Cell Cycle . 2005 Feb 3;4(2) {Epub ahead of print} How the Cell Deals with DNA Nicks; Garg P et al.; During lagging strand DNA replication, the Okazaki fragment maturation machinery is required to degrade the initiator RNA with high speed and efficiency, and to generate with great accuracy a proper DNA nick for closure by DNA ligase . Several operational parameters are important in generating and maintaining a ligatable nick . These are the strand opening capacity of the lagging strand DNA polymerase delta (Pol delta ), and its ability to limit strand opening to that of a few nucleotides . In the presence of the flap endonuclease FEN1, Pol delta rapidly hands off the strand-opened product for cutting by FEN1, while in its absence, the ability of DNA polymerase delta to switch to its 3'-->5'-exonuclease domain in order to degrade back to the nick position is important in maintaining a ligatable nick . This regulatory system has a built-in redundancy so that dysfunction of one of these activities can be tolerated in the cell . However, further dysfunction leads to uncontrolled strand displacement synthesis with deleterious consequences, as is revealed by genetic studies of exonuclease-defective mutants of S . cerevisiae Pol delta . These same parameters are also important for other DNA metabolic processes, such as base excision repair, that depend on Pol delta for synthesis. Mem Inst Oswaldo Cruz, 2004 Nov, 99(7), 733 - 7 Epub 2005 Jan 12. Similarity between the association factor of ribosomal subunits and the protein Stm1p from Saccharomyces cerevisiae; Correia H et al.; A ribosome association factor (AF) was isolated from the yeast Sacchharomyces cerevisiae . Partial amino acid sequence of AF was determined from its fragment of 25 kDa isolated by treating AF with 2-(2-nitrophenylsulfenyl)-3-methyl-3'-Bromoindolenine (BNPS-skatole) . This sequence has a 86% identity to the product of the single-copy S . cerevisiae STM1 gene that is apparently involved in several events like binding to quadruplex and triplex nucleic acids and participating in apoptosis, stability of telomere structures, cell cycle, and ribosomal function . Here we show that AF and Stm1p share some characteristics: both bind to quadruplex and Pu triplex DNA, associates ribosomal subunits, and are thermostable . These observations suggest that these polypeptides belong to a family of proteins that may have roles in the translation process. Genetics . 2005 Jan 16; {Epub ahead of print} Meiosis-specific regulation of the S . cerevisiae S-phase cyclin CLB5 is dependent upon MCB elements in its promoter but is independent of MBF activity; Raithatha SA et al.; In proliferating S . cerevisiae, genes whose products function in DNA replication are regulated by the MBF transcription factor composed of Mbp1 and Swi6 that binds to consensus MCB sequences in target promoters . We find that during meiotic development a subset of DNA replication genes exemplified by TMP1 and RNR1 are regulated by Mbp1 . Deletion of Mbp1 deregulated TMP1 and RNR1 but did not interfere with premeiotic S-phase, meiotic recombination or spore formation . Surprisingly deletion of MBP1 had no effect on the expression of CLB5, which is purportedly controlled by MBF . Extensive analysis of the CLB5 promoter revealed that the gene is largely regulated by elements within a 100 bp fragment containing a cluster of MCB sequences . Surprisingly induction of the CLB5 promoter requires MCB sequences, but not Mbp1, implying that another MCB binding factor may exist in cells undergoing meiosis . In addition full activation of CLB5 during meiosis requires Clb5 activity suggesting that CLB5 may be regulated by a positive feedback mechanism . We further demonstrate that during meiosis MCBs function as effective transcriptional activators independent of MBP1. Cell, 2005 Jan 14, 120(1), 25 - 36 Sterile 20 Kinase Phosphorylates Histone H2B at Serine 10 during Hydrogen Peroxide-Induced Apoptosis in S . cerevisiae; Ahn SH et al.; Apoptosis is a highly coordinated cell suicide mechanism in vertebrates . Phosphorylation of serine 14 of histone H2B, catalyzed by Mst1 kinase, has been linked to chromatin compaction during apoptosis . We extend these results to unicellular eukaryotes by demonstrating that H2B is specifically phosphorylated at serine 10 (S10) in a hydrogen peroxide-induced cell death pathway in S . cerevisiae . H2B S10A mutants are resistant to cell death elicited by H(2)O(2) while H2B S10E phospho-site mimics promote cell death and induce the "constitutive" formation of condensed chromatin . Ste20 kinase, a yeast homolog of mammalian Mst1 kinase, translocates into the nucleus in a caspase-independent fashion and directly phosphorylates H2B at S10 . Conservation of targeted H2B phosphorylation and the enzyme system responsible for the process point to an ancient mechanism of chromatin remodeling that likely plays an important role in governing cellular homeostasis in a wide range of organisms. Chem Res Toxicol, 2005 Jan, 18(1), 87 - 94 Effects of substrate specificity on initiating the base excision repair of N-methylpurines by variant human 3-methyladenine DNA glycosylases; Connor EE et al.; The human 3-methyladenine (AAG, ANPG, MPG) DNA glycosylase excises alkylated purines from DNA . In previous studies, we determined the importance of an active site amino acid (asparagine 169) in the recognition of substrates by AAG . In this study, we characterize the consequences of expressing the AAG variants bearing amino acid substitutions at position 169 in Saccharomyces cerevisiae that lack endogenous 3-methyladenine DNA glycosylase . Survival, mutation induction, and DNA double strand break formation were determined in response to methyl methanesulfonate . The ability of purified wild-type and AAG variants to remove 3-methyladenine and 7-methylguanine, the two most abundant adducts produced by methyl methanesulfonate, was also determined . The N169D AAG variant displayed a approximately 100-fold lower activity for 3-methyladenine as compared to wild-type and did not detectably remove 7-methylguanine . When expressed in S . cerevisiae, the N169D variant provided better protection against methyl methanesulfonate toxicity than wild-type . Fewer strand breaks in vivo were also seen in the presence of the N169D variant following MMS exposure . In contrast, the N169A and N169S AAG variants displayed approximately 30-fold lower activity for 3-methyladenine and 7-methylguanine . Expression of the N169A and N169S AAG variants in S . cerevisiae during methyl methanesulfonate exposure resulted in greater sensitivity, greater mutation induction following MMS exposure, and more strand breaks in vivo . Strand breaks seen in S . cerevisiae that express wild-type AAG or the N169 variants were resolved to varying extents during recovery . In contrast, strand breaks formed in S . cerevisiae that expressed a catalytically inactive AAG variant were not resolved during the recovery times examined . Taken together, the results provide evidence that 3-methyladenine adducts not repaired by base excision repair cause double strand breaks that are not rapidly resolved . Evidence is also provided that the BER intermediates resulting from excision of 7-methylguanine by wild-type AAG contributes to the mutagenicity and cytotoxicity of alkylating agents. Proteins . 2005 Jan 13; {Epub ahead of print} Role of trehalose and heat in the structure of the C-terminal activation domain of the heat shock transcription factor; Bulman AL et al.; The heat shock transcription factor (HSF) is the primary transcriptional regulator of the heat shock response in eukaryotes . Saccharomyces cerevisiae HSF1 has two functional transcriptional activation domains, located N- and C-terminal to the central core of the protein . These activation domains have a low level of transcriptional activity prior to stress, but they acquire a high level of transcriptional activity in response to stresses such as heat . Previous studies on the N-terminal activation domain have shown that it can be completely disordered.1 In contrast, we show that the C-terminal activation domain of S . cerevisiae HSF1 does contain a certain amount of secondary structure as measured by circular dichroism (CD) and protease resistance . The alpha-helical content of the domain can be increased by the addition of the disaccharide trehalose but not by sucrose . Trehalose, but not sucrose, causes a blue shift in the fluorescence emission spectra, which is suggestive of an increase in tertiary structure . Trehalose, which is known to be a chemical chaperone, also increases proteases' resistance and promotes heat-induced increases in alpha-helicity . The latter is particularly intriguing because of the physiological role of trehalose in yeast . Trehalose levels are increased dramatically after heat shock, and this is thought to protect protein structure prior to the increase of heat shock protein levels . Our results suggest that the dramatic changes in S . cerevisiae HSF1 transcriptional activity in response to stress might be linked to the combined effects of trehalose and elevated temperatures in modifying the overall structure of HSF1's C-terminal activation domain . Proteins 2005 . (c) 2005 Wiley-Liss, Inc. Hum Mol Genet . 2005 Jan 13; {Epub ahead of print} Functional and genomic approaches reveal an ancient CHEK2 allele associated with breast cancer in the Ashkenazi Jewish population; Shaag A et al.; Functional and genomic approaches can be integrated to screen efficiently for pathogenic alleles in founder populations . We applied such approaches to analysis of the cancer-associated cell cycle regulator CHEK2 in the Ashkenazi Jewish population . We first identified two extended haplotypes at CHEK2 that co-segregated with breast cancer in high-risk families . We sequenced CHEK2 in a case representing each haplotype and discovered two novel amino acid substitutions, CHEK2.S428F in the kinase domain and CHEK2.P85L in the N-terminus . To assay these alleles for loss of CHEK2 function, we tested their capacity to complement Rad53 deletion in S . cerevisiae . CHEK2.S428F failed to complement Rad53 and thus largely abrogates normal CHEK2 function, whereas CHEK2.P85L complemented Rad53 as well as did wildtype CHEK2 . Epidemiologic analyses were concordant with the functional tests . Frequencies of CHEK2.S428F heterozygotes were 2.88% (47/1632) among female breast cancer patients not selected for family history or age at diagnosis and 1.37% (23/1673) among controls (OR = 2.13, 95% CI {1.26, 3.69}, P = 0.004), whereas frequencies of CHEK2.P85L were 0.92% among cases and 0.83% among controls . Based on the experience of mothers, sisters, and daughters of probands, breast cancer risk due to CHEK2.S428F was estimated as 0.17 (+/-0.08) by age 60 . We conclude that CHEK2.S428F increases breast cancer risk approximately two-fold among Ashkenazi Jewish women, whereas CHEK2.P85L is a neutral allele . In general, these results suggest that selecting probands with extended haplotypes that co-segregate with disease can improve the efficiency of resequencing efforts, and that quantitative complementation tests in yeast can be used to evaluate variants in genes with highly conserved function. J Biol Chem . 2005 Jan 13; {Epub ahead of print} A structural disulfide of yeast protein disulfide isomerase destabilizes the active site disulfide of the N-terminal thioredoxin domain; Wilkinson B et al.; Protein Disulfide Isomerase (PDI) is an essential catalyst of disulfide formation and isomerization in the eukaryotic endoplasmic reticulum (ER) . PDI has two active sites at either end of the molecule, each containing two cysteines that facilitate thiol-disulfide exchange . In addition to its four catalytic cysteines, PDI possesses two non-active site cysteines, whose location and separation distance varies by organism . In higher eukaryotes, the non-active site cysteines are located in the C-terminal half of the protein sequence and are separated by 30 amino acids . In contrast, the internal cysteines of PDI from lower eukaryotes are located near the N-terminal active site and are much closer together in sequence . The function of these cysteines and the significance of their unique location in yeast PDI have been unclear . Previous data (Xiao, et al, J . Biol . Chem., 279, 49780-6, 2004) suggest that the internal cysteines exist as a disulfide in the ER of S . cerevisiae . By coupling mass spectrometry with a gel-shift technique that allows us to measure the redox potentials of the PDI active sites in the presence and absence of the non-active site cysteines, we find that the non-active site cysteines form a disulfide that is stable in even a very reducing environment and demonstrate that this disulfide exists to destabilize the N-terminal active site disulfide, making it a better oxidant by 18-fold . Consistent with this finding, we show that mutating the non-active site cysteines to alanines disrupts both the oxidase and isomerase activities of PDI in vitro. BMC Bioinformatics . 2005 Jan 14;6(1):8 {Epub ahead of print} Structural comparison of metabolic networks in selected single cell organisms; Zhu D et al.; Background There has been tremendous interest in the study of biological network structure . An array of measurements has been conceived to assess the topological properties of these networks . In this study, we compared the metabolic network structures of eleven single cell organisms representing the three domains of life using these measurements, hoping to find out whether the intrinsic network design principle(s), reflected by these measurements, are different among species in the three domains of life . Results Three groups of topological properties were used in this study: network indices, degree distribution measures and motif profile measure . All of which are higher-level topological properties except for the marginal degree distribution . Metabolic networks in Archaeal species are found to be different from those in S . cerevisiae and the six Bacterial species in almost all measured higher-level topological properties . Our findings also indicate that the metabolic network in Archaeal species is similar to the exponential random network . Conclusion If these metabolic network properties of the organisms studied can be extended to other species in their respective domains (which is likely), then the design principle(s) of Archaea are fundamentally different from those of Bacteria and Eukaryote . Furthermore, the functional mechanisms of Archaeal metabolic networks revealed in this study differentiate significantly from those of Bacterial and Eukaryotic organisms, which warrant further investigation. Mol Biol Evol . 2005 Jan 12; {Epub ahead of print} Comparative Genomics of Hemiascomycete Yeasts: Genes Involved in DNA Replication, Repair and Recombination; Richard GF et al.; Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA . Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair and recombination in C . glabrata, K . lactis, D . hansenii and Y . lipolytica, and compared them with their S . cerevisiae orthologues . We found that proteins belonging to the replication fork and to the Nucleotide Excision Repair pathway were -on the average- more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination . The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs) are conserved, as is Mus81p, involved in resolving meiotic recombination intermediates . Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis, are also found in C . glabrata, K . lactis and D . hansenii, but not in Y . lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts . In addition, we found that SGS1 and TOP1, respectively a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C . glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K . lactis . Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy. Proc Natl Acad Sci U S A . 2005 Jan 12; {Epub ahead of print} Elevated evolutionary rates in the laboratory strain of Saccharomyces cerevisiae; Gu Z et al.; By using the maximum likelihood method, we made a genome-wide comparison of the evolutionary rates in the lineages leading to the laboratory strain (S288c) and a wild strain (YJM789) of Saccharomyces cerevisiae and found that genes in the laboratory strain tend to evolve faster than in the wild strain . The pattern of elevated evolution suggests that relaxation of selection intensity is the dominant underlying reason, which is consistent with recurrent bottlenecks in the S . cerevisiae laboratory strain population . Supporting this conclusion are the following observations: (i) the increases in nonsynonymous evolutionary rate occur for genes in all functional categories; (ii) most of the synonymous evolutionary rate increases in S288c occur in genes with strong codon usage bias; (iii) genes under stronger negative selection have a larger increase in nonsynonymous evolutionary rate; and (iv) more genes with adaptive evolution were detected in the laboratory strain, but they do not account for the majority of the increased evolution . The present discoveries suggest that experimental and possible industrial manipulations of the laboratory strain of yeast could have had a strong effect on the genetic makeup of this model organism . Furthermore, they imply an evolution of laboratory model organisms away from their wild counterparts, questioning the relevancy of the models especially when extensive laboratory cultivation has occurred . In addition, these results shed light on the evolution of livestock and crop species that have been under human domestication for years. Yeast . 2005 Jan 11;22(2):79-90 {Epub ahead of print} Genome-wide survey of non-essential genes required for slowed DNA synthesis-induced filamentous growth in yeast; Kang CM et al.; We recently discovered that slowed DNA synthesis induces filamentous differentiation in S . cerevisiae . We screened the BY yeast deletion strains and identified four classes of non-essential genes that are required for both slowed DNA-induced filamentous growth and classic forms of filamentous growth: (a) genes encoding regulators of the actin cytoskeleton and cell polarity, ABP1, CAP2 and HUF1 (= YOR300W), in addition to the previously known BNI1, BUD2, PEA2, SPA2 and TPM1; (b) genes that are likely involved in cell wall biosynthesis, ECM25, GAS1 and PRS3; (c) genes encoding possible regulators of protein secretion, SEC66, RPL21A and RPL34B; (d) genes encoding factors for normal mitochondrial function, IML1 and UGO1 . These results showed that pseudohyphal formation involves not the only previously known regulation of the actin cytoskeleton/cell polarity but also regulation of cell wall synthesis, protein secretion and mitochondrial function . Identification of multiple classes of genes that are required for both slowed DNA synthesis-induced and classic forms of filamentous growth confirms that slowed DNA synthesis-induced filamentous growth is bone fide filamentous differentiation . Copyright (c) 2005 John Wiley & Sons, Ltd. Mol Biol Cell . 2005 Jan 5; {Epub ahead of print} Three-Dimensional Ultrastructure of Saccharomyces cerevisiae Meiotic Spindles; Winey M et al.; Monitoring Editor: Ted Salmon Meiotic chromosome segregation leads to the production of haploid germ cells . During meiosis I (MI) the paired homologous chromosomes are separated . Meiosis II (MII) segregation leads to the separation of paired sister chromatids . In the budding yeast, S . cerevisiae, both of these divisions take place in a single nucleus, giving rise to the four spored ascus . We have modeled the microtubules in 20 MI and 15 MII spindles using reconstruction from electron micrographs of serially sectioned meiotic cells . Meiotic spindles contain more microtubules than their mitotic counterparts, with the highest number in MI spindles . It is possible to differentiate between MI versus MII spindles based on microtubule numbers and organization . Similar to mitotic spindles, kinetochores in either MI or MII are attached by a single microtubule . The models indicate that the kinetochores of paired homologous chromosomes in MI or sister chromatids in MII are separated at metaphase, similar to mitotic cells . Examination of both MI and MII spindles reveals that anaphase A likely occurs in addition to anaphase B, and that these movements are concurrent . This analysis offers a structural basis for considering meiotic segregation in yeast and for the analysis of mutants defective in this process. Mol Biol Cell . 2005 Jan 5; {Epub ahead of print} Mammalian PIG-X and Yeast Pbn1p Are the Essential Components of Glycosylphosphatidylinositol-mannosyltransferase I; Ashida H et al.; Monitoring Editor: Reid Gilmore Within the endoplasmic reticulum (ER), mannoses and glucoses, donated from dolichol-phosphate-mannose and -glucose, are transferred to N-glycan and GPI-anchor precursors, and serine/threonine residues in many proteins . Glycosyltransferases that mediate these reactions are ER-resident multi-transmembrane proteins with common characteristics, forming a superfamily of more than ten enzymes . Here, we report an essential component of glycosylphosphatidylinositol-mannosyltransferase I (GPI-MT-I), which transfers the first of the four mannoses in the GPI-anchor precursors . We isolated a Chinese hamster ovary (CHO) cell mutant defective in GPI-MT-I but not its catalytic component PIG-M . The mutant gene, termed PIG-X, encoded a 252 amino-acid ER-resident type I transmembrane protein with a large lumenal domain . PIG-X and PIG-M formed a complex, and PIG-M expression was <10% in the absence of PIG-X, indicating that PIG-X stabilizes PIG-M . We found that S . cerevisiae Pbn1p/YCL052Cp, which was previously reported to be involved in autoprocessing of proproteinase B, is the functional homologue of PIG-X; Pbn1p is critical for Gpi14p/YJR013Wp function, the yeast homologue of PIG-M . This is the first report of an essential subcomponent of glycosyltransferases utilizing dolichol-phosphate-monosaccharide. FASEB J . 2005 Jan 4; {Epub ahead of print} Functional characterization of human proapoptotic molecules in yeast S . cerevisiae; Guscetti F et al.; The presence of a complete (BH1-3) proapoptotic molecule is necessary for the induction of the intrinsic apoptotic cascade in mammalian cells . It is unclear, however, what distinct roles the members of the large family of BH3-only proapoptotic molecules play in apoptosis . Although biochemical analysis of these molecules can characterize binding efficiencies of BH3 family members, the biologic consequences of these interactions are difficult to predict . We have, therefore, established three functional categories of BH3-only human proapoptotic proteins based on their toxicity after expression in budding yeast: directly killing (tBid), sensitizing in Bax/Bcl-2 expressing cells (Bad or Puma), and non-toxic (BNip3, BNip3L, and Noxa) . The mechanism of killing by the proapoptotic molecules in yeast, however, is not due to activation of the recently described yeast metacaspase MCA1. Biophys J . 2004 Dec 30; {Epub ahead of print} k-Cone Analysis: Determining All Candidate Values for Kinetic Parameters on a Network-scale; Famili I et al.; The absence of comprehensive measured kinetic values and the observed inconsistency in the available in vitro kinetic data has hindered the formulation of network-scale kinetic models of biochemical reaction networks . To meet this challenge we present an approach to construct a convex space, termed the k-cone, that contains all the allowable numerical values of the kinetic constants in large-scale biochemical networks . The definition of the k-cone relies on the incorporation of in vivo concentration data and a simplified approach to represent enzyme kinetics within an established constraint-based modeling approach . The k-cone approach was implemented to define the allowable combination of numerical values for a full kinetic model of human red blood cell metabolism and to study its correlated kinetic parameters . The k-cone approach can be used to determine the consistency between in vitro measured kinetic values and in vivo concentration and flux measurements when used in a network-scale kinetic model . k-cone analysis was successful in determining whether in vitro measured kinetic values used in the reconstruction of a kinetic-based model of Saccharomyces cerevisiae central metabolism could reproduce in vivo measurements . Further, the k-cone can be used to determine which numerical values of in vitro measured parameters are required to be changed in a kinetic model if in vivo measured values are not reproduced . k-cone analysis could identify what minimum number of in vitro determined kinetic parameters needed to be adjusted in the S . cerevisiae model in order to be consistent with the in vivo data . Applying the k-cone analysis a priori to kinetic model development may reduce the time and effort involved in model building and parameter adjustment . With the recent developments in high-throughput profiling of metabolite concentrations at a whole-cell scale and advances in metabolomics technologies, the k-cone approach presented here may hold the promise for kinetic characterization of metabolic networks as well as other biological functions at a whole-cell level. Acta Biochim Pol, 2004, 51(4), 953 - 62 Benzodiazepine binding to mitochondrial membranes of the amoeba Acanthamoeba castellanii and the yeast Saccharomyces cerevisiae; Slocinska M et al.; Benzodiazepine binding sites were studied in mitochondria of unicellular eukaryotes, the amoeba Acathamoeba castellanii and the yeast Saccharomyces cerevisiae, and also in rat liver mitochondria as a control . For that purpose we applied Ro5-4864, a well-known ligand of the mitochondrial benzodiazepine receptor (MBR) present in mammalian mitochondria . The levels of specific {(3)H}Ro5-4864 binding, the dissociation constant (K(D)) and the number of {(3)H}Ro5-4864 binding sites (B(max)) determined for fractions of the studied mitochondria indicate the presence of specific {(3)H}Ro5-4864 binding sites in the outer membrane of yeast and amoeba mitochondria as well as in yeast mitoplasts . Thus, A . castellanii and S . cerevisiae mitochondria, like rat liver mitochondria, contain proteins able to bind specifically {(3)H}Ro5-4864 . Labeling of amoeba, yeast and rat liver mitochondria with {(3)H}Ro5-4864 revealed proteins identified as the voltage dependent anion selective channel (VDAC) in the outer membrane and adenine nucleotide translocase (ANT) in the inner membrane . Therefore, the specific MBR ligand binding is not confined only to mammalian mitochondria and is more widespread within the eukaryotic world . However, it can not be excluded that MBR ligand binding sites are exploited efficiently only by higher multicellular eukaryotes . Nevertheless, the MBR ligand binding sites in mitochondria of lower eukaryotes can be applied as useful models in studies on mammalian MBR. Huan Jing Ke Xue, 2004 Sep, 25(5), 127 - 32 {Cloning and expression of the endo-beta-glucanase III cDNA gene from Trichoderma viride AS3.3711}; Liu BD et al.; To study the construction of yeast bioengineering strain which can degrade cellulosic waste, an endo-beta-glucanase III (EG III) cDNA gene of Trichoderma viride AS3.3711 was isolated with RT-PCR protocol . After sequencing it was constructed on S . cerevisiae induceable expression vector pYES2 . A L9 (3(4)) orthogonal design was used to optimize yeast sonication assistant transformation . The expression of EG III gene was induced by 2% beta-D-glactose, the transcription and expression of it was detected by Northern blotting and Congo Red method respectively . The endo-beta-glucanase activity was assayed as CMCase activity with CMC-Na as a substrate . The results show that the ORF of EG III was 1254 bp, encoding 418 aa, deducing molecular weight 44.1 x 10(3), group 5 (sonication treat time 60 s, incubate 40 min, SS-DNA 150 microg, heat shock 5 min) was the optimum one of the orthogonal experiment, and EG III transformants can produced clear hydrolysis halos on the Congo-Red-CMC plate . The measure of the enzyme activity show that the expression product can be expressed in active forms and secreted to the medium . The enzyme activity was approached the highest level (0.041 U/mL) when the culture time was 60 h . The optimized enzyme reaction temperature was 50 degrees C and the optimized pH was 5.8. Curr Biol, 2004 Dec 29, 14(24), 2283 - 8 Ubiquitin-like protein Hub1 is required for pre-mRNA splicing and localization of an essential splicing factor in fission yeast; Wilkinson CR et al.; Hub1/Ubl5 is a member of the family of ubiquitin-like proteins (UBLs) . The tertiary structure of Hub1 is similar to that of ubiquitin; however, it differs from known modifiers in that there is no conserved glycine residue near the C terminus which, in ubiquitin and UBLs, is required for covalent modification of target proteins . Instead, there is a conserved dityrosine motif proximal to the terminal nonconserved amino acid . In S . cerevisiae, high molecular weight adducts can be formed in vivo from Hub1, but the structure of these adducts is not known, and they could be either covalent or noncovalent . The budding yeast HUB1 gene is not essential, but Delta hub1 mutants display defects in mating . Here, we report that fission yeast hub1 is an essential gene, whose loss results in cell cycle defects and inefficient pre-mRNA splicing . A screen for Hub1 interactors identified Snu66, a component of the U4/U6.U5 tri-snRNP splicing complex . Furthermore, overexpression of Snu66 suppresses the lethality of a hub1ts mutant . In cells lacking functional hub1, the nuclear localization of Snu66 is disrupted, suggesting that an important role for Hub1 is the correct subcellular targeting of Snu66, although our data suggest that Hub1 is likely to perform other roles in splicing as well. Mol Biol Cell . 2004 Dec 22; {Epub ahead of print} Functional Analysis of Cytoplasmic Dynein Heavy Chain in Caenorhabditis elegans with Fast-acting Temperature-sensitive Mutations; Schmidt DJ et al.; Monitoring Editor: Martin Chalfie Cytoplasmic dynein, a minus-end-directed microtubule motor, has been implicated in many cellular and developmental processes . Identification of specific cellular processes that rely directly on dynein would be facilitated by a means to induce specific and rapid inhibition of its function . We have identified conditional variants of C . elegans dynein heavy chain (DHC-1) that lose function within a minute of a modest temperature upshift . Mutant embryos generated at elevated temperature show defects in centrosome separation, pronuclear migration, rotation of the centrosome/nucleus complex, bipolar spindle assembly, anaphase chromosome segregation, and cytokinesis . Our analyses of mutant embryos generated at permissive temperature and then upshifted quickly just before events of interest indicate that DHC-1 is required specifically for rotation of the centrosome/nucleus complex, for chromosome congression to a well-ordered metaphase plate, and for timely initiation of anaphase . Our results do not support the view that DHC-1 is required for anaphase B separation of spindle poles and chromosomes . A P-loop mutation identified in two independent dominant temperature-sensitive alleles of dhc-1, when engineered into the DHC1 gene of S . cerevisiae, conferred a dominant temperature-sensitive dynein loss-of-function phenotype . This suggests that temperature-sensitive mutations can be created for time-resolved function analyses of dyneins and perhaps other P-loop proteins in a variety of model systems. J Biol Chem . 2004 Dec 22; {Epub ahead of print} Two proteins homologous to the N- and C-terminal domains of the bacterial glycosyltransferase MurG are required for the second step of dolichyl-linked oligosaccharide synthesis in S . cerevisiae; Chantret I et al.; Two highly conserved eukaryotic gene products of unknown function showing homology to glycosyltransferases involved in the second steps of bacterial peptidoglycan (MurG), and capsular polysaccharide (Cps14F/Cps14G) biosynthesis have been identified in silico . Amino acid sequence of the eukaryotic protein that is homologous to the lipid acceptor- and membrane-associating N-terminal domain of MurG and the Cps14F beta4-galactosyltransferase enhancer protein, is predicted to possess a cleavable signal peptide and transmembrane helices . The other eukaryotic protein is predicted to possess neither transmembrane regions nor a signal peptide, but is homologous to the UDP-sugar binding C-terminal domain of MurG and the Cps14G beta4-galactosyltransferase . Both the eukaryotic proteins are encoded by essential genes in S . cerevisiae, and down regulation of either causes growth retardation, reduced N-glycosylation of carboxypeptidase Y, and accumulation of dolichyl-PP-GlcNAc . In vitro studies demonstrate that these proteins are required for transfer of {(3)H}GlcNAc from UDP-{(3)H}GlcNAc into dolichyl-PP-GlcNAc(2) . To conclude, two gene products showing homology to bacterial glycosyltransferases are required for the second step in dolichyl-PP-oligosaccharide biosynthesis. Curr Genet, 2004 Dec, 46(6), 317 - 30 Epub 2004 Nov 13. How heterologously expressed Escherichia coli genes contribute to understanding DNA repair processes in Saccharomyces cerevisiae; Brozmanova J et al.; DNA-damaging agents constantly challenge cellular DNA; and efficient DNA repair is therefore essential to maintain genome stability and cell viability . Several DNA repair mechanisms have evolved and these have been shown to be highly conserved from bacteria to man . DNA repair studies were originally initiated in very simple organisms such as Escherichia coli and Saccharomyces cerevisiae, bacteria being the best understood organism to date . As a consequence, bacterial DNA repair genes encoding proteins with well characterized functions have been transferred into higher organisms in order to increase repair capacity, or to complement repair defects, in heterologous cells . While indicating the contribution of these repair functions to protection against the genotoxic effects of DNA-damaging agents, heterologous expression studies also highlighted the role of the DNA lesions that are substrates for such processes . In addition, bacterial DNA repair-like functions could be identified in higher organisms using this approach . We heterologously expressed three well characterized E . coli repair genes in S . cerevisiae cells of different genetic backgrounds: (1) the ada gene encoding O(6)-methylguanine DNA-methyltransferase, a protein involved in the repair of alkylation damage to DNA, (2) the recA gene encoding the main recombinase in E . coli and (3) the nth gene, the product of which (endonuclease III) is responsible for the repair of oxidative base damage . Here, we summarize our results and indicate the possible implications they have for a better understanding of particular DNA repair processes in S . cerevisiae. Curr Genet . 2004 Dec 22; {Epub ahead of print} Biosynthesis of hydroxymethylpyrimidine pyrophosphate in Saccharomyces cerevisiae; Kawasaki Y et al.; Two redundant genes, THI20 and THI21, of Saccharomyces cerevisiae encode a 2-methyl-4-amino-5-hydroxymethylpyrimidine monophosphate (HMP-P) kinase required for thiamin biosynthesis . Using functional complementation analysis with an Escherichia coli mutant strain and a defined biochemical system containing partially purified proteins for the reconstitution of thiamin monophosphate synthesis, we demonstrate that both Thi20p and Thi21p proteins also have HMP kinase activity . Although each isoform independently can synthesize HMP pyrophosphate (HMP-PP) from HMP, there is a marked difference in efficiency between the two proteins . The thi20 deletion strain grows at the same rate as the parental strain in minimal medium without thiamin, but its ability to synthesize HMP-PP from HMP is significantly decreased . We discuss the possibility that HMP is not involved in the pathway of de novo thiamin synthesis in S . cerevisiae. J Agric Food Chem, 2004 Dec 29, 52(26), 7814 - 21 Identification of phenolics for control of Aspergillus flavus using Saccharomyces cerevisiae in a model target-gene bioassay; Kim JH et al.; The yeast Saccharomyces cerevisiae was used in a high-throughput bioassay to identify phenolic agents for control of the aflatoxigenic fungus Aspergillus flavus . Veratraldehyde, 1, cinnamic acid, 5, and the respective benzoic acid derivatives vanillin, 2, vanillic acid, 3, and vanillylacetone, 4, and cinnamic acid derivatives o-coumaric acid, 6, m-coumaric acid, 7, and p-coumaric acid, 8, showed significant antifungal activities (from highest to lowest, 2, 5 > 1 > 6, 7 > 4 > 3, 8) in the yeast system, with caffeic acid, 9, having little to no effect . Antifungal activity levels against A . flavus were similar . This similarity in antifungal activity demonstrated the usefulness of the S . cerevisiae bioassay for screening antifungal compounds . Assays using deletion mutants of yeast identified signal transduction and antioxidative stress response genes important to fungal tolerance . Targeting the antioxidative stress response system with certain compounds (e.g., 4) in combination with strobilurin fungicides had a synergistic effect against both fungi. Curr Genet . 2004 Dec 21; {Epub ahead of print} Sed1p interacts with Arn3p physically and mediates ferrioxamine B uptake in Saccharomyces cerevisiae; Park YS et al.; Two-hybrid analysis can be used to study protein function and metabolic pathways . Using yeast two-hybrid analysis to identify a siderophore uptake pathway in the yeast Saccharomyces cerevisiae, we found that the C-terminal part of the cell-wall protein Sed1p interacts with the N-terminal region of Arn3p . To confirm the physical interaction between the Sed1p C-terminal fragment and the hydrophilic N-terminal fragment of Arn3p, we used an in vitro co-immunoprecipitation assay and a growth test of the strain with bait and SED1 plasmids in quadruple amino acid-depleted medium . The expression of SED1 was upregulated by overexpression of AFT1-1(up) under the control of the GAL promoter . This occurred despite the lack of an Aft1p-binding consensus region on the upstream region of SED1 or a high concentration of free iron . Although free-iron uptake activity in the Deltased1 strain did not differ from that in the parental strain, ferrioxamine bound-iron uptake activity was reduced in the Deltased1 strain . Moreover, the Deltased1 strain showed low viability at high iron concentrations . Taken together, these results suggest that Sed1p mediates siderophore transport and confers iron resistance in S . cerevisiae. Genetics, 2004 Dec, 168(4), 1877 - 89 Genetic and Biochemical Interactions Among Yar1, Ltv1 and RpS3 Define Novel Links Between Environmental Stress and Ribosome Biogenesis in Saccharomyces cerevisiae; Loar JW et al.; In the yeast S . cerevisiae, ribosome assembly is linked to environmental conditions by the coordinate transcriptional regulation of genes required for ribosome biogenesis . In this study we show that two nonessential stress-responsive genes, YAR1 and LTV1, function in 40S subunit production . We provide genetic and biochemical evidence that Yar1, a small ankyrin-repeat protein, physically interacts with RpS3, a component of the 40S subunit, and with Ltv1, a protein recently identified as a substoichiometric component of a 43S preribosomal particle . We demonstrate that cells lacking YAR1 or LTV1 are hypersensitive to particular protein synthesis inhibitors and exhibit aberrant polysome profiles, with a reduced absolute number of 40S subunits and an excess of free 60S subunits . Surprisingly, both mutants are also hypersensitive to a variety of environmental stress conditions . Overexpression of RPS3 suppresses both the stress sensitivity and the ribosome biogenesis defect of Deltayar1 mutants, but does not suppress either defect in Deltaltv1 mutants . We propose that YAR1 and LTV1 play distinct, nonessential roles in 40S subunit production . The stress-sensitive phenotypes of strains lacking these genes reveal a hitherto unknown link between ribosome biogenesis factors and environmental stress sensitivity. J Biol Chem . 2004 Dec 20; {Epub ahead of print} psi 35 in the branch site recognition region of U2 snRNA is important for pre-mRNA splicing in S . cerevisiae; Yang C et al.; Pseudouridine 35 (psi35) in the branch site recognition region of yeast U2 snRNA is absolutely conserved in all eukaryotes examined . Pus7p catalyzes pseudouridylation at position 35 in S . cerevisiae U2 . The pus7-deletion strain, although viable in rich medium, is growth-disadvantaged under certain conditions . To clarify the function of U2 psi35 in yeast, we used this pus7-deletion strain to screen a collection of mutant U2 snRNAs, each containing a point mutation near the branch site recognition sequence, for a synthetic growth defect phenotype . The screen identified two U2 mutants, one containing a U40 to G40 substitution (U40G) and another having a U40 deletion (U40D) . Yeast strains carrying either of these U2 mutations grew as well as the wild-type strain in the selection medium, but they exhibited a temperature-sensitive growth defect phenotype when coupled with the pus7-deletion (pus7D) . A subsequent temperature-shift assay and a conditional pus7-depletion (via GAL promoter shutoff) in the U2-U40 mutant genetic background caused pre-mRNA accumulation, suggesting that psi35 is required for pre-mRNA splicing under certain conditions. J Biol Chem . 2004 Dec 20; {Epub ahead of print} Purification, functional reconstitution and characterization of the Saccharomyces Cerevisiae isoprenylcysteine carboxyl methyltransferase Ste14p; Anderson JL et al.; Numerous proteins, including Ras, contain a C-terminal CaaX motif that directs a series of three sequential post-translational modifications: isoprenylation of the cysteine residue, endoproteolysis of the three terminal amino acids and a-carboxyl methylesterification of the isoprenylated cysteine . This study focuses on the isoprenylcysteine carboxyl methyltransferase (Icmt) enzyme from Saccharomyces cerevisiae, Ste14p, the founding member of a homologous family of endoplasmic reticulum membrane proteins present in all eukaryotes . Ste14p, like all Icmt's, has multiple membrane spanning domains, presenting a significant challenge to its purification in an active form . Here, we have detergent-solubilized, purified, and reconstituted enzymatically active His-tagged Ste14p from S . cerevisiae, thus providing conclusive proof that Ste14p is the sole component necessary for the carboxylmethylation of isoprenylated substrates . Among the extensive panel of detergents that was screened, optimal solubilization and retention of Ste14p activity occurred with ss-D-dodecylmaltoside . The activity of Ste14p could be further optimized upon reconstitution into liposomes . Our expression and purification schemes generate milligram quantities of pure and active Ste14p, that is highly stable under many conditions . Using pure reconstituted Ste14p, we demonstrate quantitatively that Ste14p does not have a preference for the farnesyl or geranylgeranyl moieties in the model substrates AFC and AGGC in vitro . In addition to catalyzing methylation of AFC, we also show that purified Ste14p methylates a known in vivo substrate, Ras2p . Evidence that metals ions are required for activity of Ste14p is also presented . These results pave the way for further characterization of pure Ste14p, as well as determination of its three-dimensional structure. Mol Cell, 2004 Dec 22, 16(6), 943 - 54 Pre-18S ribosomal RNA is structurally compacted into the SSU processome prior to being cleaved from nascent transcripts in Saccharomyces cerevisiae; Osheim YN et al.; Recent studies have revealed multiple dynamic complexes that are precursors to eukaryotic ribosomes . EM visualization of nascent rRNA transcripts provides in vivo temporal and structural context for these events . In exponentially growing S . cerevisiae, pre-18S rRNA is dramatically compacted into a large particle (SSU processome) within seconds of completion of its transcription and is released cotranscriptionally by cleavage in ITS1 . After cleavage, a new terminal knob is formed on the nascent large subunit rRNA, compacting it progressively in a 5'-3' direction . Depletion of individual components shows that cotranscriptional SSU processome formation is a sensitive indicator of the occurrence or timing of the early A0-A2 cleavages and depends on factors not isolated in preribosome complexes, as well as on favorable growth conditions . The results show that the approximately 40 components of the SSU processome/90S preribosome can complete their tasks within approximately 85 s in optimal conditions. Dev Growth Differ, 2004 Dec, 46(6), 545 - 54 Identification of differentially expressed genes in yeast Saccharomyces cerevisiae cells with inactivated Mmf1p and Hmf1p, members of proteins family YERO57c/YJGF; Pozdniakovaite N et al.; We used differential display analysis of mRNA to investigate the differences between gene expression in wild-type (wt) yeast Saccharomyces cerevisiae cells and mutated ones with disrupted activity of genes MMF1 and HMF1, members of the YERO57c/YJGF family . Reverse transcription-polymerase chain reaction (RT-PCR) analysis was performed to determine the differences in the degree of expression of 14 specific transcripts in normal and mutated yeast cells . Obtained data demonstrate that disruption of genes encoding proteins Mmf1p, Hmf1p (or both of them) result in the correlative variation of expression level of the target 12 genes both in the cells with changed phenotype (mmf1 and mmf1 hmf1) and in the cells retaining w.t . shape and growth rate (wt cells, hmf1) . Metabolic processes and cellular pathways have been indicated for Mmf1p and Hmf1p based on the different profiles of the expression of 14 genes in mmf1, hmf1 yeast S . cerevisiae cells. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D598 - 604 Organelle DB: a cross-species database of protein localization and function; Wiwatwattana N et al.; To efficiently utilize the growing body of available protein localization data, we have developed Organelle DB, a web-accessible database cataloging more than 25,000 proteins from nearly 60 organelles, subcellular structures and protein complexes in 154 organisms spanning the eukaryotic kingdom . Organelle DB is the first on-line resource devoted to the identification and presentation of eukaryotic proteins localized to organelles and subcellular structures . As such, Organelle DB is a strong resource of data from the human proteome as well as from the major model organisms Saccharomyces cerevisiae, Arabidopsis thaliana, Drosophila melanogaster, Caenorhabditis elegans and Mus musculus . In particular, Organelle DB is a central repository of yeast data, incorporating results--and actual fluorescent imagesfrom ongoing large-scale studies of protein localization in S.cerevisiae . Each protein in Organelle DB is presented with its sequence and, as available, a detailed description of its function; functions were extracted from relevant model organism databases, and links to these databases are provided within Organelle DB . To facilitate data interoperability, we have annotated all protein localizations using vocabulary from the Gene Ontology consortium . We also welcome new data for inclusion in Organelle DB, which may be freely accessed at http://organelledb.lsi.umich.edu. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D374 - 7 Fungal BLAST and Model Organism BLASTP Best Hits: new comparison resources at the Saccharomyces Genome Database (SGD); Balakrishnan R et al.; The Saccharomyces Genome Database (SGD; is a scientific database of gene, protein and genomic information for the yeast Saccharomyces cerevisiae . SGD has recently developed two new resources that facilitate nucleotide and protein sequence comparisons between S.cerevisiae and other organisms . The Fungal BLAST tool provides directed searches against all fungal nucleotide and protein sequences available from GenBank, divided into categories according to organism, status of completeness and annotation, and source . The Model Organism BLASTP Best Hits resource displays, for each S.cerevisiae protein, the single most similar protein from several model organisms and presents links to the database pages of those proteins, facilitating access to curated information about potential orthologs of yeast proteins. Nucleic Acids Res, 2005 Jan 1, 33 Database Issue, D364 - 8 CYGD: the Comprehensive Yeast Genome Database; Guldener U et al.; The Comprehensive Yeast Genome Database (CYGD) compiles a comprehensive data resource for information on the cellular functions of the yeast Saccharomyces cerevisiae and related species, chosen as the best understood model organism for eukaryotes . The database serves as a common resource generated by a European consortium, going beyond the provision of sequence information and functional annotations on individual genes and proteins . In addition, it provides information on the physical and functional interactions among proteins as well as other genetic elements . These cellular networks include metabolic and regulatory pathways, signal transduction and transport processes as well as co-regulated gene clusters . As more yeast genomes are published, their annotation becomes greatly facilitated using S.cerevisiae as a reference . CYGD provides a way of exploring related genomes with the aid of the S.cerevisiae genome as a backbone and SIMAP, the Similarity Matrix of Proteins . The comprehensive resource is available under http://mips.gsf.de/genre/proj/yeast/. Free Radic Biol Med, 2005 Jan 15, 38(2), 226 - 34 RNA interference toward UMP1 induces proteasome inhibition in Saccharomyces cerevisiae: evidence for protein oxidation and autophagic cell death; Chen Q et al.; The proteasome is a large intracellular protease that is responsible for a large portion of intracellular proteolysis, in particular the degradation of a majority of short-lived and oxidized proteins . Inhibition of proteasome function occurs in response to multiple stressors, with proteasome inhibition sufficient for the induction of a wide range of cytotoxic processes . Although considerable advances have been made in the understanding of the proteasome, and the effects of proteasome inhibition, our understanding of these topics in Saccharomyces cerevisiae has been slowed by the inability of proteasome inhibitors to penetrate and/or be retained in S . cerevisiae . Expression of UMP1 is necessary for proteasome assembly in S . cerevisiae, and in the present study we examined the effectiveness of RNA interference for UMP1 as a means of achieving proteasome inhibition in S . cerevisiae . Induction of RNA interference for UMP1 resulted in a dramatic decrease in UMP1 at the protein level, which was not observed in cells transformed with control vector . RNA interference caused an impairment in proteasome function, and increase in protein oxidation, with proteins involved in both stress response and energy metabolism showing increased oxidation . Interestingly, RNA interference induced cell death that seemed to be autophagic in nature, suggesting possible cross talk between the proteasome and the autophagic proteolytic pathways . Taken together, these data indicate that RNA interference may be a useful model with which to study the effects of proteasome inhibition in S . cerevisiae and demonstrate the ability of proteasome inhibition to induce cytotoxic alterations in S . cerevisiae. J Chromatogr B Analyt Technol Biomed Life Sci, 2005 Jan 5, 814(1), 53 - 9 Characterization of N(alpha)-acetyl methionyl human growth hormone formed during expression in Saccharomyces cerevisiae with liquid chromatography and mass spectrometry; Jung C et al.; We found a new variant of human growth hormone (hGH) from the recombinant hGH expression process in Saccharomyces cerevisiae . The variant was identified as N(alpha)-acetyl methionyl hGH which may be formed by N(alpha)-acetylation of met-hGH during the intracellular expression of hGH in S . cerevisiae . The variant was isolated from manufacturing process of LG Life Sciences' hGH product . The variant was subjected to trypsin digestion and RP-HPLC analysis, resulting in a delayed retention time and an increased mass (173Da) of T1 tryptic peptide . The amino acid composition and amino acid sequence of the peptide showed the same result with T1 peptide of met-hGH except the N-terminal modification on methionine in the variant peptide . With collision induced dissociation (CID) experiments of the variant T1 tryptic peptide, we found the sequence and the a(1) fragment of N-terminal residue matched with those of acetyl-methionyl hGH . Within our production process, we produce the methionyl hGH first and then use the aminopeptidase to cut the N-terminal methionine . So the acetylation may inhibit the aminopeptidase to remove methionine and produces N(alpha)-acetyl methionyl hGH . And the biological activity of the variant was comparable to one of the unmodified hGH when tested by rat weight gain bioassay. Eur J Biochem, 2004 Dec, 271(23-24), 4788 - 97 Enhanced peptide secretion by gene disruption of CYM1, a novel protease in Saccharomyces cerevisiae; Jonson L et al.; Saccharomyces cerevisiae is a widely used host in the production of therapeutic peptides and proteins . Here we report the identification of a novel endoprotease in S . cerevisiae . It is encoded by the CYM1 gene and is specific for the C-terminus of basic residues of heterologously expressed peptides . Gene disruption of CYM1 not only reduced the intracellular proteolysis, but also enhanced the secretion of heterologously expressed peptides such as growth hormone, pro-B-type natriuretic peptide and pro-cholecystokinin . Cym1p resembles metalloendoproteases of the pitrilysin family with the HXXEH(X)E(71-77) catalytic domain as seen in insulysin, nardilysin and human metalloprotease 1 . It is a nuclear encoded protease that localizes to mitochondria without a hydrophobic N-terminal signal sequence or a C-terminal tail-anchor . The protease does not require post-translational processing prior to activation and it contains cytosolic activity that processes peptides designated for the secretory pathway prior to translocation into the endoplasmic reticulum. Environ Mol Mutagen, 2005, 45(1), 36 - 43 RAD6 gene is involved in heat shock induction of bleomycin resistance in Saccharomyces cerevisiae; Keszenman DJ et al.; Cells react to environmental and endogenous challenges such as high temperature, reactive oxygen species, DNA damage, and nutrient starvation by activating several defense mechanisms known as stress responses . An important feature is the overlap between different stress responses that contributes at least in part to the phenomenon of cross-protection . We previously demonstrated that pretreatment with a heat shock (HS) induces resistance to the lethal and mutagenic effects of the antineoplastic drug Bleomycin (BLM) in wild-type Saccharomyces cerevisiae . At the DNA level, the HS resulted in more efficient repair of BLM-induced DNA damage . In the present study, we have investigated the mechanisms involved in this HS-induced BLM resistance . Since the RAD6 gene is involved in the ubiquitin system and DNA repair, we analyzed the effects of HS on the lethality of BLM in a rad6Delta (ubc2) mutant strain of S . cerevisiae . The rad6Delta mutant was more sensitive to the lethal effects of BLM than wild-type yeast and HS had no effect on the lethality of BLM in the mutant . Analysis of cell proliferation kinetics indicated that the HS-induced cell cycle delay observed in the wild-type yeast was absent in the rad6Delta mutant strain . BLM treatment impaired mutant cell proliferation, and HS had no effect on the delayed cell kinetics of the mutant . In addition, pulsed-field electrophoresis of chromosomes damaged by BLM indicated that there was very little recovery from damage in the mutant after 24 hr of incubation in BLM-free nutrient medium, and that HS had little effect on the recovery . These data indicate that the RAD6 gene is involved in the HS-induced BLM resistance observed in the isogenic wild-type strain . Environ . Mol . Mutagen., 2005 . (c) 2004 Wiley-Liss, Inc. Proc Natl Acad Sci U S A, 2004 Dec 28, 101(52), 18069 - 74 Epub 2004 Dec 16. Genomic dissection of the cell-type-specification circuit in Saccharomyces cerevisiae; Galgoczy DJ et al.; The budding yeast Saccharomyces cerevisiae has three cell types (a cells, alpha cells, and a/alpha cells), each of which is specified by a unique combination of transcriptional regulators . This transcriptional circuit has served as an important model for understanding basic features of the combinatorial control of transcription and the specification of cell type . Here, using genome-wide chromatin immunoprecipitation, transcriptional profiling, and phylogenetic comparisons, we describe the complete cell-type-specification circuit for S . cerevisiae . We believe this work represents a complete description of cell-type specification in a eukaryote. Biol Cell, 2005 Jan, 97(1), 75 - 86 RNA localization in yeast: moving towards a mechanism; Gonsalvez GB et al.; RNA localization is a widely utilized strategy employed by cells to spatially restrict protein function . In Saccharomyces cerevisiae asymmetric sorting of mRNA to the bud has been reported for at least 24 mRNAs . The mechanism by which the mRNAs are trafficked to the bud, illustrated by ASH1 mRNA, involves recognition of cis-acting localization elements present in the mRNA by the RNA-binding protein, She2p . The She2p/mRNA complex subsequently associates with the myosin motor protein, Myo4p, through an adapter, She3p . This ribonucleoprotein complex is transported to the distal tip of the bud along polarized actin cables . While the mechanism by which ASH1 mRNA is anchored at the bud tip is unknown, current data point to a role for translation in this process, and the rate of translation of Ash1p during the transport phase is regulated by the cis-acting localization elements . Subcellular sorting of mRNA in yeast is not limited to the bud; certain mRNAs corresponding to nuclear-encoded mitochondrial proteins are specifically sorted to the proximity of mitochondria . Analogous to ASH1 mRNA localization, mitochondrial sorting requires cis-acting elements present in the mRNA, though trans-acting factors involved with this process remain to be identified . This review aims to discuss mechanistic details of mRNA localization in S . cerevisiae. Proc Natl Acad Sci U S A, 2004 Dec 28, 101(52), 18018 - 23 Epub 2004 Dec 13. Golgi inheritance in small buds of Saccharomyces cerevisiae is linked to endoplasmic reticulum inheritance; Reinke CA et al.; According to the cisternal maturation hypothesis, endoplasmic reticulum (ER)-derived membranes nucleate new Golgi cisternae . The yeast Saccharomyces cerevisiae offers a unique opportunity to test this idea because small buds contain both ER and Golgi structures early in the cell cycle . We previously predicted that mutants defective in ER inheritance also would show defects in Golgi inheritance . Surprisingly, studies of S . cerevisiae have not revealed the expected link between ER and Golgi inheritance . Here, we revisit this issue by generating mutant strains in which many of the small buds are devoid of detectable ER . These strains also show defects in the inheritance of both early and late Golgi cisternae . Strikingly, virtually all of the buds that lack ER also lack early Golgi cisternae . Our results fit with the idea that membranes exported from the ER coalesce with vesicles derived from existing Golgi compartments to generate new Golgi cisternae . This basic mechanism of Golgi inheritance may be conserved from yeast to vertebrate cells. Mol Biol Cell . 2004 Dec 9; {Epub ahead of print} The Yeast GTPase Mtg2p Is Required for Mitochondrial Translation and Partially Suppresses an rRNA Methyltransferase Mutant, mrm2; Datta K et al.; Monitoring Editor: Marvin P . Wickens The assembly of ribosomes involves the coordinate processing and modification of rRNAs with the temporal association of ribosomal proteins . This process is regulated by assembly factors such as helicases, modifying enzymes and GTPases . In contrast to the assembly of cytoplasmic ribosomes, there is a paucity of information concerning the role of assembly proteins in the biogenesis of mitochondrial ribosomes . In this study we demonstrate that the S . cerevisiae GTPase Mtg2p (Yhr168wp) is essential for mitochondrial ribosome function . Cells lacking MTG2 lose their mitochondrial DNA, giving rise to petite cells . In addition, cells expressing a temperature sensitive mgt2-1 allele are defective in mitochondrial protein synthesis and contain lowered levels of mitochondrial ribosomal subunits . Significantly, elevated levels of Mtg2p partially suppress the thermosensitive loss of mitochondrial DNA in a 21S rRNA methyltransferase mutant mrm2 . We propose that Mtg2p is involved in mitochondrial ribosome biogenesis . Consistent with this role, we show that Mtg2p is peripherally localized to the mitochondrial inner membrane and associates with the 54S large ribosomal subunit in a salt-dependent manner. Eukaryot Cell, 2004 Dec, 3(6), 1557 - 66 Increased genome instability and telomere length in the elg1-deficient Saccharomyces cerevisiae mutant are regulated by S-phase checkpoints; Banerjee S et al.; Gross chromosomal rearrangements (GCRs) are frequently observed in cancer cells . Abnormalities in different DNA metabolism including DNA replication, cell cycle checkpoints, chromatin remodeling, telomere maintenance, and DNA recombination and repair cause GCRs in Saccharomyces cerevisiae . Recently, we used genome-wide screening to identify several genes the deletion of which increases GCRs in S . cerevisiae . Elg1, which was discovered during this screening, functions in DNA replication by participating in an alternative replication factor complex . Here we further characterize the GCR suppression mechanisms observed in the elg1Delta mutant strain in conjunction with the telomere maintenance role of Elg1 . The elg1Delta mutation enhanced spontaneous DNA damage and resulted in GCR formation . However, DNA damage due to inactivation of Elg1 activates the intra-S checkpoints, which suppress further GCR formation . The intra-S checkpoints activated by the elg1Delta mutation also suppress GCR formation in strains defective in the DNA replication checkpoint . Lastly, the elg1Delta mutation increases telomere size independently of other previously known telomere maintenance proteins such as the telomerase inhibitor Pif1 or the telomere size regulator Rif1 . The increase in telomere length caused by the elg1Delta mutation was suppressed by a defect in the DNA replication checkpoint, which suggests that DNA replication surveillance by Dpb11-Mec1/Tel1-Dun1 also has an important role in telomere length regulation. Eukaryot Cell, 2004 Dec, 3(6), 1533 - 43 The histone fold domain of Cse4 is sufficient for CEN targeting and propagation of active centromeres in budding yeast; Morey L et al.; Centromere-specific H3-like proteins (CenH3s) are conserved across the eukaryotic kingdom and are required for packaging centromere DNA into a specialized chromatin structure required for kinetochore assembly . Cse4 is the CenH3 protein of the budding yeast Saccharomyces cerevisiae . Like all CenH3 proteins, Cse4 consists of a conserved histone fold domain (HFD) and a divergent N terminus (NT) . The Cse4 NT contains an essential domain designated END (for essential N-terminal domain); deletion of END is lethal . To investigate the role of the Cse4 NT in centromere targeting, a series of deletion alleles (cse4DeltaNT) were analyzed . No part of the Cse4 NT was required to target mutant proteins to centromere DNA in the presence of functional Cse4 . A Cse4 degron strain was used to examine targeting of a Cse4DeltaNT protein in the absence of wild-type Cse4 . The END was not required for centromere targeting under these conditions, confirming that the HFD confers specificity of Cse4 centromere targeting . Surprisingly, overexpression of the HFD bypassed the requirement for the END altogether, and viable S . cerevisiae strains in which the cells express only the Cse4 HFD and six adjacent N-terminal amino acids (Cse4Delta129) were constructed . Despite the complete absence of the NT, mitotic chromosome loss in the cse4Delta129 strain increased only 6-fold compared to a 15-fold increase in strains overexpressing wild-type Cse4 . Thus, when overexpressed, the Cse4 HFD is sufficient for centromere function in S . cerevisiae, and no posttranslational modification or interaction of the NT with other kinetochore component(s) is essential for accurate chromosome segregation in budding yeast. J Biol Chem . 2004 Dec 6; {Epub ahead of print} Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae; Hannich JT et al.; SUMO, or Smt3 in S . cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo . Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes . However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways . Here we use a combination of yeast two-hybrid screening, a high-copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast . These three approaches yielded overlapping sets of proteins, with the most extensive set by far being those identified by mass spectrometry . The two-hybrid data also yielded a potential SUMO-binding motif . Functional categories of SUMO-modified proteins include SUMO conjugation-system enzymes, chromatin- and gene silencing-related factors, DNA-repair and genome-stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes . The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms. J Biol Chem . 2004 Dec 8; {Epub ahead of print} A novel ER membrane protein Rcr1 regulates chitin deposition in the cell wall of Saccharomyces cerevisiae; Imai K et al.; Congo red binds to the cell wall and inhibits the growth of yeast . In a screening for multicopy suppressor genes of Congo red hypersensitivity of erd1-Delta mutant, we found that a previously uncharacterized gene YBR005w makes most of the S . cerevisiae strains resistant to Congo red . This gene was named RCR1 (resistance to Congo red 1) . An rcr1-Delta null mutant showed an increased sensitivity to Congo red . RCR1 encodes a novel ER membrane protein with a single transmembrane domain . Molecular dissection suggested that the transmembrane domain and a part of the C-terminal polypeptide are sufficient for the activity . We examined the effect of RCR1 in various null mutants of genes related to the cell wall . The resistance of mutants to Congo red correlates with a reduction of chitin content . Multicopy RCR1 caused a significant decrease in the chitin content while the amount of alkali-soluble glucan did not change . The binding of Calcofluor white to the cell wall significantly decreased in these cells . Our results show that RCR1 regulates the chitin deposition and add firm genetic and biochemical evidences that the primary target of Congo red is chitin in S . cerevisiae. DNA Repair (Amst), 2005 Feb 3, 4(2), 243 - 51 Substrate specificity of the Saccharomyces cerevisiae Mus81-Mms4 endonuclease; Fricke WM et al.; Mus81-Mms4/Eme1 is a conserved structure-specific endonuclease that functions in mitotic and meiotic recombination . It has been difficult to identify a single preferred substrate of this nuclease because it is active on a variety of DNA structures . In addition, it has been suggested that the specificity of the recombinant protein may differ from that of the native enzyme . Here, we addressed these issues with respect to Mus81-Mms4 from S . cerevisiae . At low substrate concentrations, Mus81-Mms4 was active on any substrate containing a free end adjacent to the branchpoint . This includes 3'-flap (3'F), regressed leading strand replication fork (RLe), regressed lagging strand replication fork (RLa), and nicked Holliday junction (nHJ) substrates . Kinetic analysis was used to quantitate differences between substrates . High K(cat)/K(m) values were obtained only for substrates with a 5'-end near the branchpoint (i.e., 3'F, RLe, and nHJ); 10-fold lower values were obtained for nicked duplex (nD) and RLa substrates . Substrates lacking any free ends at the branch point generated K(cat)/K(m) values that were four orders of magnitude lower than those of the preferred substrates . Native Mus81-Mms4 was partially purified from yeast cells and found to retain its preference for 3'F over intact HJ substrates . Taken together, these results narrow the range of optimal substrates for Mus81-Mms4 and indicate that, at least for S . cerevisae, the native and recombinant enzymes display similar substrate specificities. J Cell Biol, 2004 Dec 6, 167(5), 819 - 30 Human Rif1 protein binds aberrant telomeres and aligns along anaphase midzone microtubules; Xu L et al.; We identified and characterized a human orthologue of Rif1 protein, which in budding yeast interacts in vivo with the major duplex telomeric DNA binding protein Rap1p and negatively regulates telomere length . Depletion of hRif1 by RNA interference in human cancer cells impaired cell growth but had no detectable effect on telomere length, although hRif1 overexpression in S . cerevisiae interfered with telomere length control, in a manner specifically dependent on the presence of yeast Rif1p . No localization of hRif1 on normal human telomeres, or interaction with the human telomeric proteins TRF1, TRF2, or hRap1, was detectable . However, hRif1 efficiently translocated to telomerically located DNA damage foci in response to the synthesis of aberrant telomeres directed by mutant-template telomerase RNA . The hRif1 level rose during late S/G2 but hRif1 was not visible on chromosomes in metaphase and anaphase; however, notably, specifically during early anaphase, hRif1 aligned along a subset of the midzone microtubules between the separating chromosomes . In telophase, hRif1 localized to chromosomes, and in interphase, it was intranuclear . These results define a novel subcellular localization behavior for hRif1 during the cell cycle. Comb Chem High Throughput Screen, 2004 Nov, 7(7), 661 - 8 Identification and characterization of Sir2 inhibitors through phenotypic assays in yeast; Posakony J et al.; Yeast Sir2 is a defining member of a large family of protein deacetylases found in organisms ranging from bacteria to humans . SIR2 was discovered as a gene required for mating in S . cerevisiae 25 years ago, but it was only recently that Sir2's activity as an NAD-dependent protein deacetylase was established . However, years of extensive research did generate a large body of knowledge about the cellular roles of Sir2 in yeast long before its biochemical function was discovered . In addition to Sir2, yeast have four additional NAD-dependent histone deacetylases Hst1-4 (for homologue of Sir2), with distinct cellular roles . Detailed knowledge of the phenotypes of SIR2 and HST loss of function mutants has allowed design of a series of cell based screens that yielded the first inhibitors of NAD-dependent protein deacetylases . These phenotypic assays, amenable to high throughput screening, and coupled with transcript array analysis for evaluation of compound specificity, allowed the identification and detailed characterization of a series of Sir2 inhibitors, entirely bypassing traditional biochemical approaches. Mol Biol Cell . 2004 Dec 1; {Epub ahead of print} Synthetic Genetic Array Analysis of the PtdIns 4-kinase Pik1p Identifies Components in a Golgi-specific Ypt31/rab-GTPase Signaling Pathway; Sciorra VA et al.; Monitoring Editor: Randy Schekman Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells . In S . cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function, Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi . Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway . We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p . We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function. RNA, 2005 Jan, 11(1), 70 - 6 Epub 2004 Dec 01. A pre-tRNA carrying intron features typical of Archaea is spliced in yeast; Di Segni G et al.; Archaeal pre-tRNAs are characterized by the presence of the bulge-helix-bulge (BHB) structure in the intron stem-and-loop region . A chimeric pre-tRNA was constructed bearing an intron of the archaeal type and the mature domain of the Saccharomyces cerevisiae suppressor SUP4 tRNA(Tyr) . This pre-tRNA(ArchEuka) is correctly cleaved in several cell-free extracts and by purified splicing endonucleases . It is also cleaved and ligated in S . cerevisiae cells, providing efficient suppression of nonsense mutations in various genes. Mol Cell Biol, 2004 Dec, 24(24), 10975 - 85 Amino acid substitutions in yeast TFIIF confer upstream shifts in transcription initiation and altered interaction with RNA polymerase II; Ghazy MA et al.; Transcription factor IIF (TFIIF) is required for transcription of protein-encoding genes by eukaryotic RNA polymerase II . In contrast to numerous studies establishing a role for higher eukaryotic TFIIF in multiple steps of the transcription cycle, relatively little has been reported regarding the functions of TFIIF in the yeast Saccharomyces cerevisiae . In this study, site-directed mutagenesis, plasmid shuffle complementation assays, and primer extension analyses were employed to probe the functional domains of the S . cerevisiae TFIIF subunits Tfg1 and Tfg2 . Analyses of 35 Tfg1 alanine substitution mutants and 19 Tfg2 substitution mutants identified 5 mutants exhibiting altered properties in vivo . Primer extension analyses revealed that the conditional growth properties exhibited by the tfg1-E346A, tfg1-W350A, and tfg2-L59K mutants were associated with pronounced upstream shifts in transcription initiation in vivo . Analyses of double mutant strains demonstrated functional interactions between the Tfg1 mutations and mutations in Tfg2, TFIIB, and RNA polymerase II . Importantly, biochemical results demonstrated an altered interaction between mutant TFIIF protein and RNA polymerase II . These results provide direct evidence for the involvement of S . cerevisiae TFIIF in the mechanism of transcription start site utilization and support the view that a TFIIF-RNA polymerase II interaction is a determinant in this process. J Biol Chem . 2004 Nov 30; {Epub ahead of print} Random mutagenesis of the M3 muscarinic acetylcholine receptor expressed in yeast: Identification of second-site mutations that restore function to a coupling-deficient mutant M3 receptor; Li B et al.; The M3 muscarinic receptor is a prototypical member of the class A family of G protein-coupled receptors (GPCRs) . To gain insight into the structural mechanisms governing agonist-mediated M3 receptor activation, we recently developed a genetically modified yeast strain (S . cerevisiae) which allows the efficient screening of large libraries of mutant M3 receptors in order to identify mutant receptors with altered/novel functional properties . Class A GPCRs contain a highly conserved Asp residue located in transmembrane domain II (TM II; corresponding to D113 in the rat M3 muscarinic receptor) which is of fundamental importance for receptor activation . As observed previously with other GPCRs analyzed in mammalian expression systems, the D113N point mutation abolished agonist-induced receptor/G protein coupling in yeast . We then subjected the D113N mutant M3 receptor to PCR-based random mutagenesis, followed by a yeast genetic screen to recover point mutations that can restore G protein coupling to the D113N mutant receptor . A large scale screening effort led to the identification of three such second-site suppressor mutations, R165W, R165M, and Y250D . When expressed in the wild-type receptor background, these three point mutations did not lead to an increase in basal activity and reduced the efficiency of receptor/G protein coupling . Similar results were obtained when the various mutant receptors were expressed and analyzed in transfected mammalian cells (COS-7 cells) . Interestingly, like D113, R165 and Y250, which are located at the cytoplasmic ends of TM III and TM V, respectively, are also highly conserved among class A GPCRs . Our data suggest a conformational link between the highly conserved D113, R165, and Y250 residues that is critical for receptor activation. Biochemistry, 2004 Dec 7, 43(48), 15210 - 6 Alpha-anomeric deoxynucleotides, anoxic products of ionizing radiation, are substrates for the endonuclease IV-type AP endonucleases; Ishchenko AA et al.; Alpha-anomeric 2'-deoxynucleosides (alphadN) are one of the products formed by ionizing radiation (IR) in DNA under anoxic conditions . Alpha-2'-deoxyadenosine (alphadA) and alpha-thymidine (alphaT) are not recognized by DNA glycosylases, and are likely removed by the alternative nucleotide incision repair (NIR) pathway . Indeed, it has been shown that alphadA is a substrate for the Escherichia coli Nfo and human Ape1 proteins . However, the repair pathway for removal of alphadA and other alphadN in yeast is unknown . Here we report that alphadA when present in DNA is recognized by the Saccharomyces cerevisiae Apn1 protein, a homologue of Nfo . Furthermore, alphaT is a substrate for Nfo and Apn1 . Kinetic constants indicate that alphadA and alphaT are equally good substrates, as a tetrahydrofuranyl (THF) residue, for Nfo and Apn1 . Using E . coli and S . cerevisiae cell-free extracts, we have further substantiated the role of the nfo and apn1 gene products in the repair of alphadN . Surprisingly, we found that bacteria and yeast NIR-deficient mutants are not sensitive to IR, suggesting that DNA strand breaks with terminal 3'-blocking groups rather than alphadN might contribute to cell survival . We propose that the novel substrate specificities of Nfo and Apn1 play an important role in counteracting oxidative DNA base damage. BMC Evol Biol . 2004 Nov 27;4(1):51. Structure and evolution of protein interaction networks: a statistical model for link dynamics and gene duplications; Berg J et al.; BACKGROUND: The structure of molecular networks derives from dynamical processes on evolutionary time scales . For protein interaction networks, global statistical features of their structure can now be inferred consistently from several large-throughput datasets . Understanding the underlying evolutionary dynamics is crucial for discerning random parts of the network from biologically important properties shaped by natural selection . RESULTS: We present a detailed statistical analysis of the protein interactions in Saccharomyces cerevisiae based on several large-throughput datasets . Protein pairs resulting from gene duplications are used as tracers into the evolutionary past of the network . From this analysis, we infer rate estimates for two key evolutionary processes shaping the network: (i) gene duplications and (ii) gain and loss of interactions through mutations in existing proteins, which are referred to as link dynamics . Importantly, the link dynamics is asymmetric, i.e., the evolutionary steps are mutations in just one of the binding parters . The link turnover is shown to be much faster than gene duplications . Both processes are assembled into an empirically grounded, quantitative model for the evolution of protein interaction networks . CONCLUSIONS: According to this model, the link dynamics is the dominant evolutionary force shaping the statistical structure of the network, while the slower gene duplication dynamics mainly affects its size . Specifically, the model predicts (i) a broad distribution of the connectivities (i.e., the number of binding partners of a protein) and (ii) correlations between the connectivities of interacting proteins, a specific consequence of the asymmetry of the link dynamics . Both features have been observed in the protein interaction network of S . cerevisiae. Mol Biol Cell . 2004 Dec 1; {Epub ahead of print} The Yeast Par-1 Homologs, Kin1 and Kin2, Show Genetic and Physical Interactions with Components of the Exocytic Machinery; Elbert M et al.; Monitoring Editor: Keith Mostov Kin1 and Kin2 are S . cerevisiae counterparts of Par-1, the C . elegans kinase essential for the establishment of polarity in the one cell embryo . Here, we present evidence for a novel link between Kin1, Kin2 and the secretory machinery of the budding yeast . We isolated KIN1 and KIN2 as suppressors of a mutant form of Rho3, a Rho-GTPase acting in polarized trafficking . Genetic analysis suggests that KIN1 and KIN2 act downstream of the Rab-GTPase Sec4, its exchange factor Sec2, and several components of the vesicle tethering complex, the Exocyst . We show that Kin1 and Kin2 physically interact with the t-SNARE Sec9 and the Lgl homologue Sro7, proteins acting at the final stage of exocytosis . Structural analysis of Kin2 reveals that its catalytic activity is essential for its function in the secretory pathway, and implicates the conserved 42 amino acid tail at the carboxy-terminal of the kinase in auto-inhibition . Finally, we find that Kin1 and Kin2 induce phosphorylation of t-SNARE Sec9 in vivo and stimulate its release from the plasma membrane . In summary, we report the finding that yeast Par-1 counterparts are associated with and regulate the function of the exocytic apparatus via phosphorylation of Sec9. Mol Biol Cell . 2004 Nov 24; {Epub ahead of print} Munc18-1 Regulates Early and Late Stages of Exocytosis via Syntaxin-independent Protein Interactions; Ciufo LF et al.; Monitoring Editor: Keith Mostov Sec1/Munc18 (SM) proteins are involved in various intracellular membrane trafficking steps . Many SM proteins bind to appropriate syntaxin homologues involved in these steps, suggesting that SM proteins function as syntaxin chaperones . Organisms with mutations in SM genes, however, exhibit defects in either early (docking) or late (fusion) stages of exocytosis, implying that SM proteins may have multiple functions . To gain insight into the role of SM proteins, we introduced mutations modeled on those identified in C . elegans, D . melanogaste, and S . cerevisiae into mammalian Munc18-1 . As expected, several mutants exhibited reduced binding to syntaxin1A . However, 3 mutants displayed wild-type syntaxin binding affinities, indicating syntaxin-independent defects . Expression of these mutants in chromaffin cells either increased the rate and extent of exocytosis or altered the kinetics of individual release events . This latter effect was associated with a reduced Mint binding affinity in one mutant, implying a potential mechanism for the observed alteration in release kinetics . Furthermore, this phenotype persisted when the mutation was combined with a second mutation that greatly reduced syntaxin binding affinity . These results clarify the data on the function of SM proteins in mutant organisms, and indicate that Munc18-1 controls multiple stages of exocytosis via both syntaxin-dependent and -independent protein interactions. J Biol Chem . 2004 Nov 23; {Epub ahead of print} Characterizing the sphingolipid signaling pathway that remediates defects associated with loss of the yeast amphiphysin-like orthologs, Rvs161p and Rvs167p; Germann M et al.; Loss of function of either the RVS161 or RVS167 S . cerevisiae amphiphysin-like genes confers similar growth phenotypes that can be suppressed by mutations in sphingolipid biosynthesis . We performed a yeast two-hybrid screen using Rvs161p as bait in order to uncover proteins involved in this sphingolipid-dependent suppressor pathway . In the process, we have demonstrated a direct physical interaction between Rvs167p and the two-hybrid interacting proteins, Acf2p, Gdh3p, and Ybr108wp (Table I), while also elucidating the Rvs167p amino acid domains to which these proteins bind . By using subcellular fractionation, we demonstrate that Rvs167p, Ybr108wp, Gdh3p, and Acf2p all localize to Rvs161p-containing lipid rafts, thus placing them within a single compartment that should facilitate their interactions . Moreover, our results suggest that Acf2p and Gdh3p functions are needed for suppressor pathway activity . To further determine pathway mechanisms, we examined the localization of Rvs167p in suppressor mutants . These studies reveal roles for Rvs161p and the very long chain fatty acid elongase, Sur4p, in the localization and/or stability of Rvs167p . Previous yeast studies showed that rvs defects could be suppressed by changes in sphingolipid metabolism brought about by deleting SUR4 (Desfarges, L., Durrens, P., Juguelin, H., Cassagne, C., Bonneu, M., and Aigle, M . (1993) Yeast 9, 267-277) . Using rvs167 sur4 and rvs161 sur4 double null cells as models to study suppressor pathway activity, we demonstrate that loss of SUR4 does not remediate the steady-state actin cytoskeletal defects of rvs167 or rvs161 cells . Moreover, suppressor activity does not require the function of the actin-binding protein, Abp1p, or Sla1p, a protein that is thought to regulate assembly of the cortical actin cytoskeleton . Based on our results, we suggest that sphingolipid-dependent suppression of rvs defects may not work entirely through regulating changes in actin organization. BMC Bioinformatics . 2004 Nov 22;5(1):181. Discriminative topological features reveal biological network mechanisms; Middendorf M et al.; BACKGROUND: Recent genomic and bioinformatic advances have motivated the development of numerous network models intending to describe graphs of biological, technological, and sociological origin . In most cases the success of a model has been evaluated by how well it reproduces a few key features of the real-world data, such as degree distributions, mean geodesic lengths, and clustering coefficients . Often pairs of models can reproduce these features with indistinguishable fidelity despite being generated by vastly different mechanisms . In such cases, these few target features are insufficient to distinguish which of the different models best describes real world networks of interest; moreover, it is not clear a priori that any of the presently-existing algorithms for network generation offers a predictive description of the networks inspiring them . RESULTS: We present a method to assess systematically which of a set of proposed network generation algorithms gives the most accurate description of a given biological network . To derive discriminative classifiers, we construct a mapping from the set of all graphs to a high-dimensional (in principle infinite-dimensional) "word space" . This map defines an input space for classification schemes which allow us to state unambiguously which models are most descriptive of a given network of interest . Our training sets include networks generated from 17 models either drawn from the literature or introduced in this work . We show that different duplication-mutation schemes best describe the E . coli genetic network, the S . cerevisiae protein interaction network, and the C . elegans neuronal network, out of a set of network models including a linear preferential attachment model and a small-world model . CONCLUSIONS: Our method is a first step towards systematizing network models and assessing their predictability, and we anticipate its usefulness for a number of communities. J Food Prot, 2004 Nov, 67(11), 2596 - 602 Inactivation of Saccharomyces cerevisiae suspended in orange juice using high-intensity pulsed electric fields; Elez-Martinez P et al.; Saccharomyces cerevisiae is often associated with the spoilage of fruit juices . The purpose of this study was to evaluate the effect of high-intensity pulsed electric field (HIPEF) treatment on the survival of S . cerevisiae suspended in orange juice . Commercial heat-sterilized orange juice was inoculated with S . cerevisiae (CECT 1319) (10(8) CFU/ml) and then treated by HIPEFs . The effects of HIPEF parameters (electric field strength, treatment time, pulse polarity, frequency, and pulse width) were evaluated and compared to those of heat pasteurization (90 degrees C/min) . In all of the HIPEF experiments, the temperature was kept below 39 degrees C . S . cerevisiae cell damage induced by HIPEF treatment was observed by electron microscopy . HIPEF treatment was effective for the inactivation of S . cerevisiae in orange juice at pasteurization levels . A maximum inactivation of a 5.1-log (CFU per milliliter) reduction was achieved after exposure of S . cerevisiae to HIPEFs for 1,000 micros (4-micros pulse width) at 35 kV/cm and 200 Hz in bipolar mode . Inactivation increased as both the field strength and treatment time increased . For the same electric field strength and treatment time, inactivation decreased when the frequency and pulse width were increased . Electric pulses applied in the bipolar mode were more effective than those in the monopolar mode for destroying S . cerevisiae . HIPEF processing inactivated S . cerevisiae in orange juice, and the extent of inactivation was similar to that obtained during thermal pasteurization . HIPEF treatments caused membrane damage and had a profound effect on the intracellular organization of S . cerevisiae. J Biol Chem . 2004 Nov 17; {Epub ahead of print} In vitro phosphorylation by cAMP-dependent protein kinase upregulates recombinant S . cerevisiae mannosylphospho dolichol synthase; Banerjee DK et al.; Dpm1 is the structural gene for mannosylphospho dolichol synthase (i.e., Dol-P-Man synthase, DPMS) in S . cerevisiae . Earlier studies with cDNA cloning and sequence analysis have established that Mr 31-kDa DPMS of S . cerevisiae contains a consensus sequence (YRRVIS141) that can be phosphorylated by cAMP-dependent protein kinase (PKA) . We have been studying the upregulation of DPMS activity by PKA-mediated phosphorylation in higher eukaryotes, and used the recombinant DPMS from S . cerevisiae in this study to advance our knowledge further . DPMS catalytic activity was indeed enhanced several folds when the recombinant protein was phosphorylated in vitro . The rate as well as the magnitude of catalysis was higher with the phosphorylated enzyme . A similar increase in the catalytic activity was also observed when the in vitro phosphorylated recombinant DPMS was assayed as a function of increasing concentrations of exogenous dolichylmonophosphate (Dol-P) . Kinetic studies indicated that there was no change in the Km for GDP-mannose between the in vitro phosphorylated and control recombinant DPMS, but the Vmax was increased by 6-fold with the phosphorylated enzyme . In vitro phosphorylated recombinant DPMS also exhibited higher enzyme turnover (kcat) and the enzyme efficiency (kcat/Km) . SDS-PAGE followed by autoradiography of the 32P-labeled DPMS detected a Mr 31-kDa phosphoprotein, and immunoblotting with anti-phosphoserine antibody established the presence of a phosphoserine residue in in vitro phosphorylated recombinant DPMS . To confirm the phosphorylation activation of recombinant DPMS, serine-141 in the consensus sequence was replaced with alanine by PCR-site directed mutagenesis . S141A DPMS mutant exhibited 50% reduction in catalytic activity compared to the wild type when both were analyzed after in vitro phosphorylation . Thus, confirming that S . cerevisiae DPMS activity is indeed regulated by the cAMP-dependent protein phosphorylation signal, and the phosphorylation target is serine-141. J Biol Chem . 2004 Nov 16; {Epub ahead of print} Effect of amino acid substitutions in the Rad50 ATP binding domain on DNA double-strand break repair in yeast; Chen L et al.; The S . cerevisiae Rad50/Mre11/Xrs2 complex plays a central role in the cellular response to DNA double-strand breaks . Rad50 has a globular ATPase head domain with a long coiled-coil tail . DNA binding by Rad50 is ATP-dependent and the Rad50/Mre11/Xrs2 complex possesses DNA unwinding and endonuclease activities that are regulated by ATP . Here we have examined the role of the Rad50 Walker type A ATP binding motif in DNA double-strand break repair by a combination of genetic and biochemical approaches . Replacement of the conserved lysine residue within the Walker A motif with alanine, glutamate, or arginine results in the same DNA damage sensitivity and homologous recombination defect as the rad50 deletion mutation . The Walker A mutations also cause a deficiency in non-homologous end joining . As expected, complexes containing the rad50 Walker A mutant proteins are defective in ATPase, ATP-dependent DNA unwinding, and ATP-stimulated endonuclease activities . Although the DNA end-bridging activity of the Rad50/Mre11/Xrs2 complex is ATP-independent, the end bridging activity of complexes containing the rad50 Walker A mutant proteins is salt sensitive . These results provide a molecular explanation for the observed in vivo defects of the rad50 Walker mutant strains and reveal a novel ATP-independent function for Rad50 in DNA end-bridging. Mol Cell Biol, 2004 Dec, 24(23), 10300 - 12 Dependence of ORC silencing function on NatA-mediated Nalpha acetylation in Saccharomyces cerevisiae; Geissenhoner A et al.; N(alpha) acetylation is one of the most abundant protein modifications in eukaryotes and is catalyzed by N-terminal acetyltransferases (NATs) . NatA, the major NAT in Saccharomyces cerevisiae, consists of the subunits Nat1p, Ard1p, and Nat5p and is necessary for the assembly of repressive chromatin structures . Here, we found that Orc1p, the large subunit of the origin recognition complex (ORC), required NatA acetylation for its role in telomeric silencing . NatA functioned genetically through the ORC binding site of the HMR-E silencer . Furthermore, tethering Orc1p directly to the silencer circumvented the requirement for NatA in silencing . Orc1p was N(alpha) acetylated in vivo by NatA . Mutations that abrogated its ability to be acetylated caused strong telomeric derepression . Thus, N(alpha) acetylation of Orc1p represents a protein modification that modulates chromatin function in S . cerevisiae . Genetic evidence further supported a functional link between NatA and ORC: (i) nat1Delta was synthetically lethal with orc2-1 and (ii) the synthetic lethality between nat1Delta and SUM1-1 required the Orc1 N terminus . We also found Sir3p to be acetylated by NatA . In summary, we propose a model by which N(alpha) acetylation is required for the binding of silencing factors to the N terminus of Orc1p and Sir3p to recruit heterochromatic factors and establish repression. Int J Food Microbiol, 2004 Dec 15, 97(2), 157 - 69 Metabolism of extracellular inositol hexaphosphate (phytate) by Saccharomyces cerevisiae; Andlid TA et al.; Iron and zinc deficiencies are global problems, frequently leading to severe illness in vulnerable human populations . Addition of phytases can improve the bioavailability of iron and zinc in food . Saccharomyces cerevisiae would be an ideal candidate as a bioavailability improving food additive if it demonstrates significant phytase activity . The purpose of the paper was to study yeast phytase activity to obtain information required to improve strains . All yeasts tested readily degraded extracellular inositol hexaphosphate (phytate; IP6) in media with IP6 as the sole phosphorous source . Phosphate (Pi) addition yielded repression consistent with the PHO system . However, repression of IP6-degrading enzymes was not only dependent on level of Pi, but also on pH and medium composition . In complex medium, containing Pi at a concentration previously suggested to yield full repression of the secretory acid phosphatases (SAPs; e.g., {Mol . Biol . Cell 11 (2000) 4309}), and at relatively high pH, repression of phytate-degrading enzymes was weak . The capacity to degrade phytate, irrespective of Pi addition or not, was highest at the pH most distant from the pH optimum of the SAPs {Microbiol . Res . 151 (1996) 291}, suggesting that expression rather than enzyme activity was affected by pH . In synthetic medium, repression was strong and pH-independent (no IP6 degradation within the range tested) . The distinct difference between media shows that, in addition to known regulatory role of Pi for the PHO system, additional factors may be involved . Using a deletion strain, we further demonstrate that the main secretory acid phosphatase Pho5p is not essential for intact phytate-degrading capacity and growth without Pi, neither is Pho3p . However, when constitutively overexpressing PHO5 an increased net phytase activity was obtained, in repressing and non-repressing conditions . This proves that, although redundant in a wild type, Pho5p can catalyze hydrolysis of IP6 and that at least one more enzyme is capable of effective hydrolysis of IP6 (sufficient to provide the cell with phosphorous at a rate yielding maximum growth) . Finally, a bread dough experiment showed that the typical concentrations of Pi during leavening exceed levels shown to repress phytate degradation by a wild-type S . cerevisiae. J Cell Sci, 2004 Dec 1, 117(Pt 25), 6031 - 41 Epub 2004 Dec 1. Distribution of Can1p into stable domains reflects lateral protein segregation within the plasma membrane of living S . cerevisiae cells; Malinska K et al.; Recently, lipid-raft-based subdomains within the plasma membrane of living Saccharomyces cerevisiae cells were visualized using green fluorescent protein fusions, and non-overlapping subdomains containing either Pma1p or Can1p were distinguished . In this study, the long-term stability of the subdomains was investigated . Experiments with latrunculin A and nocodazole ruled out the involvement of cytoskeletal components in the stabilization of the subdomains . Also a putative role of the cell wall was excluded, because protoplasting of the cells changed neither the pattern nor the stability of the subdomains . By contrast, the expected inner dynamics of the membrane subdomains was documented by FRAP experiments . Finally, two other proteins were localized within the frame of the Can1p/Pma1p plasma-membrane partition . We show that Fur4p (another H(+) symporter) and Sur7p (a protein of unknown function) occupy the Can1p subdomain. Bioinformatics . 2004 Nov 16; {Epub ahead of print} Predicting gene function through systematic analysis and quality assessment of high-throughput data; Kemmeren P et al.; MOTIVATION: Determining gene function is an important challenge arising from the availability of whole genome sequences . Until recently, approaches based on sequence homology were the only high-throughput method for predicting gene function . Use of high-throughput generated experimental datasets for determining gene function has been limited for several reasons . RESULTS: Here a new approach is presented for integration of high-throughput datasets, leading to prediction of function based on relationships supported by multiple types and sources of data . This is achieved with a database containing 125 different high-throughput datasets describing phenotypes, cellular localizations, protein interactions and mRNA expression levels from S . cerevisiae, using a bit-vector representation and information content based ranking . The approach takes characteristic and qualitative differences between the datasets into account, is highly flexible, efficient and scalable . Database queries result in predictions for 543 uncharacterized genes, based on multiple functional relationships each supported by at least three types of experimental data . Some of these are experimentally verified, further demonstrating their reliability . The results also generate insights into the relative merits of different data types and provide a coherent framework for functional genomic datamining . AVAILABILITY: Free availability over the internet . SUPPLEMENTARY INFORMATION: http://www.genomics.med.uu.nl/pub/pk/comb_gen_network. Dev Cell, 2004 Nov, 7(5), 755 - 62 The S . pombe Cdc14-like phosphatase Clp1p regulates chromosome biorientation and interacts with Aurora kinase; Trautmann S et al.; The S . pombe Cdc14-related phosphatase Clp1p/Flp1p regulates G2/M transition by antagonizing CDK activity and is essential for coordinating the nuclear division cycle with cytokinesis through the cytokinesis checkpoint . At the G2/M transition, Clp1p/Flp1p is released from the nucleolus and SPB and distributes throughout the nucleus to the spindle and the contractile ring . This early relocalization is analogous to vertebrate Cdc14 homologs and stands in contrast to S . cerevisiae Cdc14p, which is not released from the nucleolus until metaphase/anaphase transition . Here, we report that Clp1p/Flp1p localizes to kinetochores in prometaphase and functions in chromosome segregation, since deletion of clp1/flp1 causes cosegregation of sister chromatids, when sister kinetochores are prone to mono-orientation . Genetic, cytological, and biochemical experiments suggest that Clp1p/Flp1p functions together with Aurora kinase at kinetochores . Together, these results suggest that Clp1p/Flp1p has a role in repairing mono-orientation of sister kinetochores. Opt Lett, 2004 Oct 1, 29(19), 2270 - 2 Real-time three-dimensional optical micromanipulation of multiple particles and living cells; Rodrigo PJ et al.; Counterpropagating light fields provide a stationary optical potential well for a Brownian particle . Introducing variability in the relative strengths of the counterpropagating beams allows us to create a more general configuration-the optical elevator . An optical elevator dynamically controls the axial location of the potential minimum where the particle finds a stable equilibrium position . We describe the implementation of multiple real-time reconfigurable optical elevators with the generalized phase contrast method for dynamic manipulation of polystyrene spheres and yeast cells S . cerevisiae in three dimensions. Biotechnol Bioeng, 2004 Dec 5, 88(5), 664 - 70 Fluorescence-based sensing system for copper using genetically engineered living yeast cells; Shetty RS et al.; A whole cell-based optical sensing system for copper was developed based on Saccharomyces cerevisiae cells harboring plasmid pYEX-GFPuv . The basis of this system was the ability of the transcriptional activator protein Ace1 present in S . cerevisiae to control the expression of the reporter protein, GFPuv . When copper ions are present in the sample, the Ace1 protein activates the cup1 promoter located upstream from the gfpuv gene in plasmid pYEX-GFPuv, thus inducing the production of GFPuv . The concentration of copper ions in the sample can then be related to the GFPuv expressed in the yeast . The amount of GFPuv produced in the system was determined by monitoring the fluorescence emitted at 507 nm after excitation at 397 nm . This system can detect copper at concentrations as low as 5 x 10(-7) M, and is selective for copper over a variety of metal ions, with the exception of silver . The applicability of this sensing system to different analytical platforms and in real samples is demonstrated. Yeast, 2004 Oct 30, 21(14), 1205 - 17 Comparison of three expression systems for heterologous xylanase production by S . cerevisiae in defined medium; Gorgens JF et al.; The influence of the auxotrophic deficiencies of the host strain and expression vector selection on the production of a heterologous protein was investigated . Heterologous xylanase production by two prototrophic S . cerevisiae transformants, containing either a plasmid-based, YEp-type expression system or an integrative, YIp-type expression system, were compared with production by an auxotrophic transformant, containing an identical YEp-type expression system, in batch and continuous cultivation, using a chemically defined medium . Heterologous xylanase production by the auxotrophic strains in defined medium was critically dependent on the availability of amino acids, as extracellular xylanase production increased dramatically when amino acids were over-consumed from the medium to the point of saturating the cell . Saturation with amino acids, indicated by an increased leakage of amino acids from the cell, was thus a prerequisite for high level of heterologous protein production by the auxotrophic strain . Maximal xylanase production levels by the auxotrophic strain corresponded to the levels obtained with a similar prototrophic strain during cultivation in defined medium without amino acids . Superfluous auxotrophic markers thus had a strong deleterious effect on heterologous protein production by recombinant yeasts, and the use of such strains should be limited to initial exploratory investigations . The increased copy number and foreign gene dosage of the YEp-based expression vector, stabilized by the ura3 fur1 autoselection system, significantly improved production levels of heterologous xylanase, compared to the YIp system, which is based on a single integration into the yeast genome . No evidence was found of the possible saturation of the host secretory capacity by multicopy overexpression . Stable production of heterologous xylanase at high levels by the prototrophic YEp-based recombinant strain, compared to the YIp system, was demonstrated . copyright 2004 John Wiley & Sons, Ltd. Plant J, 2004 Nov, 40(4), 488 - 99 AtPTR1, a plasma membrane peptide transporter expressed during seed germination and in vascular tissue of Arabidopsis; Dietrich D et al.; For the efficient translocation of organic nitrogen, small peptides of two to three amino acids are posited as an important alternative to amino acids . A new transporter mediating the uptake of di- and tripeptides was isolated from Arabidopsis thaliana by heterologous complementation of a peptide transport-deficient Saccharomyces cerevisiae mutant . AtPTR1 mediated growth of S . cerevisiae cells on different di- and tripeptides and caused sensitivity to the phytotoxin phaseolotoxin . The spectrum of substrates recognized by AtPTR1 was determined in Xenopus laevis oocytes injected with AtPTR1 cRNA under voltage clamp conditions . AtPTR1 not only recognized a broad spectrum of di- and tripeptides, but also substrates lacking a peptide bond . However, amino acids, omega-amino fatty acids or peptides with more than three amino acid residues did not interact with AtPTR1 . At pH 5.5 AtPTR1 had an apparent lower affinity (K(0.5) = 416 microm) for Ala-Asp compared with Ala-Ala (K(0.5) = 54 microm) and Ala-Lys (K(0.5) = 112 microm) . Transient expression of AtPTR1/GFP fusion proteins in tobacco protoplasts showed that AtPTR1 is localized at the plasma membrane . In addition, transgenic plants expressing the beta-glucuronidase (uidA) gene under control of the AtPTR1 promoter demonstrated expression in the vascular tissue throughout the plant, indicative of a role in long-distance transport of di- and tripeptides. Curr Biol, 2004 Oct 26, 14(20), 1812 - 21 SIR2 is required for polycomb silencing and is associated with an E(Z) histone methyltransferase complex; Furuyama T et al.; BACKGROUND: SIR2 was originally identified in S . cerevisiae for its role in epigenetic silencing through the creation of specialized chromatin domains . It is the most evolutionarily conserved protein deacetylase, with homologs in all kingdoms . SIR2 orthologs in multicellular eukaryotes have been implicated in lifespan determination and regulation of the activities of transcription factors and other proteins . Although SIR2 has not been widely implicated in epigenetic silencing outside yeast, Drosophila SIR2 mutations were recently shown to perturb position effect variegation, suggesting that the role of SIR2 in epigenetic silencing may not be restricted to yeast . RESULTS: Evidence is presented that Drosophila SIR2 is also involved in epigenetic silencing by the Polycomb group proteins . Sir2 mutations enhance the phenotypes of Polycomb group mutants and disrupt silencing of a mini-white reporter transgene mediated by a Polycomb response element . Consistent with this, SIR2 is physically associated with components of an E(Z) histone methyltransferase complex . SIR2 binds to many euchromatic sites on polytene chromosomes and colocalizes with E(Z) at most sites . CONCLUSIONS: SIR2 is involved in the epigenetic inheritance of silent chromatin states mediated by the Drosophila Polycomb group proteins and is physically associated with a complex containing the E(Z) histone methyltransferase. Biochem J . 2004 Oct 21; {Epub ahead of print} Manganese toxicity and Saccharomyces cerevisiae Mam3p, a member of the ACDP (Ancient Conserved Domain Protein) family of proteins; Yang M et al.; Manganese is an essential but potentially toxic trace metal in biological systems . Overexposure to manganese is known to cause neurological deficits in humans, but the pathways that lead to manganese toxicity are largely unknown . We have employed the bakers' yeast S . cerevisiae as a model system to identify genes that contribute to manganese related damage . In a genetic screen for yeast manganese resistance mutants, we identified S . cerevisiae MAM3 as a gene which when deleted would increase cellular tolerance to toxic levels of manganese and also increased cell's resistance towards cobalt and zinc . By sequence analysis, Mam3p shares strong homology with the mammalian ACDP (ancient conserved domain protein) family of polypeptides . Mutations in human ACDP1 have been associated with urofacial (Ochoa) syndrome . However the functions of eukaryotic ACDPs remain unknown . We show here that S . cerevisiae MAM3 encodes an integral membrane protein of the yeast vacuole whose expression levels directly correlate with the degree of manganese toxicity . Surprisingly, Mam3p contributes to manganese toxicity without any obvious changes in vacuolar accumulation of metals . Furthermore, through genetic epistasis studies, we demonstrate that MAM3 operates independently of the well-established manganese trafficking pathways in yeast involving the manganese transporters Pmr1p, Smf2p and Pho84p . This is the first report of a eukaryotic ACDP family protein involved in metal homeostasis. Biochem J . 2004 Oct 21; {Epub ahead of print} Demonstration of N/C intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity; Faye A et al.; We have previously proposed that changes in malonyl-CoA sensitivity of rat liver carnitine palmitoyltransferase 1 (L-CPT1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains . By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we now prove experimentally the existence of such N/C intramolecular interactions both in wild-type L-CPT1 expressed in S . cerevisiae and in the native L-CPT1 in fed rat liver mitochondria . These N/C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity . Moreover, increasing the outer-membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitisation, attenuated the N/C interactions . This indicates that the changes in malonyl-CoA sensitivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N/C interactions . Finally, we show that mutations in the regulatory regions of the N-domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased (S24AQ30A) or abrogated (E3A) malonyl-CoA sensitivity . Moreover, we have identified the region immediately N-terminal to TM1 (residues 40-47) as being involved in the chemical N/C cross-linking . These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational