln1p and Ypd1p may act as phosphodonors for Skn7p . We also observed that loss of PTC1 (a protein serine/threonine phosphatase implicated in negative control of the HOG pathway) in a skn7delta background results in severely retarded growth and in morphological defects . Deletion of either PBS2 or HOG1 alleviates the slow growth phenotype of ptc1delta skn7delta cells, suggesting that Skn7p may participate, in concert with known regulatory components, in modulating HOG pathway activity . The contribution of Skn7p to HOG pathway regulation appears to be modulated by the receiver domain, since non-phosphorylatable Skn7pD427N is unable to fully restore growth to ptc1/skn7 cells.

Acta Physiol Scand Suppl, 1998 Aug, 643, 297 - 300
The inventory of all ion and drug ATPases encoded by the yeast genome; Goffeau A; The 5,885 members of the yeast proteome have been screened for amino acid sequence signatures of either P-type ATPases or ABC transporters . A total of 16 P-type ATPases have been classified into six phylogenetic families which each seem to transport a specific class of substrates . In addition, a total of 16 ABC transporters comprising two nucleotide binding folds and two membrane domains were classified in two distinct phylogenetic families . Two ABC transporters of Family I (Pdr5p and Snq2p) share overlapping promiscuity for numerous hydrophobic drugs with a member of Family II (Yor1p) . In this case, substrate specificity seems to have differentiated more slowly during evolution than typical phylogenetic traits reflected by amino acid sequence similarity or predicted membrane topography.

Acta Physiol Scand Suppl, 1998 Aug, 643, 115 - 22
Molecular genetic probing of energy coupling by the yeast plasma membrane proton pump; Soteropoulos P et al.; Genetic probing of PMA1, which encodes the plasma membrane H(+)-ATPase, has highlighted the putative role of the N-terminal half of the enzyme in the coupling process . Recent second-site suppressor studies indicate that significant interactions occur between the region near the site of phosphorylation, stalk segment 3 (S3), and the N-terminal transmembrane segments . Saturation mutagenesis was used to explore I183 in S2, which partially uncouples proton transport when converted to alanine . Numerous substitutions could be made at this position . However, stable substitutions with Arg, Tyr or Asn were often accompanied by second-site mutations at the extreme C-terminus, suggesting a close interaction between these regions . Several mutations in the putative stalk domain are known to alter coupling, and scanning glycine and proline mutagenesis was used to probe the predicted alpha-helical character of the stalk segments . The results indicate that the introduction of proline or glycine in S2, S4 or S5, was highly disruptive to enzyme function often resulting in cell death . Similar substitutions in stalk 3 yielded viable but significantly altered enzymes . These results suggest that the helical properties of these segments may be important for catalysis . Finally, the stalk region has been modeled as a helical bundle, which helps account for the effects of specific perturbations in this region.

Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 12860 - 5
Folding in vivo of a newly translated yeast cytosolic enzyme is mediated by the SSA class of cytosolic yeast Hsp70 proteins; Kim S et al.; The nature of chaperone action in the eukaryotic cytosol that assists newly translated cytosolic proteins to reach the native state has remained poorly defined . Actin, tubulin, and Galpha transducin are assisted by the cytosolic chaperonin, CCT, but many other proteins, for example, ornithine transcarbamoylase (OTC), a cytosolic homotrimeric enzyme of yeast, do not require CCT action . Here, we observe that yeast cytosolic OTC is assisted to its native state by the SSA class of yeast cytosolic Hsp70 proteins . In vitro, refolding of OTC diluted from denaturant was assisted by crude yeast cytosol and ATP and found to be directed by SSA1/2 . In vivo, when OTC was induced in a temperature-sensitive SSA-deficient strain, it exhibited reduced specific activity, and nonnative subunits were detected in the soluble fraction . These findings indicate that, in vivo, the Hsp70 system assists in folding at least some newly translated cytosolic enzymes, most likely functioning in a posttranslational manner.

J Cell Sci, 1998 Nov, 111 ( Pt 22), 3347 - 56
Synaptojanin family members are implicated in endocytic membrane traffic in yeast; Singer-Kruger B et al.; The synaptojanins represent a subfamily of inositol 5'-phosphatases that contain an NH2-terminal Sac1p homology domain . A nerve terminal-enriched synaptojanin, synaptojanin 1, was previously proposed to participate in the endocytosis of synaptic vesicles and actin function . The genome of Saccharomyces cerevisiae contains three synaptojanin-like genes (SJL1, SJL2 and SJL3), none of which is essential for growth . We report here that a yeast mutant lacking SJL1 and SJL2 (Deltasjl1 Deltasjl2) exhibits a severe defect in receptor-mediated and fluid-phase endocytosis . A less severe endocytic defect is present in a Deltasjl2 Deltasjl3 mutant, while endocytosis is normal in a Deltasjl1 Deltasjl3 mutant . None of the mutants are impaired in invertase secretion . The severity of the endocytic impairment of the sjl double mutants correlates with the severity of actin and polarity defects . Furthermore, the deletion of SJL1 suppresses the temperature-sensitive growth defect of sac6, a mutant in yeast fimbrin, supporting a role for synaptojanin family members in actin function . These findings provide a first direct evidence for a role of synaptojanin family members in endocytosis and provide further evidence for a close link between endocytosis and actin function.

Nat Biotechnol, 1998 Oct, 16(10), 946 - 50
Identification of a calcium channel modulator using a high throughput yeast two-hybrid screen; Young K et al.; The interaction of the N-type calcium channel beta3 subunit with the alpha1B subunit alters the activation/inactivation kinetics and the maximal conductance of the channel . The defined protein-protein interaction of the human alpha1B and beta3 subunits provides a target for small-molecule modulation of N-type channel activity . We describe a high throughput screen based on a counterselection yeast two-hybrid assay, which was used to identify small molecules that disrupt alpha1B-beta3 subunit interactions and inhibit N-type calcium channel activity . These small molecules may be a new class of calcium channel antagonists with therapeutic potential.

J Cell Biol, 1998 Oct 19, 143(2), 359 - 73
Mitochondrial fusion in yeast requires the transmembrane GTPase Fzo1p; Hermann GJ et al.; Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment . In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis . Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserved role in mitochondrial fusion . A conditional fzo1 mutation causes the mitochondrial reticulum to fragment and blocks mitochondrial fusion during yeast mating . Fzo1p is a mitochondrial integral membrane protein with its GTPase domain exposed to the cytoplasm . Point mutations that alter conserved residues in the GTPase domain do not affect Fzo1p localization but disrupt mitochondrial fusion . Suborganellar fractionation suggests that Fzo1p spans the outer and is tightly associated with the inner mitochondrial membrane . This topology may be required to coordinate the behavior of the two mitochondrial membranes during the fusion reaction . We propose that the fuzzy onions family of transmembrane GTPases act as molecular switches to regulate a key step in mitochondrial membrane docking and/or fusion.

J Cell Biol, 1998 Oct 19, 143(2), 333 - 49
The dynamin-related GTPase, Dnm1p, controls mitochondrial morphology in yeast; Otsuga D et al.; The Saccharomyces cerevisiae Dnm1 protein is structurally related to dynamin, a GTPase required for membrane scission during endocytosis . Here we show that Dnm1p is essential for the maintenance of mitochondrial morphology . Disruption of the DNM1 gene causes the wild-type network of tubular mitochondrial membranes to collapse to one side of the cell but does not affect the morphology or distribution of other cytoplasmic organelles . Dnm1 proteins containing point mutations in the predicted GTP-binding domain or completely lacking the GTP-binding domain fail to rescue mitochondrial morphology defects in a dnm1 mutant and induce dominant mitochondrial morphology defects in wild-type cells . Indirect immunofluorescence reveals that Dnm1p is distributed in punctate structures at the cell cortex that colocalize with the mitochondrial compartment . These Dnm1p-containing structures remain associated with the spherical mitochondria found in an mdm10 mutant strain . In addition, a portion of Dnm1p cofractionates with mitochondrial membranes during differential sedimentation and sucrose gradient fractionation of wild-type cells . Our results demonstrate that Dnm1p is required for the cortical distribution of the mitochondrial network in yeast, a novel function for a dynamin-related protein.

J Biol Chem, 1998 Oct 30, 273(44), 29106 - 12
Contribution to substrate recognition of two aromatic amino acid residues in putative transmembrane segment 10 of the yeast sugar transporters Gal2 and Hxt2; Kasahara M et al.; The comprehensive study of chimeras between the Gal2 galactose transporter and the Hxt2 glucose transporter of Saccharomyces cerevisiae has shown that Tyr446 is essential and Trp455 is important for galactose recognition by Gal2 . Consistent with this finding, replacement of the corresponding Phe431 and Tyr440 residues of Hxt2 with Tyr and Trp, respectively, allowed Hxt2 to transport galactose, suggesting that the two amino acid residues in putative transmembrane segment 10 play a definite role in galactose recognition (Kasahara, M., Shimoda, E., and Maeda, M . (1997) J . Biol . Chem . 272, 16721-16724) . Replacement of Trp455 of Gal2 with any of the other 19 amino acids was shown to reduce galactose transport activity to between 0 and <20% of that of wild-type Gal2 . The role of Phe431 in Hxt2 was similarly studied . Other than Phe, only Tyr at position 431 was able to support glucose transport activity, at the reduced level of <20% . In contrast, replacement of Tyr440 of Hxt2 with other amino acids revealed that most replacements, with the exception of Pro and charged amino acids, supported glucose transport activity . The importance of residue 431 in sugar recognition was more pronounced in a modified Hxt2 in which Tyr440 was replaced with Trp . Glucose transport was supported only by the aromatic amino acids Phe, Tyr, and Trp at position 431, and galactose transport was supported only by Tyr . These results suggest that an aromatic amino acid located in the middle of transmembrane segment 10 (Tyr446 in Gal2 and Phe431 in Hxt2) plays a critical role in substrate recognition in the yeast sugar transporter family to which Gal2 and Hxt2 belong.

J Biol Chem, 1998 Oct 30, 273(44), 29086 - 92
A mutant yeast topoisomerase II (top2G437S) with differential sensitivity to anticancer drugs in the presence and absence of ATP; Sabourin M et al.; To further characterize the mechanistic basis for cellular resistance/hypersensitivity to anticancer drugs, a yeast genetic system was used to select a mutant type II topoisomerase that conferred cellular resistance to CP-115,953, amsacrine, etoposide, and ellipticine . The mutant enzyme contained a single point mutation that converted Gly437 --> Ser (top2G437S) . Purified top2G437S displayed wild-type enzymatic activity in the absence of drugs but exhibited two properties that were not predicted by the cellular resistance phenotype . First, in the absence of ATP, it was hypersensitive to all of the drugs examined and hypersensitivity correlated with increased drug affinity . Second, in the presence of ATP, top2G437S lost its hypersensitivity and displayed wild-type drug sensitivity . Since the resistance of yeast harboring top2G437S could not be explained by alterations in enzyme-drug interactions, physiological levels of topoisomerase II were determined . The Gly437 --> Ser mutation reduced the stability of topoisomerase II and decreased the cellular concentration of the enzyme . These findings suggest that the physiological drug resistance phenotype conferred by top2G437S results primarily from its decreased stability . This study highlights the need to analyze both the biochemistry and the physiology of topoisomerase II mutants with altered drug sensitivity in order to define the mechanistic bridge that links enzyme function to cellular phenotype.

J Biol Chem, 1998 Oct 30, 273(44), 28752 - 8
Site-directed mutagenesis of yeast eEF1A . Viable mutants with altered nucleotide specificity; Cavallius J et al.; Site-directed mutants of eEF1A (formerly eEF-1alpha) were generated using a modification of a highly versatile yeast shuttle vector (Cavallius, J., Popkie, A . P., and Merrick, W . C . (1997) Biochim . Biophys . Acta 1350, 345-358) . The nucleotide specificity sequence NKMD (residues number 153-156) was targeted for mutagenesis, and the following mutants were obtained: N153D (DKMD), N153T (TKMD), D156N (NKMN), D156W (NKMW), and the double mutant N153T,D156E (TKNE) . All of the yeast strains containing the mutant eEF1As as the sole source of eEF1A were viable except for the N153D mutant . Most of the purified mutant eEF1As had specific activities in the poly(U)-directed synthesis of polyphenylalanine similar to wild type, although with a Km for GTP increased by 1-2 orders of magnitude . The mutants showed a reduced rate of GTP hydrolysis, and most displayed misincorporation rates greater than wild type . The mutant NKMW eEF1A showed unusual properties . The yeast strain was temperature sensitive for growth, although the purified protein was not . Second, this form of eEF1A was 10-fold more accurate in protein synthesis, and its rate of GTP hydrolysis was about 20% of wild type . In total, the wild-type protein contains the most optimal nucleotide specificity sequence, NKMD, and even subtle changes in this sequence have drastic consequences on eEF1A function in vitro or yeast viability.

J Biol Chem, 1998 Oct 30, 273(44), 28713 - 20
Regulation of zinc homeostasis in yeast by binding of the ZAP1 transcriptional activator to zinc-responsive promoter elements; Zhao H et al.; Zinc homeostasis in yeast is controlled primarily through the regulation of zinc uptake . Transcription of the ZRT1 and ZRT2 zinc transporters increases in zinc-limited cells, and this induction is dependent on the ZAP1 gene . We hypothesized previously that ZAP1 encodes a zinc-responsive transcriptional activator . Expression of ZAP1 itself increases in zinc-limited cells . This response is also dependent on ZAP1 function through a potential positive autoregulatory mechanism . In this report, we describe the characterization of zinc-responsive elements (ZREs) in the promoters of the ZRT1, ZRT2, and ZAP1 genes . A ZRE consensus sequence, 5'-ACCYYNAAGGT-3', was identified and found to be both necessary and sufficient for zinc-responsive transcriptional regulation . We also demonstrate that ZREs are DNA binding sites for ZAP1 . First, a dominant ZAP1 mutation, ZAP1-1(up), which causes increased expression of ZAP1-regulated genes in zinc-replete cells, exerted its effects specifically through the ZREs . Second, electrophoretic mobility shift assays and in vitro DNase I footprint analyses indicated that ZAP1 binds to ZREs in a sequence-specific fashion . These studies demonstrate that ZAP1 plays a direct role in controlling zinc-responsive gene expression in yeast by binding to zinc-responsive elements in the promoters of genes that it regulates.

J Biol Chem, 1998 Oct 30, 273(44), 28617 - 24
Zinc-induced inactivation of the yeast ZRT1 zinc transporter occurs through endocytosis and vacuolar degradation; Gitan RS et al.; The ZRT1 gene encodes the transporter responsible for high affinity zinc uptake in yeast . ZRT1 is transcribed in zinc-limited cells and its transcription is repressed in zinc-replete cells . In this report, we describe a second, post-translational mechanism that regulates ZRT1 activity . In zinc-limited cells, ZRT1 is a stable, N-glycosylated plasma membrane protein . Exposure to high levels of extracellular zinc triggers a rapid loss of ZRT1 uptake activity . Our results demonstrate that this inactivation occurs through zinc-induced endocytosis of the protein and its subsequent degradation in the vacuole . Mutations that inhibit the internalization step of endocytosis also inhibited zinc-induced ZRT1 inactivation and the major vacuolar proteases were required to degrade ZRT1 in response to zinc . Furthermore, immunofluorescence microscopy showed that ZRT1 is localized to the plasma membrane in zinc-limited cells and that the protein is transferred to the vacuole via an endosome-like compartment upon exposure to zinc . ZRT1 inactivation is a relatively specific response to zinc; cadmium and cobalt ions trigger the response but less effectively than zinc . Moreover, zinc does not alter the stability of several other plasma membrane proteins . Therefore, zinc-induced ZRT1 inactivation is a specific regulatory system to shut off zinc uptake activity in cells exposed to high extracellular zinc levels thereby preventing overaccumulation of this potentially toxic metal.

Mikrobiologiia, 1998 Jul-Aug, 67(4), 476 - 82
{Effect of 2,4-dinitrophenol on stability of a turbidostat yeast culture to heat shock}; Kaliuzhin VA; The resistance of a turbidostat culture of Saccharomyces cerevisiae 14 to heat shock was investigated . The growth rate of the turbidostat culture after a cultivation temperature shift from a suboptimal (20 degrees C) or optimal (30 degrees C) value to a supraoptimal value of 37.5 degrees C was taken as an index of heat-shock resistance . Experiments were performed in both the two variants: with 2,4-dinitrophenol (DNP) present in cells and medium and with DNP present only in the medium . Cells were found to be resistant to heat shock when they contained no DNP; intracellular DNP did not prevent the formation of the system responsible for thermotolerance, but hindered the functioning of this system . The resistance of yeast to heat shock is presumably determined by the efficiency of oxidative phosphorylation.

Biochem Biophys Res Commun, 1998 Sep 29, 250(3), 791 - 7
The p16(INK4A) protein and flavopiridol restore yeast cell growth inhibited by Cdk4; Moorthamer M et al.; Cyclin-dependent kinase 4 (Cdk4) activity is misregulated in most cancers . Loss of Cdk4 regulation can occur through overexpression of Cdk4 catalytic subunit or its regulatory partner cyclin D1, or if the Cdk4-specific inhibitory protein p16(INK4A) is inactive . We have attempted to express the two human subunits, Cdk4 and cyclin D1, in the yeast Saccharomyces cerevisiae . Surprisingly, expression of Cdk4 alone, under control of the strong GAL promoter, inhibits cell growth . Coexpression of both subunits allows formation of an active Cdk4-cyclin D1 complex which accentuates growth arrest . In cells expressing Cdk4 only, growth is restored by overexpressing human Cdc37, a Cdk4-binding molecular chaperone . Interestingly, the effect of Cdk4 on yeast is also overcome by both p16- and p21-families of Cdk-inhibitory proteins . Moreover, flavopiridol, a compound which inhibits Cdk4 enzyme activity, restores cell division . The fact that p16(INK4A) and flavopiridol negate Cdk4-mediated suppression of yeast cell growth implies that this simple system can be used as a screen for identifying Cdk4-specific antagonists which may mimic p16(INK4A) in the cancer cell cycle .

Biochem Biophys Res Commun, 1998 Sep 29, 250(3), 593 - 7
The requirement of yeast Ssl2 (Rad25) for the repair of cisplatin-damaged DNA; Yang WL et al.; Cisplatin is one of the most widely used anticancer agents . Cisplatin-induced cytotoxicity results from its ability to form cisplatin-DNA adducts within the cellular genome which can inhibit the transcription of genes and the replication of DNA . Cisplatin-adducts are primarily removed by the nucleotide excision repair (NER) pathway . The SSL2 (RAD25) gene of Saccharomyces cerevisiae, a homolog of the XPB (ERCC3) gene in humans, is involved in the nucleotide excision repair of UV-damaged DNA and is also required for cell viability . However, the role of Ssl2 (Rad25) in cisplatin sensitivity has not been examined . In this study, we have demonstrated that a yeast strain carrying the mutant allele SSL2-XP, a truncated form of SSL2 (RAD25) at the carboxyl terminus to mimic the human XPB (ERCC3) mutation, has increased cellular sensitivity to cisplatin in comparison to wild type cells . Analysis by host cell reactivation (HCR) assay further shows that Ssl2 (Rad25) is required for the repair of cisplatin-damaged DNA .

Science, 1998 Oct 23, 282(5389), 699 - 705
The transcriptional program of sporulation in budding yeast; Chu S et al.; Diploid cells of budding yeast produce haploid cells through the developmental program of sporulation, which consists of meiosis and spore morphogenesis . DNA microarrays containing nearly every yeast gene were used to assay changes in gene expression during sporulation . At least seven distinct temporal patterns of induction were observed . The transcription factor Ndt80 appeared to be important for induction of a large group of genes at the end of meiotic prophase . Consensus sequences known or proposed to be responsible for temporal regulation could be identified solely from analysis of sequences of coordinately expressed genes . The temporal expression pattern provided clues to potential functions of hundreds of previously uncharacterized genes, some of which have vertebrate homologs that may function during gametogenesis.

Microbiology, 1998 Sep, 144 ( Pt 9), 2589 - 97
The vanadate-tolerant yeast Hansenula polymorpha undergoes cellular reorganization during growth in, and recovery from, the presence of vanadate; Mannazzu I et al.; When present at intracellular concentrations above micromolar, vanadate becomes toxic to most organisms . However, the yeast Hansenula polymorpha is able to grow on vanadate concentrations in the millimolar range, showing at the same time modifications in cellular ultrastructure and polyphosphate metabolism . Here, the development of the ultrastructural changes, and of vacuolar and secretory activities, during exponential growth on vanadate and upon a return to vanadate-free conditions was investigated . External invertase secretion was inhibited by vanadate, as shown by a decrease in external invertase activity, an intracellular accumulation of small vesicles and a cytoplasmic accumulation of internal invertase . An aberrant appearance of the cell wall and defects in cellular surface growth, possibly linked to defects in secretion, were also observed . However, inhibition of the secretory pathway was not complete since the activity of another secreted enzyme, exoglucanase, increased in the presence of vanadate . Growth on vanadate was also accompanied by an enhancement of vacuolar proteolysis, as indicated by an increase in carboxypeptidase Y activity . However, these modifications were all reversible upon return to vanadate-free conditions, with the normalization process being complex and involving new and dramatic ultrastructural changes and activation of an autophagic mechanism . This mechanism is involved in the elimination/resorption of the observed vanadate-induced aberrant cell structures and/or sites involved in vanadate accumulation, a necessary prerequisite for restoration of conventional ultrastructure and metabolic functions.

Biochimie, 1998 May-Jun, 80(5-6), 451 - 9
1H- and 2H-NMR studies of a fragment of PMP1, a regulatory subunit associated with the yeast plasma membrane H(+)-ATPase . Conformational properties and lipid-peptide interactions; Beswick V et al.; PMP1 is a 38-residue polypeptide associated with the yeast plasma membrane H(+)-ATPase, found to regulate the enzyme activity . To investigate the molecular basis of the PMP1 biological function, the conformational properties of a synthetic PMP1 fragment, A18-F38, comprising the predicted C-terminal cytoplasmic domain and a part of the transmembrane anchor have been studied by 1H- and 2H-NMR spectroscopies . High resolution 1H-NMR experiments showed that, in deuterated DPC micelles, the A18-G34 segment adopts a well defined helix conformation . Our data suggest that the whole PMP1 molecule forms a unique helix whose axis might be slightly tilted with respect to the bilayer normal . Protonated DPC, DMPC and DMPS were incorporated in deuterated micelles containing the PMP1 fragment for studying lipid-peptide interactions . Unusually strong and selective intermolecular NOEs between lipid chain and peptide side chain protons, especially those of the unique Trp residue, were observed . Solid state 2H-NMR experiments performed on pure deuterated POPC and mixed deuterated POPC:POPS (5:1) bilayers revealed that the PMP1 fragment specifically interacts with negatively charged PS lipids.

Am J Ophthalmol, 1998 Oct, 126(4), 590 - 2
Laboratory diagnosis of Acanthamoeba keratitis using buffered charcoal-yeast extract agar; Penland RL et al.; PURPOSE: To evaluate the use of buffered charcoal-yeast extract agar for the isolation of Acanthamoeba from clinical specimens . METHODS: We retrospectively reviewed laboratory records of patients with ocular acanthamebic infection from October 1993 to September 1997 to compare the recovery of Acanthamoeba from clinical specimens inoculated onto various media . We then compared the experimental recovery of 10 corneal isolates of Acanthamoeba on buffered charcoal-yeast extract and blood agars . RESULTS: Paired data for buffered charcoal-yeast extract and blood agars were available from 24 cultures performed in 13 cases of ocular acanthamebic infection . Acanthamebic trails were detected on both buffered charcoal-yeast extract and blood agars in nine cultures, only on buffered charcoal-yeast extract agar in nine cultures, and only on blood agar in one culture (P = .027) . In the experimental study, all 10 clinical isolates produced trails on buffered charcoal-yeast extract agar, and the mean recovery after 10 days of incubation ranged from 38% to 95% of the original inoculum number . For seven of the 10 isolates, more than 70% of the original inoculum was recovered on buffered charcoal-yeast extract agar . Only two of the 10 strains produced persistent trails on the blood agar, and the mean recoveries after 10 days of incubation were 0.67% and 1.17% . Recovery was significantly better on buffered charcoal-yeast extract agar than blood agar (P < or = .0003) . CONCLUSION: Buffered charcoal-yeast extract agar is an excellent commercially available culture medium for the recovery of Acanthamoeba.

Cell, 1998 Oct 2, 95(1), 115 - 23
RNA-assisted nuclear transport of the meiotic regulator Mei2p in fission yeast; Yamashita A et al.; Fission yeast Mei2p is an RNA-binding protein required for both premeiotic DNA synthesis and meiosis I . Mei2p binds to a polyadenylated RNA molecule, meiRNA, loss of which blocks meiosis I . Mei2p forms a dot in meiotic prophase nuclei . Here, we show that meiRNA is required for the nuclear localization of Mei2p and is detectable in the dot . However, Mei2p carrying a nuclear localization signal can produce a nuclear dot and promote meiosis I in the absence of meiRNA . Mei2p expressed in cultured mammalian cells stays in the cytoplasm, but it accumulates in the nucleolus if meiRNA is coexpressed . These results indicate that meiRNA contributes to the promotion of meiosis I exclusively as a cofactor that assists nuclear transport of Mei2p.

Acta Trop, 1998 Jul 30, 70(3), 277 - 84
Triatoma infestans can be captured under natural climatic conditions using yeast-baited traps; Lorenzo MG et al.; The effectiveness of a trap for triatomines baited with yeast cultures has been previously demonstrated in laboratory assays . We report results from assays testing yeast-traps for Triatoma infestans performed under natural climatic conditions . All assays were conducted at experimental chicken-coops colonised by bugs, situated at an endemic area for Chagas disease in Argentina . Two different models of traps were tested, for use either on the floor, or attached to walls . The results obtained clearly demonstrate that yeast-baited traps are effective for capturing triatomine bugs . Floor traps, when baited, captured significantly more bugs than their controls (t-test, P = 0.0008, k = 10) . In addition, wall traps were also more effective, when compared to their controls (t-test, P = 0.011, k = 10) . The maximum capture by a single yeast-trap was 39 bugs in one night, while for the same period the maximum capture by a control-trap was two bugs . Traps captured significantly more bugs at nights when maximum temperature was above 18 degrees C (t-test, P = 0.0002) . Results indicate that yeast traps are capable of capturing significant numbers of free insects in one night . The potential use of yeast- traps for the detection of triatomine bugs is discussed.

Nucleic Acids Res, 1998 Nov 1, 26(21), 5011 - 2
Yeast artificial chromosome segregation from host chromosomes with similar lengths; Izvolsky KI et al.; We propose a new method for segregation of yeast artificial chromosomes (YACs) from endogenous yeast chromosomes with similar lengths . The method is based on recently developed PNA-assisted rare cleavage (PARC) of genomic DNA . We apply the PARC procedure to YAC-containing samples of yeast DNA in such a way that host chromosomes, which electrophoretically comigrate with the chosen YACs, are selectively digested while YACs remain intact . These data demonstrate that a pool of appropriate PNAs can be used as an efficient tool for the PARC-based isolation of intact purified YACs directly from the host cells.

Nucleic Acids Res, 1998 Nov 1, 26(21), 4965 - 74
A specific RNA-protein interaction at yeast polyadenylation efficiency elements; Chen S et al.; The specific RNA-protein interactions responsible for the production of mature 3' ends of eukaryotic mRNAs are not well understood . Sequence elements at the 3' ends of yeast genes have been identified that specify the position of the poly(A) site and the efficiency of polyadenylation . To provide additional insights into the interaction between important sequences that direct 3'-end formation in vivo and nuclear proteins, we utilized gel mobility shift assays and UV-crosslinking studies . The data indicate that a protein, with an apparent molecular weight of 80 kDa, interacts specifically with pre-mRNA at the (UA)3efficiency element . Although the interaction is specific, it can be competed by RNA sequences that do not contain the same type of efficiency element; that is, a sequence lacking a (UA)3repeat . This result implies that the protein binding site is flexible . Using immunoprecipitation techniques, the protein has been identified as Hrp1, a heteronuclear RNA binding protein . The role of Hrp1p in 3'-end formation including RNA processing and transcription termination is addressed.

Environ Mol Mutagen, 1998, 32(2), 173 - 8
Defects in base excision repair combined with elevated intracellular dCTP levels dramatically reduce mutation induction in yeast by ethyl methanesulfonate and N-methyl-N'-nitro-N-nitrosoguanidine; Kunz BA et al.; Previously, we determined that elimination of deoxycytidylate (dCMP) deaminase (DCD1) in the yeast Saccharomyces cerevisiae increases the intracellular dCTP:dTTP ratio and reduces the induction of G x C --> A x T transitions in the SUP4-o gene by ethyl methanesulfonate (EMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Simultaneously, the G x C --> C x G transversion frequency rises substantially . We attributed the first response to dCTP outcompeting dTTP for incorporation opposite O6-alkylguanine, and the second outcome to the increased dCTP pool causing error-prone repair of apurinic (AP) sites resulting from the removal or lability of N7-alkylguanine . To test the latter hypothesis, we used isogenic dcd1 strains deleted for either of two genes (MAG1: 3-methyladenine glycosylase; APN1: apurinic endonuclease) involved in the repair of N7-alkylguanine . In these backgrounds, EMS or MNNG induction of total SUP4-o mutations, G x C --> A x T transitions and G x C --> C x G transversions were reduced by >98%, >97%, and >80%, respectively . Mutation frequencies in the dcd1 apn1 strain were close to those for spontaneous mutagenesis in the wild-type parent . These findings argue that misincorporation of dCTP during repair of alkylation-induced AP sites is responsible for the increased G x C --> C x G transversion frequency in the dcd1 strain treated with EMS or MNNG . The data also demonstrate that defective repair of AP sites coupled with an elevated dCTP:dTTP ratio eliminates most EMS and MNNG mutagenesis . In addition, the results point to a role for AP sites in the production of some EMS- and MNNG-induced G x C --> A x T transitions as well as other substitutions in the dcd1 strain.

Mol Cell Biol, 1998 Nov, 18(11), 6805 - 15
Cse1p is involved in export of yeast importin alpha from the nucleus; Solsbacher J et al.; Proteins bearing a nuclear localization signal (NLS) are targeted to the nucleus by the heterodimeric transporter importin . Importin alpha binds to the NLS and to importin beta, which carries it through the nuclear pore complex (NPC) . Importin disassembles in the nucleus, evidently by binding of RanGTP to importin beta . The importin subunits are exported separately . We investigated the role of Cse1p, the Saccharomyces cerevisiae homologue of human CAS, in nuclear export of Srp1p (yeast importin alpha) . Cse1p is located predominantly in the nucleus but also is present in the cytoplasm and at the NPC . We analyzed the in vivo localization of the importin subunits fused to the green fluorescent protein in wild-type and cse1-1 mutant cells . Srp1p but not importin beta accumulated in nuclei of cse1-1 mutants, which are defective in NLS import but not defective in NLS-independent import pathways . Purified Cse1p binds with high affinity to Srp1p only in the presence of RanGTP . The complex is dissociated by the cytoplasmic RanGTP-binding protein Yrb1p . Combined with the in vivo results, this suggests that a complex containing Srp1p, Cse1p, and RanGTP is exported from the nucleus and is subsequently disassembled in the cytoplasm by Yrb1p . The formation of the trimeric Srp1p-Cse1p-RanGTP complex is inhibited by NLS peptides, indicating that only NLS-free Srp1p will be exported to the cytoplasm.

Mol Cell Biol, 1998 Nov, 18(11), 6374 - 86
Yeast Los1p has properties of an exportin-like nucleocytoplasmic transport factor for tRNA; Hellmuth K et al.; Saccharomyces cerevisiae Los1p, which is genetically linked to the nuclear pore protein Nsp1p and several tRNA biogenesis factors, was recently grouped into the family of importin/karyopherin-beta-like proteins on the basis of its sequence similarity . In a two-hybrid screen, we identified Nup2p as a nucleoporin interacting with Los1p . Subsequent purification of Los1p from yeast demonstrates its physical association not only with Nup2p but also with Nsp1p . By the use of the Gsp1p-G21V mutant, Los1p was shown to preferentially bind to the GTP-bound form of yeast Ran . Furthermore, overexpression of full-length or N-terminally truncated Los1p was shown to have dominant-negative effects on cell growth and different nuclear export pathways . Finally, Los1p could interact with Gsp1p-GTP, but only in the presence of tRNA, as revealed in an indirect in vitro binding assay . These data confirm the homology between Los1p and the recently identified human exportin for tRNA and reinforce the possibility of a role for Los1p in nuclear export of tRNA in yeast.

J Clin Microbiol, 1998 Nov, 36(11), 3396 - 8
Efficacy of API 20C and ID 32C systems for identification of common and rare clinical yeast isolates; Ramani R et al.; The abilities of the API 20C and ID 32C yeast identification systems to identify 123 common and 120 rare clinical yeast isolates were compared . API 20C facilitated correct identification of 97% common and 88% rare isolates while ID 32C facilitated correct identification of 92% common and 85% rare isolates.

J Biol Chem, 1998 Oct 23, 273(43), 28107 - 15
Cell cycle- and Cln2p-Cdc28p-dependent phosphorylation of the yeast Ste20p protein kinase; Wu C et al.; Ste20p from Saccharomyces cerevisiae is a member of the Ste20/p21-activated protein kinase family of protein kinases . The Ste20p kinase is post-translationally modified by phosphorylation in a cell cycle-dependent manner, as judged by the appearance of phosphatase-sensitive species with reduced mobility on SDS-polyacrylamide gel electrophoresis . This modification is maximal during S phase, and correlates with the accumulation of Ste20p fused to green fluorescent protein at the site of bud emergence . Overexpression of Cln2p, but not Clb2p or Clb5p, causes a quantitative shift of Ste20p to the reduced mobility form, and this shift is dependent on Cdc28p activity . The post-translational mobility shift can be generated in vitro by incubation of Ste20p with immunoprecipitated Cln2p kinase complexes, but not by immunoprecipitated Clb2p or Clb5p kinase complexes . Ste20p is therefore a substrate for the Cdc28p kinase, and undergoes a Cln2p-Cdc28p mediated mobility shift as cells initiate budding and DNA replication . In cells that express only the Cln2p G1 cyclin, minor overexpression of Ste20p causes aberrant morphology, suggesting a proper coordination of Ste20p and Cln-Cdc28p activity may be required for the control of cell shape.

J Biol Chem, 1998 Oct 23, 273(43), 27831 - 40
Schistosoma mansoni Ca2+-ATPase SMA2 restores viability to yeast Ca2+-ATPase-deficient strains and functions in calcineurin-mediated Ca2+ tolerance; Talla E et al.; The sarco(endo)plasmic reticulum of animal cells contains an ATP-powered Ca2+ pump that belongs to the P-type family of membrane-bound cation-translocating enzymes . In Schistosoma mansoni, the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is encoded by the SMA1 and SMA2 genes . A full-length SMA2 cDNA clone was isolated, sequenced, and expressed into a yeast Ca2+-ATPase-deficient strain requiring plasmid-borne rabbit SERCA1a for viability . The S . mansoni Ca2+-ATPase supports growth of mutant cells lacking SERCA1a, indicating functional expression in yeast and a role in calcium sequestration . Subcellular fractionation showed that the SMA2 ATPase is localized in yeast internal membranes . SMA2 expression was found to be associated with thapsigargin-sensitive, Ca2+-dependent ATPase activity . The activity increased 2-fold upon calcineurin inactivation, which correlates with in vivo stimulated contribution of SMA2 in calcium tolerance . These results suggest that calcineurin controls calcium homeostasis by inhibiting Ca2+-ATPase activity in an internal compartment.

EMBO J, 1998 Oct 15, 17(20), 5868 - 76
Identification of in vivo substrates of the yeast mitochondrial chaperonins reveals overlapping but non-identical requirement for hsp60 and hsp10; Dubaquie Y et al.; The mechanism of chaperonin-assisted protein folding has been mostly analyzed in vitro using non-homologous substrate proteins . In order to understand the relative importance of hsp60 and hsp10 in the living cell, homologous substrate proteins need to be identified and analyzed . We have devised a novel screen to test the folding of a large variety of homologous substrates in the mitochondrial matrix in the absence or presence of functional hsp60 or hsp10 . The identified substrates have an Mr of 15-90 kDa and fall into three groups: (i) proteins that require both hsp60 and hsp10 for correct folding; (ii) proteins that completely fail to fold after inactivation of hsp60 but are unaffected by the inactivation of hsp10; and (iii) newly imported hsp60 itself, which is more severely affected by inactivation of hsp10 than by inactivation of pre-existing hsp60 . The majority of the identified substrates are group I proteins . For these, the lack of hsp60 function has a more pronounced effect than inactivation of hsp10 . We suggest that homologous substrate proteins have differential chaperonin requirements, indicating that hsp60 and hsp10 do not always act as a single functional unit in vivo.

Mol Endocrinol, 1998 Oct, 12(10), 1525 - 41
Functional interaction between the estrogen receptor and CTF1: analysis of the vitellogenin gene B1 promoter in yeast; Tsai-Pflugfelder M et al.; Eukaryotic gene expression depends on a complex interplay between the transcriptional apparatus and chromatin structure . We report here a yeast model system for investigating the functional interaction between the human estrogen receptor (hER) and CTF1, a member of the CTF/NFI transcription factor family . We show that a CTF1-fusion protein and the hER transactivate a synthetic promoter in yeast in a synergistic manner . This interaction requires the proline-rich transactivation domain of CTF1 . When the natural estrogen-dependent vitellogenin B1 promoter is tested in yeast, CTF1 and CTF1-fusion proteins are unable to activate transcription, and no synergy is observed between hER, which activates the B1 promoter, and these factors . Chromatin structure analysis on this promoter reveals positioned nucleosomes at -430 to -270 (+/-20 bp) and at -270 to - 100 (+/-20 bp) relative to the start site of transcription . The positions of the nucleosomes remain unchanged upon hormone-dependent transcriptional activation of the promoter, and the more proximal nucleosome appears to mask the CTF/NFI site located at - 101 to -114 . We conclude that a functional interaction of hER with the estrogen response element located upstream of a basal promoter occurs in yeast despite the nucleosomal organization of this promoter, whereas the interaction of CTF1 with its target site is apparently precluded by a nucleosome.

Cancer Genet Cytogenet, 1998 Oct 1, 106(1), 1 - 10
Isolation of yeast artificial chromosomes containing the entire transcriptional unit of the human FGF1 gene: a 720-kb contig spanning human chromosome 5q31.3-->q32; Chiu IM et al.; The q31-q33 region of chromosome 5 includes a number of genes encoding growth factors, growth factor receptors, and hormone/neurotransmitter receptors . The human fibroblast growth factor 1 locus (FGF1) resides in this region of chromosome 5, which is frequently lost in myelodysplastic syndromes and acute myeloid leukemia patients . Other disease loci, including the loci for limb-girdle muscular dystrophy and an autosomal dominant deafness, have been mapped on this region, but their genes have not been isolated . It was shown that the critical region lost in two patients with the 5q- syndrome resides between FGF1 and IL12B . We previously reported the construction of a yeast artificial chromosome (YAC) contig spanning 330 kb around the FGF1 gene . Here we report the isolation of additional YAC clones that extend 290 kb from the previous contig . Sequence-tagged sites developed from the outermost YAC ends were utilized in the contig cloning of two P1 clones P1Y2 and P1Y8 . Together, these YAC and P1 clones span 720 kb around the FGF1 locus . With the use of fluorescence in situ hybridization, a physical map has been constructed of these P1 and GRL (glucocorticoid receptor locus) probes on metaphase and interphase chromosomes . On the basis of our work and the known orientation of GRL transcription, the determined order of these loci on chromosome 5q31.3-q32 is centromere-P1Y8-3'{FGF1}5'-P1Y2-5'{GRL}3'-telome re . Knowing the transcriptional orientation of the FGF1 gene relative to the centromere will now facilitate the directional cloning of clinically important genes that may reside in this region.

Nat Genet, 1998 Oct, 20(2), 203 - 6
Circular chromosome formation in a fission yeast mutant defective in two ATM homologues; Naito T et al.; Telomeres, found at chromosomal ends, are essential for stable maintenance of linear chromosomes in eukaryotes . The ATM family of genes, including budding yeast TEL1 (refs 1,2), fission yeast rad3+ (ref . 3) and human ATM (ref . 4), have been reported to be involved in telomere length regulation, although the significance of the telomere phenotypes observed with the mutated genes remains elusive . We have cloned tel1+, another fission yeast ATM homologue, and found that a tel1rad3 double mutant lost all telomeric DNA sequences . Thus, the ATM homologues are essential in telomere maintenance . The mutant grew poorly and formed irregular-shaped colonies, probably due to chromosome instability, however, during prolonged culture of the double mutant, cells forming normal round-shaped colonies arose at a relatively high frequency . All three chromosomes in these derivative cells were circular and lacked telomeric sequences . To our knowledge, this is the first report of eukaryotic cells whose chromosomes are all circular . Upon meiosis, these derivative cells produced few viable spores . Therefore, the exclusively circular genome lacking telomeric sequences is proficient for mitotic growth, but does not permit meiosis.

Cancer Chemother Pharmacol, 1998, 42(5), 345 - 56
Yeast cells expressing differential levels of human or yeast DNA topoisomerase II: a potent tool for identification and characterization of topoisomerase II-targeting antitumour agents; van Hille B et al.; PURPOSE: To identify and characterize the specificity and potency of topoisomerase II-interacting antitumour drugs in an in vivo model utilizing the yeast Saccharomyces cerevisiae . METHODS: Four yeast transformants were selected for the expression of either human or yeast DNA topoisomerase II at different, biologically relevant, levels under the tight control of promoters of various strengths . RESULTS: Analyses of 24 drugs permitted their classification into three distinct groups, depending on whether they induced topoisomerase II-related cytotoxicity (etoposide), showed nonspecific cytotoxicity (camptothecin), or exerted no cytotoxicity at all (vinorelbine) . Within the first group different patterns of action were distinguishable: (1) classical topoisomerase II expression-dependent cytotoxicity for both species of enzyme (e.g . etoposide, amsacrine, doxorubicin, actinomycin D), although amsacrine and TOP 53 were more active, respectively, on human and yeast topoisomerase II; and (2) compounds that appeared to poison only one species of topoisomerase II with, for example, genistein and the bisdioxopiperazine ICRF-193 lethally targeting only the human type, and mitoxantrone only the yeast isozyme . Three of the 16 known topoisomerase II inhibitors tested were not correctly identified with this assay, possibly owing to restricted cell wall permeability or to the absence of correct processing pathways such as, for example, in the case of the prodrug etopophos . CONCLUSION: This methodology, in vivo in yeast, selected for a large range of potent topoisomerase II-targeting anticancer agents . Of particular interest in this yeast model, and in contrast to yeast topoisomerase II, human topoisomerase II was shown to confer dominant sensitivity in the presence of the series of bisdioxopiperazine derivatives tested . This assay therefore has the potential easily to identify and characterize the potency and specificity of synthesized anticancer drugs with modified original chemical structures or those present, for example, in natural plant extracts or marine organisms.

Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12504 - 9
Cleavage motifs of the yeast 20S proteasome beta subunits deduced from digests of enolase 1; Nussbaum AK et al.; The 436-amino acid protein enolase 1 from yeast was degraded in vitro by purified wild-type and mutant yeast 20S proteasome particles . Analysis of the cleavage products at different times revealed a processive degradation mechanism and a length distribution of fragments ranging from 3 to 25 amino acids with an average length of 7 to 8 amino acids . Surprisingly, the average fragment length was very similar between wild-type and mutant 20S proteasomes with reduced numbers of active sites . This implies that the fragment length is not influenced by the distance between the active sites, as previously postulated . A detailed analysis of the cleavages also allowed the identification of certain amino acid characteristics in positions flanking the cleavage site that guide the selection of the P1 residues by the three active beta subunits . Because yeast and mammalian proteasomes are highly homologous, similar cleavage motifs might be used by mammalian proteasomes . Therefore, our data provide a basis for predicting proteasomal degradation products from which peptides are sampled by major histocompatibility complex class I molecules for presentation to cytotoxic T cells.

Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12346 - 51
Phospholipase D activity is required for suppression of yeast phosphatidylinositol transfer protein defects; Xie Z et al.; Yeast phosphatidylinositol transfer protein (Sec14p) function is essential for production of Golgi-derived secretory vesicles, and this requirement is bypassed by mutations in at least seven genes . Analyses of such 'bypass Sec14p' mutants suggest that Sec14p acts to maintain an essential Golgi membrane diacylglycerol (DAG) pool that somehow acts to promote Golgi secretory function . SPO14 encodes the sole yeast phosphatidylinositol-4,5-bisphosphate-activated phospholipase D (PLD) . PLD function, while essential for meiosis, is dispensable for vegetative growth . Herein, we report specific physiological circumstances under which an unanticipated requirement for PLD activity in yeast vegetative Golgi secretory function is revealed . This PLD involvement is essential in 'bypass Sec14p' mutants where normally Sec14p-dependent Golgi secretory reactions are occurring in a Sec14p-independent manner . PLD catalytic activity is necessary but not sufficient for 'bypass Sec14p', and yeast operating under 'bypass Sec14p' conditions are ethanol-sensitive . These data suggest that PLD supports 'bypass Sec14p' by generating a phosphatidic acid pool that is somehow utilized in supporting yeast Golgi secretory function.

FEMS Microbiol Lett, 1998 Sep 15, 166(2), 317 - 24
Catabolite inactivation of the maltose transporter in nitrogen-starved yeast could be due to the stimulation of general protein turnover; Penalver E et al.; Addition of glucose to Saccharomyces cerevisiae inactivates the maltose transporter . The general consensus is that this inactivation, called catabolite inactivation, is one of the control mechanisms developed by this organism to use glucose preferentially whenever it is available . Using nitrogen-starved cells (resting cells), it has been shown that glucose triggers endocytosis and degradation of the transporter in the vacuole . We now show that maltose itself triggers inactivation and degradation of its own transporter as efficiently as glucose . This fact, and the observation that glucose inactivates a variety of plasma membrane proteins including glucose transporters themselves, suggests that catabolite inactivation of the maltose transporter in nitrogen-starved cells is not a control mechanism specifically directed to ensure a preferential use of glucose . It is proposed that, in this metabolic condition, inactivation of the maltose transporter might be due to the stimulation of the general protein turnover that follows nitrogen starvation.

FEMS Microbiol Lett, 1998 Sep 15, 166(2), 267 - 73
Glucose transport in a methylotrophic yeast Hansenula polymorpha; Karp H et al.; Glucose transport was studied in a methylotrophic yeast Hansenula polymorpha . Two kinetically different glucose transport systems were revealed in cells grown under different growth conditions . Glucose-repressed cells exhibited a low-affinity transport system (Km for glucose 1.75 mM) while glucose-derepressed and ethanol-grown cells had a high-affinity transport system (Km for glucose 0.05-0.06 mM) . The high- and low-affinity transport systems differed in substrate specificity, sensitivity to pH, dinitrophenol and protonophore carbonyl cyanide-m-chlorophenyl-hydrazone . The kinetic rearrangement of the glucose transport system in response to altered growth conditions was dependent on de novo protein synthesis.

Pathol Biol (Paris), 1998 Feb, 46(2), 96 - 7
{The yeast genome}; Goffeau A; The yeast genome has been completed and the almost 6000 proteins from the yeast proteome are under analysis . This analysis will contribute to elucidate the basic mechanisms of unicellular eucaryotic life . Several aspects of such studies are relevant to human health problems . In particular, it should allow the development of new fungicides which would be more efficient and specific and would escape the complex multidrug cell export systems developed by pathogenic fungi . Also several human genes determining hereditary diseases have yeast homologues . The function of these homologues can be determined using a series of genetic tools which are unique to yeast . These human genes can also be expressed in yeast and be submitted to the same tools . Several examples of these approaches will be chosen concerning yeast homologues of human membrane proteins belonging to ion channels, permeases, cation transport-ATPases or ABC transporters superfamilies.

RNA, 1998 Oct, 4(10), 1259 - 67
The RNA recognition motif of yeast translation initiation factor Tif3/eIF4B is required but not sufficient for RNA strand-exchange and translational activity; Niederberger N et al.; The Saccharomyces cerevisiae TIF3 gene encodes a 436-amino acid (aa) protein that is the yeast homologue of mammalian translation Initiation factor eIF4B . Tif3p can be divided into three parts, the N-terminal region with an RNA recognition motif (RRM) (aa 1-182), followed in the middle part by a sevenfold repeat of 26 amino acids rich in basic and acidic residues (as 183-350), and a C-terminal region without homology to any known sequence (aa 351-436) . We have analyzed several Tif3 proteins with deletions at their N and C termini for their ability (1) to complement a tif3delta strain in vivo, (2) to stimulate Tif3-dependent translation extracts, (3) to bind to single-stranded RNA, and (4) to catalyze RNA strand-exchange in vitro . Here we report that yeast Tif3/eIF4B contains at least two RNA binding domains able to bind to single-stranded RNA . One is located in the N-terminal region of the protein carrying the RRM, the other in the C-terminal two-thirds region of Tif3p . The RRM-containing domain and three of the seven repeat motifs are essential for RNA strand-exchange activity of Tif3p and translation in vitro and for complementation of a tif3delta strain, suggesting an important role for RNA strand-exchange activity in translation.

Parasite Immunol, 1998 Sep, 20(9), 413 - 20
Intranasal immunization with yeast-expressed 19 kD carboxyl-terminal fragment of Plasmodium yoelii merozoite surface protein-1 (yMSP119) induces protective immunity to blood stage malaria infection in mice; Hirunpetcharat C et al.; Variable protection against malaria blood-stage infection has been demonstrated in mice following parenteral immunization with the highly conserved 19 kD carboxylterminal fragment of the merozoite surface protein-1 (MSP119) using CFA/IFA and other adjuvants . Here we show that intranasal immunization of BALB/C mice with yeast expressed Plasmodium yoelii MSP119 plus a mixture of native and recombinant cholera toxin B subunit, could induce serum MSP119-specific antibodies at titres ranging from 20 000 to 2 560 000 . The Ig subclass responses were predominantly G1 and G2b . Intranasal immunization led to protection following challenge (peak parasitaemia < 1%) in mice with the highest MSP119-specific titre (>/= 640 000) . In two of the three protected mice, a peak parasitaemia of 0.1%-1% was followed by a boost of the antibody response whereas one of the three protected mice did not boost its antibody response after a peak parasitaemia of 0.02% . In unprotected mice, antibody levels rose, then fell, following the detection of parasites in the peripheral blood . CD4+ T cell-depletion abrogated the ability of the mice to boost their antibody response following challenge . These data demonstrate the potential for intranasal immunization with MSP119 to protect against malaria.

Br J Dermatol, 1998 Aug, 139(2), 254 - 63
Seborrhoeic dermatitis is not caused by an altered immune response to Malassezia yeast; Parry ME et al.; The immune response of patients with seborrhoeic dermatitis and healthy age- and sex-matched controls was examined to test the hypothesis that an inadequate or inappropriate immune response to Malassezia yeast leads to seborrhoeic dermatitis . Antibody responses were examined using enzyme-linked immunosorbent assays (ELISAs) and Western blots and lymphocyte responses using lymphocyte proliferation assays . The level of IgG and IgM specific for whole yeast cells or extracted proteins of two isolates of M . furfur was tested in ELISA . A wide range of antibody levels was found but the patient and control groups were indistinguishable (n = 19), and the groups could not be distinguished by the pattern of Malassezia proteins recognized by their sera in Western blots . The average affinity of the subjects' antibodies specific for Malassezia cells or proteins was measured using ammonium thiocyanate dissociation . Most of the sera had moderate affinities corresponding to 50% dissociation at thiocyanate concentrations of 0.5-1.0 mol/L . There was no difference between patients and matched controls . The proliferation of the patients' lymphocytes in response to a number of M . furfur cell preparations was measured: whole cells, cytoplasmic proteins, cell walls, soluble molecules extracted from the cell walls using sonication, and a commercial preparation . There was a wide range of responses between individuals, but there was no difference between the three groups: patients with seborrhoeic dermatitis (n = 16), healthy controls (n = 16) and a group suffering from other inflammatory skin conditions (n = 15) . The results do not support the hypothesis that an inadequate immune response to Malassezia yeast could lead to seborrhoeic dermatitis . Other possible pathological mechanisms include toxin production or lipase activity.

C R Acad Sci III, 1998 Apr, 321(4), 267 - 74
Dihydroorotate (dhout) and orotate (orout) utilizer mutants in yeast: identification of the dhout mutation and allelism of the DHO and URE2 genes; Bloch JC et al.; We induced by UV mutagenesis a series of yeast mutants that were able to utilize dihydroorotic (dhout) and orotic acid (orout) as precursors for pyrimidine biosynthesis . These recessive mutations defined three complementation groups named dhout, orout1 and orout2 . The wild-type allele of the gene responsible for dihydroorotate utilization was cloned using the sensitivity of the dhout mutant to 5-fluoroorotate . The DHO gene was sequenced and found to be identical to the URE2 gene . The dhout mutation resulted from the introduction of a stop codon instead of a glutamine at position 59, which led to the production of a truncated Ure2p . Therefore, the URE2 and DHO genes are alleles in yeast.

Res Microbiol, 1997 Dec, 148(9), 731 - 44
Trinucleotide repeats in yeast; Richard GF et al.; The yeast genome exhibits a variety of trinucleotide repeat arrays within protein-coding genes and intergenic regions . In the first situation, repeats are often not random relative to the translational frame, resulting preferably in long stretches of the two acidic amino acids or of their corresponding amine forms . Interestingly, the longest trinucleotide repeats are often found in genes encoding nuclearly located proteins . Repeats tend to be more frequent in long genes, but less frequent among members of gene families compared to unique genes . In the latter case, repeat arrays often differ in length or composition between the gene homologs, indicating their instability.

Genes Dev, 1998 Oct 1, 12(19), 3044 - 58
Yeast telomeres exert a position effect on recombination between internal tracts of yeast telomeric DNA; Stavenhagen JB et al.; In Saccharomyces cerevisiae, proximity to a telomere affects both transcription and replication of adjacent DNA . In this study, we show that telomeres also impose a position effect on mitotic recombination . The rate of recombination between directly repeated tracts of telomeric C1-3A/TG1-3 DNA was reduced severely by proximity to a telomere . In contrast, recombination of two control substrates was not affected by telomere proximity . Thus, unlike position effects on transcription or replication, inhibition of recombination was sequence specific . Moreover, the repression of recombination was not under the same control as transcriptional repression (telomere position effect; TPE), as mutations in genes essential for TPE did not alleviate telomeric repression of recombination . The reduction in recombination between C1-3A/TG1-3 tracts near the telomere was caused by an absence of Rad52p-dependent events as well as a reduction in Rad1p-dependent events . The sequence-specific repression of recombination near the telomere was eliminated in cells that overexpressed the telomere-binding protein Rap1p, a condition that also increased recombination between C1-3A/TG1-3 tracts at internal positions on the chromosome . We propose that the specific inhibition between C1-3A/TG1-3 tracts near the telomere occurs through the action of a telomere-specific end-binding protein that binds to the single-strand TG1-3 tail generated during the processing of recombination intermediates . The recombination inhibitor protein may also block recombination between endogenous telomeres.

J Cell Sci, 1998 Nov, 111 ( Pt 21), 3129 - 43
A factor required for nonsense-mediated mRNA decay in yeast is exported from the nucleus to the cytoplasm by a nuclear export signal sequence; Shirley RL et al.; In Saccharomyces cerevisiae, Upf3p is required for nonsense-mediated mRNA decay (NMD) . Although localized primarily in the cytoplasm, Upf3p contains three sequence elements that resemble nuclear localization signals (NLSs) and two sequence elements that resemble nuclear export signals (NESs) . We found that a cytoplasmic reporter protein localized to the nucleus when fused to any one of the three NLS-like sequences of Upf3p . A nuclear reporter protein localized to the cytoplasm when fused to one of the NES-like sequences (NES-A) . We present evidence that NES-A functions to signal the export of Upf3p from the nucleus . Combined alanine substitutions in the NES-A element caused a re-distribution of Upf3p to a subnuclear location identified as the nucleolus and conferred an Nmd- phenotype . Single mutations in NES-A failed to affect the distribution of Upf3p and were Nmd+ . When an NES element from HIV-1 Rev was inserted near the C terminus of a mutant Upf3p containing multiple mutations in NES-A, the cytoplasmic distribution typical of wild-type Upf3p was restored but the cells remained phenotypically Nmd- . These results suggest that NES-A is a functional nuclear export signal . Combined mutations in NES-A may cause multiple defects in protein function leading to an Nmd- phenotype even when export is restored.

Mol Biol Cell, 1998 Oct, 9(10), 2917 - 31
A mutation in a novel yeast proteasomal gene, RPN11/MPR1, produces a cell cycle arrest, overreplication of nuclear and mitochondrial DNA, and an altered mitochondrial morphology; Rinaldi T et al.; We report here the functional characterization of an essential Saccharomyces cerevisiae gene, MPR1, coding for a regulatory proteasomal subunit for which the name Rpn11p has been proposed . For this study we made use of the mpr1-1 mutation that causes the following pleiotropic defects . At 24 degreesC growth is delayed on glucose and impaired on glycerol, whereas no growth is seen at 36 degreesC on either carbon source . Microscopic observation of cells growing on glucose at 24 degreesC shows that most of them bear a large bud, whereas mitochondrial morphology is profoundly altered . A shift to the nonpermissive temperature produces aberrant elongated cell morphologies, whereas the nucleus fails to divide . Flow cytometry profiles after the shift to the nonpermissive temperature indicate overreplication of both nuclear and mitochondrial DNA . Consistently with the identification of Mpr1p with a proteasomal subunit, the mutation is complemented by the human POH1 proteasomal gene . Moreover, the mpr1-1 mutant grown to stationary phase accumulates ubiquitinated proteins . Localization of the Rpn11p/Mpr1p protein has been studied by green fluorescent protein fusion, and the fusion protein has been found to be mainly associated to cytoplasmic structures . For the first time, a proteasomal mutation has also revealed an associated mitochondrial phenotype . We actually showed, by the use of {rho degrees} cells derived from the mutant, that the increase in DNA content per cell is due in part to an increase in the amount of mitochondrial DNA . Moreover, microscopy of mpr1-1 cells grown on glucose showed that multiple punctate mitochondrial structures were present in place of the tubular network found in the wild-type strain . These data strongly suggest that mpr1-1 is a valuable tool with which to study the possible roles of proteasomal function in mitochondrial biogenesis.

Mol Biol Cell, 1998 Oct, 9(10), 2873 - 89
A yeast t-SNARE involved in endocytosis; Seron K et al.; The ORF YOL018c (TLG2) of Saccharomyces cerevisiae encodes a protein that belongs to the syntaxin protein family . The proteins of this family, t-SNAREs, are present on target organelles and are thought to participate in the specific interaction between vesicles and acceptor membranes in intracellular membrane trafficking . TLG2 is not an essential gene, and its deletion does not cause defects in the secretory pathway . However, its deletion in cells lacking the vacuolar ATPase subunit Vma2p leads to loss of viability, suggesting that Tlg2p is involved in endocytosis . In tlg2Delta cells, internalization was normal for two endocytic markers, the pheromone alpha-factor and the plasma membrane uracil permease . In contrast, degradation of alpha-factor and uracil permease was delayed in tlg2Delta cells . Internalization of positively charged Nanogold shows that the endocytic pathway is perturbed in the mutant, which accumulates Nanogold in primary endocytic vesicles and shows a greatly reduced complement of early endosomes . These results strongly suggest that Tlg2p is a t-SNARE involved in early endosome biogenesis.

FEBS Lett, 1998 Sep 18, 435(2-3), 245 - 50
Heterologous expression of human H1 histones in yeast; Albig W et al.; The complete set of seven human H1 histone subtype genes was heterologously expressed in yeast . Since Saccharomyces cerevisiae lacks standard histone H1 we could isolate each recombinantly expressed human H1 subtype in pure form without contamination by endogenous H I histones . For isolation of the H1 histones in this expression system no tagging was needed and the isoforms could be extracted with the authentic primary structure by a single extraction step with 5%(0.74 M) perchloric acid . The isolated H1 histone proteins were used to assign the subtype genes to the corresponding protein spots or peaks after two-dimensional gel electrophoresis and capillary zone electrophoresis, respectively . This allowed us to correlate transcriptional data with protein data, which was barely possible until now.

FEBS Lett, 1998 Sep 18, 435(2-3), 183 - 6
Mitochondrial Hsp70 cannot replace BiP in driving protein translocation into the yeast endoplasmic reticulum; Brodsky JL et al.; To determine whether mitochondrial hsp70 (mHsp70) could substitute for the endoplasmic retuculum (ER) Hsp70 (BiP) during protein translocation, we assembled ER-derived reconstituted proteoliposomes supplemented with either protein . We found that only BiP restored translocation in kar2 mutant vesicles and stimulated translocation approximately 3-fold in wild type proteoliposomes . mHsp70 associated poorly with both a BiP binding (DnaJ) domain of Sec63p and an ER precursor, and its ATPase activity was poorly enhanced upon incubation with the DnaJ domain . In contrast, BiP bound to the Sec63p-DnaJ domain in an ATP-dependent manner and its ATPase activity was stimulated significantly by this polypeptide . We conclude that mHsp70 is unable to support protein translocation into the ER because it fails to associate productively with Sec63p and a precursor.

Biotechniques, 1998 Sep, 25(3), 438 - 40, 442, 444
Fission yeast expression vectors adapted for positive identification of gene insertion and green fluorescent protein fusion; Zhao Y et al.; A pYZ series of fission yeast expression vectors, derivatives of the pREP series, was designed to allow positive identification of cloned gene insertion and fusion to the green fluorescent protein (GFP) gene for in vivo analysis of gene expression . To validate this new vector system, the human immunodeficiency virus type 1 (HIV-1) vpr gene of viral isolate pNL4-3 was expressed in the pYZ1N vector . Vpr-induced phenotypic changes were the same as those observed with vpr expressed from pREP1N . Consistent with observations in mammalian cells, a Vpr-GFP fusion protein localizes on the nuclear membrane of fission yeast cells . Additionally, we were able to detect a naturally occurring mixture of vpr genes from a plasma sample of an HIV-infected pediatric long-term surviving patient . These pYZ vectors expedite gene cloning for general purposes and are particularly suited for largescale random gene screening.

Anal Biochem, 1978 Dec, 91(2), 583 - 99
Colorimetric determination of succinic acid using yeast succinate dehydrogenase; Valle AB et al.; An enzymatic method for the rapid determination of succinic acid in biological fluids was developed utilizing yeast mitochondria as a source of succinate dehydrogenase . The yeast enzyme catalyzes a complete stoichiometric reduction of 2- (p-iodophenyl)-3-(p-nitrophenyl)-5-tetrazolium chloride to a red formazan . The formazan is extracted into ethylacetate and its absorbance measured at 490 nm . The method is simple, specific, reproducible, and very sensitive (0.01 to 0.14 mumol) . The yeast enzyme can be stored in liquid nitrogen for periods of at least 30 days with no significant change in specific activity . In this respect it is superior to a variety of succinate dehydrogenase preparations from animal tissues . The method was applied to measurement of succinic acid excreted by nonproliferating yeast cells metabolizing glucose . Derepressed yeast cells secreted several-fold as much succinic acid as repressed cells submitted to identical test conditions.

Annu Rev Biochem, 1998, 67, 307 - 33
Role of small G proteins in yeast cell polarization and wall biosynthesis; Cabib E et al.; In the vegetative (mitotic) cycle and during sexual conjugation, yeast cells display polarized growth, giving rise to a bud or to a mating projection, respectively . In both cases one can distinguish three steps in these processes: choice of a growth site, organization of the growth site, and actual growth and morphogenesis . In all three steps, small GTP-binding proteins (G proteins) and their regulators play essential signaling functions . For the choice of a bud site, Bud1, a small G protein, Bud2, a negative regulator of Bud1, and Bud5, an activator, are all required . If any of them is defective, the cell loses its ability to select a proper bud position and buds randomly . In the organization of the bud site or of the site in which a mating projection appears, Cdc42, its activator Cdc24, and its negative regulators play a fundamental role . In the absence of Cdc42 or Cdc24, the actin cytoskeleton does not become organized and budding does not take place . Finally, another small G protein, Rho1, is required for activity of beta (1-->3)glucan synthase, the enzyme that catalyzes the synthesis of the major structural component of the yeast cell wall . In all of the above processes, G proteins can work as molecular switches because of their ability to shift between an active GTP-bound state and an inactive GDP-bound state.

J Biol Chem, 1998 Oct 9, 273(41), 26559 - 65
Tra1p is a component of the yeast Ada.Spt transcriptional regulatory complexes; Saleh A et al.; The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation . We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/Ada3p . TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxyl-terminal regions related to phosphatidylinositol 3-kinases . The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p . Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns . Binding of Tra1p to DNA-cellulose required Ada components . The association of Tra1p with two Ada.Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column . In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak . Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada.Spt complexes.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1633 - 6
Phospholipid deacylating activities included in yeast; Morimoto T et al.; Yeast had been known to contain only one kind of phospholipid deacylating enzyme with an optimal pH in the acidic range . However, among 8 genera and 25 strains, 7 genera and 13 strains had phospholipid deacylating activity at pH 8.0, when screening of enzyme activity was done with micellar and liposomal substrates . The enzymatic properties of phospholipid deacylating enzyme existing in yeast was not related to genetic identity based on 18S rRNA gene sequence . The results suggest that yeast contains enzymes showing a variety of properties depending on the species of yeast, and further studies on this enzyme with more varieties of yeasts are necessary for understanding the physiological roles of the enzyme in yeast.

J Biol Chem, 1998 Oct 9, 273(41), 26375 - 82
Importance of phenylalanine residues of yeast calmodulin for target binding and activation; Okano H et al.; Recent genetic studies of yeast calmodulin (yCaM) have shown that alterations of different sets of Phe residues result in distinct functional defects (Ohya, Y., and Botstein, D . (1994) Science 263, 963-966) . To examine the importance of Phe residues for target binding and activation, we purified mutant yCaMs containing single or double Phe to Ala substitutions and determined their ability to bind and activate two target proteins, calcineurin and CaM-dependent protein kinase (CaMK) . Binding assays using the gel overlay technique and quantitative analyses using surface plasmon resonance measurements indicated that the binding of yCaM to calcineurin is impaired by either double mutations of F16A/F19A or a single mutation of F140A, while binding to CaMK is impaired by F89A, F92A, or F140A . These same mutant yCaMs fail to activate calcineurin and CaMK, respectively, in vitro . In addition, F19A exhibited a severe defect in activation of both enzymes . F12A activated calcineurin to only 50% of the level achieved by wild-type calmodulin but fully activated CaMK . These results suggest that each target protein requires a specific and distinct subset of Phe residues in yCaM for target binding and activation.

Genetics, 1998 Oct, 150(2), 601 - 11
Mutations in the membrane anchor of yeast cytochrome c1 compensate for the absence of Oxa1p and generate carbonate-extractable forms of cytochrome c1; Hamel P et al.; Oxa1p is a mitochondrial inner membrane protein that is mainly required for the insertion/assembly of complex IV and ATP synthase and is functionally conserved in yeasts, humans, and plants . We have isolated several independent suppressors that compensate for the absence of Oxa1p . Molecular cloning and sequencing reveal that the suppressor mutations (CYT1-1 to -6) correspond to amino acid substitutions that are all located in the membrane anchor of cytochrome c1 and decrease the hydrophobicity of this anchor . Cytochrome c1 is a catalytic subunit of complex III, but the CYT1-1 mutation does not seem to affect the electron transfer activity . The double-mutant cyt1-1,164, which has a drastically reduced electron transfer activity, still retains the suppressor activity . Altogether, these results suggest that the suppressor function of cytochrome c1 is independent of its electron transfer activity . In addition to the membrane-bound cytochrome c1, carbonate-extractable forms accumulate in all the suppressor strains . We propose that these carbonate-extractable forms of cytochrome c1 are responsible for the suppressor function by preventing the degradation of the respiratory complex subunits that occur in the absence of Oxa1p.

Genetics, 1998 Oct, 150(2), 563 - 76
Histone deacetylase homologs regulate epigenetic inheritance of transcriptional silencing and chromosome segregation in fission yeast; Grewal SI et al.; Position-effect control at the silent mat2-mat3 interval and at centromeres and telomeres in fission yeast is suggested to be mediated through the assembly of heterochromatin-like structures . Therefore, trans-acting genes that affect silencing may encode either chromatin proteins, factors that modify them, or factors that affect chromatin assembly . Here, we report the identification of an essential gene, clr6 (cryptic loci regulator), which encodes a putative histone deacetylase that when mutated affects epigenetically maintained repression at the mat2-mat3 region and at centromeres and reduces the fidelity of chromosome segregation . Furthermore, we show that the Clr3 protein, when mutated, alleviates recombination block at mat region as well as silencing at donor loci and at centromeres and telomeres, also shares strong homology to known histone deacetylases . Genetic analyses indicate that silencing might be regulated by at least two overlapping histone deacetylase activities . We also found that transient inhibition of histone deacetylase activity by trichostatin A results in the increased missegregation of chromosomes in subsequent generations and, remarkably, alters the imprint at the mat locus, causing the heritable conversion of the repressed epigenetic state to the expressed state . This work supports the model that the level of histone deacetylation has a role in the assembly of repressive heterochromatin and provides insight into the mechanism of epigenetic inheritance.

EMBO J, 1998 Oct 1, 17(19), 5805 - 10
Mechanism of inhibition of Psi+ prion determinant propagation by a mutation of the N-terminus of the yeast Sup35 protein; Kochneva-Pervukhova NV et al.; The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3 . This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Psi+ determinant . A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58-->Asp change in the Sup35p N-terminal domain eliminates Psi+ . Here we observed that the mutant Sup35p can be converted to the prion-like form in vitro, but such conversion proceeds slower than that of wild-type Sup35p . The overexpression of mutant Sup35p induced the de novo appearance of Psi+ cells containing the prion-like form of mutant Sup35p, which was able to transmit its properties to wild-type Sup35p both in vitro and in vivo . Our data indicate that this Psi+-eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Psi+-specific aggregates, but rather inhibits its subsequent prion-like rearrangement and/or binding of the next Sup35p molecule to the growing prion-like Sup35p aggregate.

EMBO J, 1998 Oct 1, 17(19), 5679 - 88
The budding yeast Rad9 checkpoint protein is subjected to Mec1/Tel1-dependent hyperphosphorylation and interacts with Rad53 after DNA damage; Vialard JE et al.; The Saccharomyces cerevisiae RAD9 checkpoint gene is required for transient cell-cycle arrests and transcriptional induction of DNA repair genes in response to DNA damage . Polyclonal antibodies raised against the Rad9 protein recognized several polypeptides in asynchronous cultures, and in cells arrested in S or G2/M phases while a single form was observed in G1-arrested cells . Treatment with various DNA damaging agents, i.e . UV, ionizing radiation or methyl methane sulfonate, resulted in the appearance of hypermodified forms of the protein . All modifications detected during a normal cell cycle and after DNA damage were sensitive to phosphatase treatment, indicating that they resulted from phosphorylation . Damage-induced hyperphosphorylation of Rad9 correlated with checkpoint functions (cell-cycle arrest and transcriptional induction) and was cell-cycle stage- and progression-independent . In asynchronous cultures, Rad9 hyperphosphorylation was dependent on MEC1 and TEL1, homologues of the ATR and ATM genes . In G1-arrested cells, damage-dependent hyperphosphorylation required functional MEC1 in addition to RAD17, RAD24, MEC3 and DDC1, demonstrating cell-cycle stage specificity of the checkpoint genes in this response to DNA damage . Analysis of checkpoint protein interactions after DNA damage revealed that Rad9 physically associates with Rad53.

EMBO J, 1998 Oct 1, 17(19), 5525 - 8
DNA damage checkpoint in budding yeast; Longhese MP et al.; Eukaryotic cells have evolved a network of control mechanisms, known as checkpoints, which coordinate cell-cycle progression in response to internal and external cues . The yeast Saccharomyces cerevisiae has been invaluable in dissecting genetically the DNA damage checkpoint pathway . Recent results on posttranslational modifications and protein-protein interactions of some key factors provide new insights into the architecture of checkpoint protein complexes and their order of function.

Indian J Biochem Biophys, 1998 Apr, 35(2), 86 - 90
Harmaline interactions with yeast invertase; Gill RK et al.; The effect of harmaline, a plant alkaloid has been studied on yeast invertase activity in the absence and presence of 50mM Na+ as a function of pH . Harmaline (1-3 mM) inhibited the invertase activity at pH 5.2, 6.8 and 8 both in the absence (44-92%) and (22-85%) of Na+ ions . Kinetic analysis revealed that harmaline is a non-competitive inhibitor of invertase, at pH 5.2 and 6.8 but at pH 8, it produced a mixed type of inhibition, Km increased by 450% and 175% and Vmax decreased by 82% and 63% in the absence and presence of 50mM Na ions respectively . The observed inhibition of invertase by harmaline was reversible in nature . These findings suggest that the presence of Na+ site is not a prerequisite for the inhibition of enzyme by harmaline.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4676 - 87
Regulation of poly(A) site choice of several yeast mRNAs; Sparks KA et al.; Several yeast genes produce multiple transcripts with different 3'-ends . Of these, four genes are known to produce truncated transcripts that end within the coding sequence of longer transcripts: CBP1 , AEP2 / ATP13 , RNA14 and SIR1 . It has been shown that the level of the truncated CBP1 transcript increases during the switch to respiratory growth while that of the full-length transcript decreases . To determine whether this phenomenon is unique to CBP1 , northern analysis was used to determine whether the levels of other truncated transcripts are regulated similarly by carbon source . The levels of the shortest transcripts of AEP2 / ATP13 and RNA14 increased during respiration while the shortest SIR1 transcript remained constant . However, two longer SIR1 transcripts were regulated reciprocally by carbon source . Mapping the 3'-ends of each transcript by sequencing partial cDNA clones revealed multiple 3'-ends for each transcript . Examination of the sequences surrounding the 3'-ends of the induced transcripts failed to identify a consensus sequence but did reveal weak putative 3'-end formation signals in all of the transcripts . Similarly, no consensus sequence was found when the sequences surrounding the 3'-ends of the longest transcripts were compared, but again weak putative 3'-end formation signals were identified . These data are suggestive of carbon source regulation of alternative poly(A) site choice in yeast.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4588 - 96
The yeast FBP1 poly(A) signal functions in both orientations and overlaps with a gene promoter; Aranda A et al.; This report provides an analysis of a region of chromosome XII in which the FBP1 and YLR376c genes transcribe in the same direction . Our investigation indicates that the Saccharomyces cerevisiae FBP1 gene contains strong signals for polyadenylation and transcription termination in both orientations in vivo . A (TA)14 element plays a major role in directing polyadenylation in both orientations . While this region has four nonoverlapping copies of a TATATA hexanucleotide, which is a very potent polyadenylation efficiency element in yeast, it alone is not sufficient for full activation in the reverse orientation of a cluster of downstream poly(A) sites, and an additional upstream sequence is required . The putative RNA hairpin formed from the (TA)14 element is not involved in 3'-end formation . Surprisingly, deletion of the entire (TA)14 stretch affects transcription termination in the reverse orientation, in contrast to our previous results with the forward orientation, indicating that the transcription termination element operating in the reverse orientation has very different sequence requirements . Promoter elements for the YLR376c gene overlap with the signal for FBP1 3'-end formation . To our knowledge, this is the first time that overlapping of both types of regulatory signals has been found in two adjacent yeast genes.

Biochem Biophys Res Commun, 1998 Sep 18, 250(2), 212 - 6
Protein-protein interactions of the yeast Golgi t-SNARE Sed5 protein distinct from its neural plasma membrane cognate syntaxin 1; Kosodo Y et al.; Targeting of vesicles to the acceptor membrane in protein transport depends on membrane proteins called SNAREs . Saccharomyces cerevisiae Golgi t-SNARE Sed5 protein and its neural cognate syntaxin 1 have similar three alpha-helices which are predicted to form coiled coils . We dissected the helices of Sed5 and found several characteristics unexpectedly distinct from those of syntaxin 1 . Most importantly, only the N-terminal helix is responsible for the binding of Sly1 protein while almost the entire molecule of syntaxin is necessary for the binding of the cognate, Munc-18 . The N-terminal region of Sed5 protein also binds to the C-terminal helix and Sly1 protein interfered this binding.

J Mol Biol, 1998 Oct 9, 282(5), 933 - 46
TOM1p, a yeast hect-domain protein which mediates transcriptional regulation through the ADA/SAGA coactivator complexes; Saleh A et al.; The hect-domain has been characterized as a conserved feature of a group of E3 ubiquitin ligases . Here we show that the yeast hect-domain protein TOM1p regulates transcriptional activation through effects on the ADA transcriptional coactivator proteins . Null mutations of tom1 result in similar defects in transcription from ADH2 and HIS3 promoters, and enhanced transcription from the GAL10 promoter as do null mutations in ngg1/ada3 . Strains with disruptions of both ngg1 and tom1 have the same phenotype as strains with a disruption of only ngg1 implying that these genes are acting through the same pathway . In the absence of TOM1p, the normal associations of the ADA proteins with SPT3p and the TATA-binding protein are reduced . The action of TOM1p is most likely mediated through ubiquitination since mutation of Cys3235 to Ala, corresponding residues of which are required for thioester bond formation with ubiquitin in other hect-domain proteins, results in similar changes in transcription as the null mutation . A direct role for TOM1p in regulation of ADA-associated proteins is further supported by the finding that SPT7p is ubiquitinated in a TOM1p-dependent fashion and that TOM1p coimmunoprecipitates with the ADA proteins .

Cell, 1998 Sep 18, 94(6), 795 - 807
Signal sequence recognition in posttranslational protein transport across the yeast ER membrane; Plath K et al.; We have analyzed how the signal sequence of prepro-alpha-factor is recognized during the first step of posttranslational protein transport into the yeast endoplasmic reticulum . Cross-linking studies indicate that the signal sequence interacts in a Kar2p- and ATP-independent reaction with Sec61p, the multispanning membrane component of the protein-conducting channel, by intercalation into transmembrane domains 2 and 7 . While bound to Sec61p, the signal sequence forms a helix that is contacted on one side by Sec62p and Sec71p . The binding site is located at the interface of the protein channel and the lipid bilayer . Signal sequence recognition in cotranslational translocation in mammals appears to occur similarly . These results suggest a general mechanism by which the signal sequence could open the channel for polypeptide transport.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 12043 - 8
The plant cDNA LCT1 mediates the uptake of calcium and cadmium in yeast; Clemens S et al.; Nonessential metal ions such as cadmium are most likely transported across plant membranes via transporters for essential cations . To identify possible pathways for Cd2+ transport we tested putative plant cation transporters for Cd2+ uptake activity by expressing cDNAs in Saccharomyces cerevisiae and found that expression of one clone, LCT1, renders the growth of yeast more sensitive to cadmium . Ion flux assays showed that Cd2+ sensitivity is correlated with an increase in Cd2+ uptake . LCT1-dependent Cd2+ uptake is saturable, lies in the high-affinity range (apparent KM for Cd2+ = 33 microM) and is sensitive to block by La3+ and Ca2+ . Growth assays demonstrated a sensitivity of LCT1-expressing yeast cells to extracellular millimolar Ca2+ concentrations . LCT1-dependent increase in Ca2+ uptake correlated with the observed phenotype . Furthermore, LCT1 complements a yeast disruption mutant in the MID1 gene, a non-LCT1-homologous yeast gene encoding a membrane Ca2+ influx system required for recovery from the mating response . We conclude that LCT1 mediates the uptake of Ca2+ and Cd2+ in yeast and may therefore represent a first plant cDNA encoding a plant Ca2+ uptake or an organellar Ca2+ transport pathway in plants and may contribute to transport of the toxic metal Cd2+ across plant membranes.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11721 - 6
Isolation of yeast mutants defective for localization of vacuolar vital dyes; Zheng B et al.; An application of flow cytometric sorting is used for isolation of Saccharomyces cerevisiae mutants that mislocalize vacuolar vital dyes . This screen is based on the ability of a lipophilic styryl compound, N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrie nyl )pyridinium dibromide (FM4-64), to label endocytic intermediates from the plasma membrane to the vacuole membrane at 15 degreesC . Cells stained at 15 degreesC for both FM4-64 and carboxydichlorofluorescein diacetate (a vacuolar luminal vital stain), had a pronounced shift in red/green fluorescence from cells stained at 30 degrees or 38 degreesC . Flow cytometric selection based on this characteristic shift allowed the isolation of 16 mutants . These comprised 12 complementation groups, which we have designated SVL for styryl dye vacuolar localization . These groups were put into three classes . Class I mutants contain very large vacuoles; class II mutants have very fragmented vacuoles; and class III mutants show the strongest svl phenotype with punctate/diffuse FM4-64 staining . Limited genetic overlap was observed with previously isolated mutants, namely svl2/vps41, svl6/vps16, and svl7/fab1 . The remaining svl mutants appear to represent novel genes, two of which showed temperature-sensitive vacuole staining morphology . Another mutant, svl8, displayed defects in uptake and sorting of phosphatidylcholine and phosphatidylethanolamine . Our flow cytometric strategy may be useful for isolation of other mutants where mislocalization of fluorescent compounds can be detected.

Genome Res, 1998 Sep, 8(9), 916 - 28
Compositional heterogeneity within, and uniformity between, DNA sequences of yeast chromosomes; Li W et al.; The heterogeneity within, and similarities between, yeast chromosomes are studied . For the former, we show by the size distribution of domains, coding density, size distribution of open reading frames, spatial power spectra, and deviation from binomial distribution for C + G% in large moving windows that there is a strong deviation of the yeast sequences from random sequences . For the latter, not only do we graphically illustrate the similarity for the above mentioned statistics, but we also carry out a rigorous analysis of variance (ANOVA) test . The hypothesis that all yeast chromosomes are similar cannot be rejected by this test . We examine the two possible explanations of this interchromosomal uniformity: a common origin, such as genome-wide duplication (polyploidization), and a concerted evolutionary process.

Anal Biochem, 1998 Oct 1, 263(1), 62 - 6
Use of the yeast three-hybrid system as a tool to study caspases; Van Criekinge W et al.; Caspases are a family of heteromeric (p20/p10) cysteine proteases with important functions in the regulation of apoptosis and inflammation . Up to now, tools to identify new substrates for caspases have mostly been limited to the random screening of in vitro translated proteins that are known, or assumed, to play a role in apoptosis . We describe the use of a yeast three-hybrid approach as a tool that adapts the classical two-hybrid system to the needs of heteromeric caspases for functional dissection of known interactions or screening for physiological substrates and inhibitors . Functional heteromeric caspase-1 was obtained by coexpression of p20(Cys285Ser) and p10 caspase-1 subunits that were each fused to the Gal4 DNA-binding domain . Upon coexpression of a third hybrid of the Gal4 activation domain and the viral caspase-1 pseudosubstrate inhibitors CrmA or p35, or the prototype physiological caspase-1 substrate prointerleukin-1beta, a functional Gal4 transcription factor could be reconstituted . In contrast, no interaction was found between CrmA or p35 and the immature p45 or p30 precursor forms of caspase-1 . Therefore, the three-hybrid system might allow screening for new physiological substrates and inhibitors of heteromeric caspases .

J Steroid Biochem Mol Biol, 1998 Sep, 66(5-6), 303 - 18
Using yeast to study glucocorticoid receptor phosphorylation; Pocuca N et al.; The glucocorticoid receptor (GR) is a phosphoprotein and a member of the steroid/thyroid receptor superfamily of ligand dependent transcription factors . When the glucocorticoid receptor is expressed in yeast (Saccharomyces cerevisiae), it is competent for signal transduction and transcriptional regulation . We have studied the glucocorticoid receptor phosphorylation in yeast and demonstrated that the receptor is phosphorylated in both the absence and presence of hormone, on serine and threonine residues . This phosphorylation occurs within 15 min upon addition of radioactivity in both hormone treated and untreated cells . As reported for mammalian cells, additional phosphorylation occurs upon hormone binding and this phosphorylation is dependent on the type of the ligand . We have followed the hormone dependent receptor phosphorylation by electrophoretic mobility shift assay, and have shown that this mobility change is sensitive to phosphatase treatment . In addition, the appearance of hormone dependent phosphoisoforms of the receptor depends on the potency of the agonist used . Using this method we show that the residues contributing to the hormone dependent mobility shift are localized in one of the transcriptional activation domains, between amino acids 130-247 . We altered the phosphorylation sites within this domain that correspond to the amino acids phosphorylated in mouse hormone treated cells . Using phosphopeptide maps we show that hormone changes the peptide pattern of metabolically labelled receptor, and we identify peptides which are phosphorylated in hormone dependent manner . Then we determine that phosphorylation of residues S224 and S232 is increased in the presence of hormone, whereas phosphorylation of residues T171 and S246 is constitutive . Finally, we show that in both yeast and mammalian cells the same residues on the glucocorticoid receptor are phosphorylated . Our results suggest that yeast cells would be a suitable system to study glucocorticoid receptor phosphorylation . The genetic manipulability of yeast cells, together with conservation of the phosphorylation of GR in yeast and mammalian cells and identification of hormone dependent phosphorylation, would facilitate the isolation of molecules involved in the glucocorticoid receptor phosphorylation pathway and further our understanding of this process.

Mol Gen Genet, 1998 Aug, 259(3), 282 - 93
mRNA translation in yeast during entry into stationary phase; Dickson LM et al.; The expression of some Saccharomyces cerevisiae genes is induced as cells enter stationary phase . Their mRNAs are translated during a period in the growth cycle when the translational apparatus is relatively inert, thereby raising the possibility that these mRNAs compete effectively for a limiting pool of translation factors . To test this idea, the translation of mRNAs carrying different 5'-leaders was compared during exponential growth and after entry into stationary phase upon glucose starvation . Closely related sets of lacZ mRNAs, carrying 5'-leaders from the PYK1, PGK1, RpL3, Rp29, HSP12, HSP26 or THI4 mRNAs, were studied . These mRNAs displayed differing translational efficiencies during exponential growth, but their relative translatabilities were not significantly affected by entry into stationary phase, indicating that they compete just as effectively under these conditions . Polysome analysis revealed that the wild-type PYK1, ACT1 and HSP26 mRNAs are all translated efficiently during stationary phase, when the translational apparatus is relatively inert . Also, significant levels of the translation initiation factors eIF-2alpha, eIF-4E and eIF-4A were maintained during the growth cycle . These data are consistent with the idea that, while translational activity decreases dramatically during entry into stationary phase, yeast cells maintain excess translational capacity under these conditions.

Biochim Biophys Acta, 1998 Oct 1, 1400(1-3), 289 - 300
Yeast as a model organism for studying the actions of DNA topoisomerase-targeted drugs; Reid RJ et al.; The budding yeast Saccharomyces cerevisiae has been exploited to investigate the cytotoxic mechanisms of drugs that target DNA topoisomerases . This model organism has been used to establish eukaryotic DNA topoisomerase I or II as the cellular target of specific antineoplastic agents, to define mutations in these enzymes that confer drug resistance and to elucidate the cellular factors that modulate cell sensitivity to DNA topoisomerase-targeted drugs . These findings have provided valuable insights into the critical activities of these enzymes and how perturbing their functions produces DNA damage and cell death.

What Is Protein?, What Is Growth Medium?, What Is Staphylococcus Aureus?, What Is Molecular Microbiology?, What Is Listeria Monocytogenes?, c, Bacteria, n, Microbes, e, Microbiology, a, Bacterium, c, Microorganism, n, Botulism, r, Microbial, r, Escherichia coli, i, Bacillus, a, Streptococcal, i, Escherichia coli, a, Shigella, e, Paracocci, i, Bacteria, r, S. cerevisiae, o, Salmonella, a, S. cerevisiae, e, Salmonella, o, Microflora, e, Cell cultures, c, Escherichia coli, s, Antimicrobial, s, Antimicrobials, o, Botulism, o, Escherichia coli, n, Salmonella

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005