S. cerevisiae

Cancer Res, 1999 Dec 1, 59(23), 5908 - 11
Effects of p51/p63 missense mutations on transcriptional activities of p53 downstream gene promoters; Kato S et al.; The p51/p63 gene is a homologue of p53, the product of which acts as a transcriptional activator by binding to p53-responsive elements in the promoter regions of several p53 downstream genes . Recently, we identified four distinct mutations in the p51/p63 gene after screening >200 human tumors and cell lines . Because all of the detected p51/p63 mutations were missense mutations, the pathogenic effect of these mutations is difficult to determine without performing a functional analysis . In this study, we examined the transcriptional activity of tumor-derived p51/p63 missense mutations using a yeast-based assay and compared the data with that of artificial p51/p63 missense mutations at residues corresponding to the positions and substituted residues of p53 mutation "hotspots." Although most of the p51/p63 missense mutations at the p53 hotspot residues were unable to transactivate the promoters used in this study, the tumor-derived p51/p63 missense mutations retained their ability to transactivate the MDM2 and/or the BAX promoter but not the p21/WAF1 promoter . These results suggest that the p51/p63 mutation might be involved in an unknown tumor suppression pathway distinct from that of p53.

Proc Nutr Soc, 1999 Aug, 58(3), 621 - 3
Regulation of gene expression by glucose; Ferre P; Fatty acid synthase (EC 2.3.1.85) is an enzyme involved in the lipogenic pathway allowing fatty acid synthesis from glucose . Glucose up-regulates the transcription of the fatty acid synthase gene in both adipocytes and hepatocytes, with insulin having only an indirect role . The signal metabolite could be glucose-6-phosphate rather than glucose itself . The glucose response element of the fatty acid synthase gene has not yet been precisely identified, although a -2 kb region of the fatty acid synthase promoter is sufficient to confer nutritional responsiveness to a reporter gene . ADD1/SREBP1, a b-HLH-LZ transcription factor belonging to the sterol regulatory element-binding protein family might be involved in the transduction of the glucose effect . Finally, the stimulatory effect of glucose on the expression of the fatty acid synthase gene is inhibited by the activation of AMP-activated protein kinase . Interestingly enough, AMP-activated protein kinase is structurally and functionally related to the yeast SNF1 protein kinase complex which is essential for the transcriptional activation of glucose-repressed genes in Saccharomyces cerevisiae.

Trends Cell Biol, 2000 Jan, 10(1), 25 - 31
Protein translocation into mitochondria: the role of TIM complexes; Bauer MF et al.; Import of nuclear-encoded mitochondrial preproteins is mediated by a general translocase in the outer membrane, the TOM complex, and by two distinct translocases in the mitochondrial inner membrane, the TIM23 complex and the TIM22 complex . Both TIM complexes cooperate with the TOM complex but facilitate import of different classes of precursor proteins . Precursors with an N-terminal presequence are imported via the TIM23 complex, whereas mitochondrial carrier proteins require the TIM22 complex for insertion into the inner membrane . This review discusses recent advances in understanding the structure and function of the translocases of the inner membrane and the possible role of Tim proteins in the development of the Mohr-Tranebjaerg syndrome, a mitochondrial disorder leading to neurodegeneration.

Oncogene, 1999 Dec 9, 18(52), 7566 - 75
Induction of apoptosis by SLK, a Ste20-related kinase; Sabourin LA et al.; We have cloned and characterized a novel murine Ste20-related kinase designated SLK . SLK displays high homology to the Ste20-related kinase LOK, and is more distantly related to MST1 and 2, both Ste20-like kinases . In addition, SLK displays high homology to microtubule and nuclear associated protein (M-NAP) and AT1-46, both of unknown function . SLK is ubiquitously expressed as multiple mRNAs in tissues and cell lines and is downregulated by mitogen depletion in differentiating myoblasts . Biochemical characterization showed that SLK overexpression activates c-Jun amino-terminal kinase 1 (JNK1) . However, in vitro kinase assays indicated that SLK was not activated in response to various growth factors or stress-inducing agents . Immunofluorescence studies revealed that SLK colocalized to distinct cytosolic domains, preferentially at the periphery of the cells . In addition, prolonged overexpression of SLK in cultured fibroblasts resulted in apoptosis as demonstrated by annexin-V and TUNEL staining . Our results suggest that SLK belongs to a new family of protein kinases, mediating activation of the stress response pathway through a novel signaling cascade.

Oncogene, 1999 Dec 9, 18(52), 7495 - 505
Ku80 can translocate to the nucleus independent of the translocation of Ku70 using its own nuclear localization signal; Koike M et al.; Ku antigen is a complex of Ku70 and Ku80 subunits and plays an important role in not only DNA double-strand breaks (DSB) repair and V(D)J recombination, but also in growth regulation . Ku is generally believed to always form and function as heterodimers on the basis of in vitro observations . Here we demonstrate that the localization of Ku80 does not completely coincide with that of Ku70 . Ku70 and Ku80 were colocalized in the nucleus in the interphase but not in the late telophase/early G1 phase of the cell cycle . Since the in vivo function of Ku might be partially regulated by the control of its transport, we attempted to investigate the molecular mechanisms underlying the nuclear translocation of Ku . The nuclear translocation of Ku80 started during the late telophase/early G1 phase after the nuclear envelope was formed and this was preceded by the nuclear translocation of Ku70 . Furthermore, we found that the Ku80 protein was transported to the nucleus without heterodimerization with Ku70 . To understand in detail the mechanism of transport of Ku80, we attempted to identify the nuclear localization signal (NLS) of Ku80 and defined to a region spanning nine amino acid residues (positions 561 - 569) . The Ku80 NLS was demonstrated to be mediated to the nuclear rim by two components of PTAC58 and PTAC97 . All these findings support the idea that Ku80 can translocate to the nucleus using its own NLS independent of the translocation of Ku70.

Chirality, 2000 Jan, 12(1), 26 - 9
Chemo-enzymatic synthesis of the antidepressant duloxetine and its enantiomer; Liu H et al.; Sodium borohydride reduction of 3-chloro-1-(2-thienyl)-1-propanone gave the corresponding racemic alcohol which was kinetically resolved with lipase B from Candida antarctica as catalyst to yield the chiral building blocks (S)-3-chloro-1-(2-thienyl)-1-propanol and the corresponding (R)-butanoate . The enantiopure chiral building blocks were converted into Duloxetine and its enantiomer .

J Biol Chem, 1999 Dec 24, 274(52), 36987 - 94
The role of the Smad3 protein in phorbol ester-induced promoter expression; Biggs JR et al.; The Sp1 transcription factor plays an important role in mediating the p53-independent activation of the p21(WAF1) (WAF1) promoter by phorbol 12-myristate13-acetate (PMA) in hematopoietic cells . Using GAL4-Sp1 fusion proteins and a luciferase reporter, PMA is shown to activate the transcriptional activity of Sp1 independent of the WAF1 promoter . This activation does not require the Ser/Thr-rich region of Sp1 and can be mediated by 41 amino acids (152-193) of Sp1 that are important for the interaction with human TAF130 . Because transforming growth factor-beta enhances WAF1 promoter activity through both Sp1 and Smad proteins, the role of Smads in PMA transcriptional activation was examined . PMA addition to hematopoietic cells was found to activate a GAL4/Smad-dependent promoter and the transforming growth factor-beta-responsive promoter, p3TP-lux . Immunofluorescence data demonstrate that PMA addition to hematopoietic cells induces the translocation of Smad3 to the nucleus . However, Smad3 does not stimulate the WAF1 promoter, but rather slightly inhibits the PMA-mediated induction of transcription from this upstream region . Additionally, transfection of Smad3 did not enhance the activation of GAL4/Sp1 by PMA . These results demonstrate that, while PMA can activate Smad-mediated transcription, Smad proteins do not appear to play a major role in the PMA induction of the WAF1 promoter.

J Biol Chem, 1999 Dec 24, 274(52), 36839 - 42
The xenopus Suc1/Cks protein promotes the phosphorylation of G(2)/M regulators; Patra D et al.; The entry into mitosis is controlled by Cdc2/cyclin B, also known as maturation or M-phase promoting factor (MPF) . In Xenopus egg extracts, the inhibitory phosphorylations of Cdc2 on Tyr-15 and Thr-14 are controlled by the phosphatase Cdc25 and the kinases Myt1 and Wee1 . At mitosis, Cdc25 is activated and Myt1 and Wee1 are inactivated through phosphorylation by multiple kinases, including Cdc2 itself . The Cdc2-associated Suc1/Cks1 protein (p9) is also essential for entry of egg extracts into mitosis, but the molecular basis of this requirement has been unknown . We find that p9 strongly stimulates the regulatory phosphorylations of Cdc25, Myt1, and Wee1 that are carried out by the Cdc2/cyclin B complex . Overexpression of the prolyl isomerase Pin1, which binds to the hyperphosphorylated forms of Cdc25, Myt1, and Wee1 found at M-phase, is known to block the initiation of mitosis in egg extracts . We have observed that Pin1 specifically antagonizes the stimulatory effect of p9 on phosphorylation of Cdc25 by Cdc2/cyclin B . This observation could explain why overexpression of Pin1 inhibits mitotic initiation . These findings suggest that p9 promotes the entry into mitosis by facilitating phosphorylation of the key upstream regulators of Cdc2.

Genes Dev, 1999 Dec 1, 13(23), 3115 - 24
Oppositely imprinted genes p57(Kip2) and igf2 interact in a mouse model for Beckwith-Wiedemann syndrome; Caspary T et al.; Beckwith-Wiedemann syndrome (BWS) is a clinically variable disorder characterized by somatic overgrowth, macroglossia, abdominal wall defects, visceromegaly, and an increased susceptibility to childhood tumors . The disease has been linked to a large cluster of imprinted genes at human chromosome 11p15.5 . A subset of BWS patients has been identified with loss-of-function mutations in p57(KIP2), a maternally expressed gene encoding a G(1) cyclin-dependent kinase inhibitor . Some patients display loss of imprinting of IGF2, a fetal-specific growth factor that is paternally expressed . To understand how the same disease can result from misregulation of two linked, but unrelated, genes, we generated a mouse model for BWS that both harbors a null mutation in p57(Kip2) and displays loss of Igf2 imprinting . These mice display many of the characteristics of BWS, including placentomegaly and dysplasia, kidney dysplasia, macroglossia, cleft palate, omphalocele, and polydactyly . Some, but not all, of the phenotypes are shown to be Igf2 dependent . In two affected tissues, the two imprinted genes appear to act in an antagonistic manner, a finding that may help explain how BWS can arise from mutations in either gene.

EMBO J, 1999 Dec 15, 18(24), 6908 - 16
The monomeric homing endonuclease PI-SceI has two catalytic centres for cleavage of the two strands of its DNA substrate; Christ F et al.; The monomeric homing endonuclease PI-SceI cleaves the two strands of its DNA substrate in a concerted manner, which raises the question of whether this enzyme harbours one or two catalytic centres . If PI-SceI has only one catalytic centre, one would expect that cross-linking enzyme and substrate should prevent reorientation of the enzyme required to perform the second cut after having made the first cut: PI-SceI, however, when cross-linked to its substrate, is able to cleave both DNA strands . If PI-SceI has two catalytic centres, one would expect that it should be possible to inactivate one catalytic centre by mutation and obtain a variant with preference for a substrate nicked in one strand; such variants have been found . The structural homology between the catalytic domain of PI-SceI having a pseudo 2-fold symmetry, and I-CreI, a homodimeric homing endonuclease, suggests that in PI-SceI active site I, which attacks the top strand, comprises Asp218, Asp229 and Lys403, while Asp326, Thr341 and Lys301 make up active site II, which cleaves the bottom strand . Cleavage experiments with modified oligodeoxynucleotides and metal ion mapping experiments demonstrate that PI-SceI interacts differently with the two strands at the cleavage position, supporting a model of two catalytic centres.

Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 405 - 10
Sas3 is a histone acetyltransferase and requires a zinc finger motif; Takechi S et al.; SAS3 was originally isolated as a gene related to SAS2, which encodes a positive regulator of transcriptional silencing in yeast . The Sas3 protein possesses an evolutionally conserved domain that is shared by a group of SAS-like factors . This conserved domain contains an atypical zinc finger motif and a putative acetyl-CoA binding motif . We showed that recombinant Sas3 exhibits histone acetyltransferase (HAT) activity toward acetylate core histones H2A, H3, and H4 . This substrate specificity is similar to those of Tip60 and Esa1 . Analysis of a series of deletion mutants revealed that the minimum region required for HAT activity is located within amino acid residues 241-577, including the domain conserved in the MYST family proteins . Amino acid substitution mutant analysis showed that both the acetyl-CoA binding motif and the zinc finger motif are required for HAT activity . These results suggest that SAS3 and its family members require the zinc finger motif for their activity .

Biochem Biophys Res Commun, 1999 Dec 20, 266(2), 347 - 51
Identification and characterization of SorCS, a third member of a novel receptor family; Hermey G et al.; A novel receptor, SorCS, was isolated from murine brain . It shows homology to the mosaic receptor SorLA and the neurotensin receptor sortilin based on a common VPS10 domain which is the hallmark of this new receptor family . In the N-terminus of SorCS two putative cleavage sites for the convertase furin mark the beginning of the VPS10 domain, followed by a module of imperfect leucine-rich repeats and a transmembrane domain . The short intracellular C-terminus contains consensus signals for rapid internalization . The identified putative binding motifs for SH2 and SH3 domains are unique in the family of VPS10 domain receptors . SorCS is predominantly expressed in brain, but also in heart, liver, and kidney . SorCS transcripts detected by in situ hybridization in the murine central nervous system point to a neuronal expression .

Protein Expr Purif, 1999 Dec, 17(3), 358 - 65
Expression and purification of the BmK M1 neurotoxin from the scorpion Buthus martensii Karsch; Shao F et al.; The gene encoding a neurotoxin (BmK M1) from the scorpion Buthus martensii Karsch was expressed in Saccharomyces cerevisiae at a high level with the alcohol dehydrogenase promoter . SDS-PAGE of the culture confirmed expression and showed secretion into medium from yeast . Recombinant BmK M1 was purified rapidly and efficiently by ion exchange and gel filtration chromatography to homogeneity, produced a single band on tricine-SDS-PAGE, and processed the homologous N-terminus . Amino acid analysis and N-terminal sequencing demonstrated that the recombinant toxin was processed correctly from the alpha-mating factor leader sequence and was chemically identical to the native form . The expressed recombinant BmK M1 was toxic for mice, which indicated that it was biologically active . Quantitative estimation showed that recombinant BmK M1 had an LD(50) similar to that of the native toxin .

J Mol Biol, 1999 Dec 17, 294(5), 1311 - 25
Crystal structure of the histone acetyltransferase Hpa2: A tetrameric member of the Gcn5-related N-acetyltransferase superfamily; Angus-Hill ML et al.; We report the crystal structure of the yeast protein Hpa2 in complex with acetyl coenzyme A (AcCoA) at 2.4 A resolution and without cofactor at 2.9 A resolution . Hpa2 is a member of the Gcn5-related N-acetyltransferase (GNAT) superfamily, a family of enzymes with diverse substrates including histones, other proteins, arylalkylamines and aminoglycosides . In vitro, Hpa2 is able to acetylate specific lysine residues of histones H3 and H4 with a preference for Lys14 of histone H3 . Hpa2 forms a stable dimer in solution and forms a tetramer upon binding AcCoA . The crystal structure reveals that the Hpa2 tetramer is stabilized by base-pair interactions between the adenine moieties of the bound AcCoA molecules . These base-pairs represent a novel method of stabilizing an oligomeric protein structure . Comparison of the structure of Hpa2 with those of other GNAT superfamily members illustrates a remarkably conserved fold of the catalytic domain of the GNAT family even though members of this family share low levels of sequence homology . This comparison has allowed us to better define the borders of the four sequence motifs that characterize the GNAT family, including a motif that is not discernable in histone acetyltransferases by sequence comparison alone . We discuss implications of the Hpa2 structure for the catalytic mechanism of the GNAT enzymes and the opportunity for multiple histone tail modification created by the tetrameric Hpa2 structure .

Biochemistry, 1999 Dec 14, 38(50), 16613 - 9
Quantitation of metal ion and DNA junction binding to the Holliday junction endonuclease Cce1; Kvaratskhelia M et al.; Cce1 is a magnesium-dependent Holliday junction endonuclease involved in the resolution of recombining mitochondrial DNA in Saccharomyces cerevisiae . Cce1 binds four-way DNA junctions as a dimer, opening the junction into an extended, 4-fold symmetric structure, and resolves junctions by the introduction of paired nicks in opposing strands at the point of strand exchange . In the present study, we have examined the interactions of wild-type Cce1 with a noncleavable four-way DNA junction and metal ions (Mg(2+) and Mn(2+)) using isothermal titration calorimetry, EPR, and gel electrophoresis techniques . Mg(2+) or Mn(2+) ions bind to Cce1 in the absence of DNA junctions with a stoichiometry of two metal ions per Cce1 monomer . Cce1 binds to four-way junctions with a stoichiometry of two Cce1 dimers per junction molecule in the presence of EDTA, and one dimer of Cce1 per junction in 15 mM magnesium . The presence of 15 mM Mg(2+) dramatically reduces the affinity of Cce1 for four-way DNA junctions, by about 900-fold . This allows an estimation of DeltaG degrees for stacking of four-way DNA junction 7 of -4.1 kcal/mol, consistent with the estimate of -3.3 to -4.5 kcal/mol calculated from branch migration and NMR experiments {Overmars and Altona (1997) J . Mol . Biol . 273, 519-524; Panyutin et al . (1995) EMBO J . 14, 1819-1826} . The striking effect of magnesium ions on the affinity of Cce1 binding to the four-way junction is predicted to be a general one for proteins that unfold the stacked X-structure of the Holliday junction on binding.

Biochemistry, 1999 Dec 14, 38(50), 16461 - 8
Guanine-rich telomeric sequences stimulate DNA polymerase activity in vitro; Ying J et al.; Guanine-rich oligonucleotides and short telomeric DNA sequences can self-associate into G-quartet stabilized complexes . We discovered that this self-association can occur in sequencing reactions and that higher-order structures stimulate DNA polymerase to synthesize extended DNA strands . Base analogues were used to identify Hoogsteen base pairings as stabilizing forces in these stimulatory DNA structures . Scanning force microscopy confirmed that quartet-DNA was formed from these oligomers and that these extended, four-stranded structures could be bound by DNA polymerase . Since guanine quartet-stabilized structures are proposed to exist in vivo, such structures may stimulate DNA polymerization in vivo.

J Clin Endocrinol Metab, 1999 Dec, 84(12), 4362 - 70
Heritable disorders of pituitary development; Parks JS et al.; Basic and translational research achievements over the past 2 decades have disclosed the molecular mechanisms underlying several genetic forms of hypopituitarism . Disorders that are limited to the hypothalamic, pituitary, GH axis are caused by mutations in individual components of that axis . Disorders involving GH and one or more additional pituitary hormones are caused by mutations in the homeodomain transcription factors that direct embryological development of the anterior pituitary gland . Pit-1 has a POU-specific and a POU-homeo DNA-binding domain . The phenotype produced by mutations in the PIT1 gene involves deficiencies of GH, PRL, and TSH . Pituitary glands are either small or normally sized . The PROP1 gene encodes a transcription factor with a single paired-like DNA-binding domain . Persons with inactivating mutations in PROP1 have deficiencies of LH and FSH, as well as GH, PRL, and TSH . Their pituitary glands may be small, normally sized, or extremely large and show suprasellar extension . Pituitary degeneration may produce acquired deficiency of ACTH . Expression of the HESX1 gene precedes expression of PROP1 and PIT1, and it is much more widespread . The protein has a paired-like domain, and it competes with the product of PROP1 for DNA-binding . Homozygosity for inactivating mutations of HESX1 produces a complex phenotype that resembles septo-optic dysplasia . Much more needs to be learned about the role of HESX1 mutations in other forms of hypopituitarism.

Hum Genet, 1999 Nov, 105(5), 489 - 95
Structure of the human Lanosterol synthase gene and its analysis as a candidate for holoprosencephaly (HPE1); Roessler E et al.; Holoprosencephaly (HPE) is the most common birth defect of the brain in humans . It involves various degrees of incomplete separation of the cerebrum into distinct left and right halves, and it is frequently accompanied by craniofacial anomalies . The HPE1 locus in human chromosome 21q22.3 is one of a dozen putative genetic loci implicated in causing HPE . Here, we report the complete gene structure of the human lanosterol synthase (LS) gene, which is located in this interval, and present its mutational analysis in HPE patients . We considered LS an excellent candidate HPE gene because of the requirement for cholesterol modification of the Sonic Hedgehog protein for the correct patterning activity of this HPE-associated protein . Despite extensive pedigree analysis of numerous polymorphisms, as well as complementation studies in yeast on one of the missense mutations, we find no evidence that the LS gene is in fact HPE1, implicating another gene located in this chromosomal region in HPE pathogenesis.

Mol Endocrinol, 1999 Dec, 13(12), 2151 - 62
Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2; Xie W et al.; In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TRE(pal)) but not the glucocorticoid response element (GRE) . This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor . In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner . Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors.

Exp Lung Res, 1999 Oct-Nov, 25(7), 561 - 76
Developmental changes in phosphatidylinositol transfer protein concentration and phospholipid transfer activities in rat type II cells; Viscardi RM et al.; The phospholipid transfer proteins (PLTPs) are cytosolic proteins that have been characterized by their ability to facilitate the transfer of phospholipids between membranes in vitro . The goals of this study were to determine whether PITP alpha concentration and phospholipid transfer activities are enriched in type II cells compared with whole lung and to determine the developmental changes in PITP alpha concentration and phospholipid transfer activities during late gestation and newborn period . The concentration of PITP alpha in type II cell cytosol measured by enzyme-linked immunosorbent assay (ELISA) increased during late fetal gestation to 2.2-fold adult levels and declined 41% during the first postnatal day . However, compared to whole adult lung cytosol, type II cell cytosol was not significantly enriched with PITP alpha . Phospholipid transfer activities were determined by a vesicle-rat lung membrane transfer assay . In adult lung, transfer activities for all the phospholipids were enriched in adult type II cell cytosol compared to whole lung cytosol (phosphatidylglycerol {PG}, 12.5-fold; phosphatidylinositol {PI}, 9.2-fold; phosphatidylcholine {PC}, 6.5-fold; and phosphatidylethanolamine {PE}, 6.6-fold; P < .05 in each case) . The rate of phospholipid transfer in type II cell cytosol increased during late fetal gestation to levels 4.9 (PG), 3.7 (PI), and 2.8 (PC) times greater than adult levels . In cytosol from cells from different stages, the order of transfer rate was PG > PI > PC > PE . PITP alpha immunodepletion of adult type II cytosol did not significantly affect phospholipid transfer activities, suggesting that other PLTPs are responsible for the majority of the observed transfer activities in these cells . Developmental increases in PITP alpha concentration and other PLTPs parallel developmental changes in type II cell surfactant phospholipid metabolism, suggesting a possible role of these transfer proteins in the unique function of the type II cell.

Plant Mol Biol, 1999 Oct, 41(3), 415 - 23
Selection of Arabidopsis genes encoding secreted and plasma membrane proteins; Goo JH et al.; Secreted and plasma membrane proteins play crucial roles in a variety of physiological and developmental processes of multicellular organisms . Systematic cloning of the genes encoding these proteins is therefore of general interest . An effective method of trapping signal sequences was first described by Tashiro et al . (1993), and a similar yet more efficient method was reported by Klein et al . (1996) and Jacobs et al . (1997) . In this study, we carried out the latter yeast-based signal sequence trap to clone genes from Arabidopsis thaliana encoding secreted and plasma membrane proteins . Of 144 sequenced cDNA clones, 18% are identical to previously cloned Arabidopsis thaliana genes, 12% are homologous to genes identified from various organisms, and 46% are novel . All of the isolated genes identical or homologous to previously reported genes are either secreted or plasma membrane proteins, and the remaining novel genes appear to contain functional signal sequences based on computer-aided sequence analysis . The full-length cDNA clones of one homologous gene and another novel gene were isolated and sequenced . The deduced amino acid sequences suggest that the former encodes a secreted protein, and the latter encodes a type 1 membrane protein . These results indicate that the signal sequence trap method is effective and useful for the isolation of plant genes encoding secreted and plasma membrane proteins.

Semin Cell Dev Biol, 1999 Oct, 10(5), 465 - 72
Role and regulation of the ER chaperone BiP; Gething MJ; BiP, an HSP70 molecular chaperone located in the lumen of the endoplasmic reticulum (ER), binds newly-synthesized proteins as they are translocated into the ER and maintains them in a state competent for subsequent folding and oligomerization . BiP is also an essential component of the translocation machinery, as well as playing a role in retrograde transport across the ER membrane of aberrant proteins destined for degradation by the proteasome . BiP is an abundant protein under all growth conditions, but its synthesis is markedly induced under conditions that lead to the accumulation of unfolded polypeptides in the ER . This attribute provides a marker for disease states that result from misfolding of secretory and transmembrane proteins.

Oncogene, 1999 Nov 18, 18(48), 6621 - 34
Mmip-2, a novel RING finger protein that interacts with mad members of the Myc oncoprotein network; Yin XY et al.; Mad proteins are basic-helix-loop-helix-leucine zipper (bHLH-ZIP)-containing members of the myc oncoprotein network . They interact with the bHLH-ZIP protein max, compete for the same DNA binding sites as myc-max heterodimers and down-regulate myc-responsive genes . Using the bHLH-ZIP domain of mad1 as a yeast two-hybrid 'bait', we identified Mmip-2, a novel RING finger protein that interacts with all mad members, but weakly or not at all with c-myc, max or unrelated bHLH or bZIP proteins . The mad1-Mmip-2 interaction is mediated by the ZIP domain in the former protein and by at least two regions in the latter which do not include the RING finger . Mmip-2 can disrupt max-mad DNA binding and can reverse the suppressive effects of mad proteins on c-myc-responsive target genes and on c-myc + ras-mediated focus formation in fibroblasts . Tagging with spectral variants of green fluorescent protein showed that Mmip-2 and mad proteins reside in separate cytoplasmic and nuclear compartments, respectively . When co-expressed, however, the proteins interact and translocate to the cellular compartment occupied by the more abundant protein . These observations suggest a novel way by which Mmip-2 can modulate the transcriptional activity of myc oncoproteins.

Mol Cells, 1999 Oct 31, 9(5), 564 - 8
Selection of Drosophila genes encoding secreted and membrane proteins; Goo JH et al.; With the use of a yeast-based signal sequence trap (YSST) method, we screened a Drosophila cDNA library to isolate genes encoding secreted and membrane proteins . Of the 136 unique cDNA clones sequenced, 11 clones (8.1%) are identical to previously known Drosophila genes, 18 clones (13.2%) are homologous to other genes identified in various organisms, and 91 clones (66.9%) are novel . Most of these genes are secreted or membrane proteins, or appear to contain putative signal sequences at their amino termini . This indicates that YSST is an effective tool for the isolation and analysis of Drosophila genes that play roles in intercellular communication.

J Agric Food Chem, 1999 Aug, 47(8), 3297 - 302
Response of the aroma fraction in sherry wines subjected to accelerated biological aging; Cortes MB et al.; The effect of an acceleration assay, carried out with a periodic aeration and an increased surface/volume ratio, on various aroma compounds of "fino" Sherry wines aging under a veil of a pure culture of Saccharomyces cerevisiae race capensis G1 flor film yeast was studied . The results were subjected to multifactor analysis of variance, and the compounds simultaneously depending on acceleration conditions and aging time at p < 0.01 were subjected to principal component analysis . The first component, accounting for 86.14% of the overall variance, was mainly defined by acetaldehyde and its derivatives 1,1-diethoxyethane and acetoin . These compounds reached higher concentrations in accelerated aging wines in a shorter time than they did in control wines, and no browning problems were detected . Taking into account that these compounds can be used as indicators for biological aging of "fino" Sherry wines, the acceleration condition assayed can be applied to shorten the time of this process.

Mol Microbiol, 1999 Dec, 34(5), 1007 - 17
The MAP kinase kpp2 regulates mating and pathogenic development in Ustilago maydis; Muller P et al.; In the phytopathogenic fungus Ustilago maydis, fusion of compatible haploid cells is a prerequisite for infection . This process is genetically controlled by the biallelic a locus, encoding pheromone precursors and receptors . These are presumed to be coupled to a heterotrimeric G protein and a MAP kinase cascade, leading to activation of the HMG domain transcription factor Prf1 . Here, we have demonstrated that putative MAP kinase sites in Prf1 are required for its activity during mating . In addition, we have identified a gene, kpp2, which encodes a putative MAP kinase related to Pmk1 of Magnaporthe grisea and Fus3p of Saccharomyces cerevisiae . kpp2 deletion mutants are attenuated in several steps of development: cell fusion, induction of pheromone-responsive genes and pathogenicity . Epistasis analysis shows that kpp2 does not affect pheromone gene expression through the cAMP signalling cascade . Pathogenicity of kpp2 mutants can be partially restored by overexpressing the b genes, indicating a regulation of Prf1 by Kpp2 . These data support the hypothesis that the MAP kinase Kpp2 transmits the pheromone signal.

Nat Struct Biol, 1999 Nov, 6(11), 1039 - 42
Solution structure of the hRPABC14.4 subunit of human RNA polymerases; del Rio-Portilla F et al.; The protein hRPABC14.4 is an essential subunit of human RNA polymerases I, II, and III and is required for the transcription of all human nuclear genes . The structure of hRPABC14.4 was determined by nuclear magnetic resonance spectroscopy . The protein fold comprises a highly conserved central domain forming two antiparallel alpha-helices flanked by the less conserved N- and C-terminal regions forming a five-stranded beta-sandwich . Amino acids from the two helices participate in the generation of a hydrophobic surface area which is conserved in all eukaryotic and archaeal homologous subunits, and likely constitutes a critical macromolecular interaction interface . The hRPABC14.4 structure accounts for mutagenesis results in Saccharomyces cerevisiae and provides a structural working model for elucidating the role of this subunit in the molecular architecture and function of the human nuclear RNA polymerases.

Nat Struct Biol, 1999 Nov, 6(11), 1029 - 32
Crystal structure of the post-chaperonin beta-tubulin binding cofactor Rbl2p; Steinbacher S; The folding pathway of tubulins includes highly specific interactions with a series of cofactors (A, B, C, D and E) after they are released from the eukaryotic chaperonin CCT . The 2.2 A crystal structure of Rbl2p, the Saccharomyces cerevisiae homolog of beta-tubulin specific cofactor A, shows alpha-helical monomers forming a flat, slightly convex dimer . The surface of the molecule is dominated by polar and charged residues and lacks hydrophobic patches typically observed for chaperones that bind unfolded or partially folded proteins . This post-chaperonin cofactor is therefore clearly distinct from typical chaperones where hydrophobicity is a hallmark of substrate recognition.

Nat Struct Biol, 1999 Nov, 6(11), 990 - 1
A chaperone with a hydrophilic surface; Cowan NJ et al.; The folding of native tubulin involves at least seven different chaperone proteins: prefoldin, the cytosolic chaperonin CCT and five tubulin-specific chaperone proteins named cofactors A-E . The structure of the yeast homolog of cofactor A, Rbl2p, shows it to be a dimer with largely hydrophilic surfaces, reflecting the fact that it interacts with quasi-native, not unfolded, beta-tubulin.

J Microsc, 1999 Dec, 196 ( Pt 3), 279 - 87
Biological ultrastructure as revealed by high resolution cryo-SEM of block faces after cryo-sectioning; Walther P et al.; Ultrastructural information was obtained by imaging the block face of high-pressure-frozen cryo-sectioned biological samples in a high-resolution cryo-SEM . Cryo-sectioning leads to a well-defined flat artificial surface in contrast to cryo-fracturing . Typical artefacts of cryo-sections such as compression and crevasses were not visible on the block face . The ultrastructural features known from resin sections and from freeze-fractures could also be found on the block faces . The cytoplasms show particles of different size which most likely represent proteins . The effects of radiation damage could be reduced considerably by applying the double layer coating technique and backscattered electron imaging . High quality cryo-sections are only obtained from vitrified material . Reasonably flat block faces were, however, also obtained from adequately frozen microcrystalline samples, thereby facilitating ultrastructural studies in the frozen hydrated state.

Plant Physiol, 1999 Dec, 121(4), 1127 - 42
Stop-and-go movements of plant Golgi stacks are mediated by the acto-myosin system; Nebenfuhr A et al.; The Golgi apparatus in plant cells consists of a large number of independent Golgi stack/trans-Golgi network/Golgi matrix units that appear to be randomly distributed throughout the cytoplasm . To study the dynamic behavior of these Golgi units in living plant cells, we have cloned a cDNA from soybean (Glycine max), GmMan1, encoding the resident Golgi protein alpha-1,2 mannosidase I . The predicted protein of approximately 65 kD shows similarity of general structure and sequence (45% identity) to class I animal and fungal alpha-1,2 mannosidases . Expression of a GmMan1::green fluorescent protein fusion construct in tobacco (Nicotiana tabacum) Bright Yellow 2 suspension-cultured cells revealed the presence of several hundred to thousands of fluorescent spots . Immuno-electron microscopy demonstrates that these spots correspond to individual Golgi stacks and that the fusion protein is largely confined to the cis-side of the stacks . In living cells, the stacks carry out stop-and-go movements, oscillating rapidly between directed movement and random "wiggling." Directed movement (maximal velocity 4.2 microm/s) is related to cytoplasmic streaming, occurs along straight trajectories, and is dependent upon intact actin microfilaments and myosin motors, since treatment with cytochalasin D or butanedione monoxime blocks the streaming motion . In contrast, microtubule-disrupting drugs appear to have a small but reproducible stimulatory effect on streaming behavior . We present a model that postulates that the stop-and-go motion of Golgi-trans-Golgi network units is regulated by "stop signals" produced by endoplasmic reticulum export sites and locally expanding cell wall domains to optimize endoplasmic reticulum to Golgi and Golgi to cell wall trafficking.

Mol Cell Biol, 2000 Jan, 20(1), 286 - 98
Essential function of the polo box of Cdc5 in subcellular localization and induction of cytokinetic structures; Song S et al.; Members of the polo subfamily of protein kinases play pivotal roles in cell proliferation . In addition to the kinase domain, polo kinases have a strikingly conserved sequence in the noncatalytic C-terminal domain, termed the polo box . Here we show that the budding-yeast polo kinase Cdc5, when fused to green fluorescent protein and expressed under its endogenous promoter, localizes at spindle poles and the mother bud neck . Overexpression of Cdc5 can induce a class of cells with abnormally elongated buds in a polo box- and kinase activity-dependent manner . In addition to localizing at the spindle poles and cytokinetic neck filaments, Cdc5 induces and localizes to additional septin ring structures within the elongated buds . Without impairing kinase activity, conservative mutations in the polo box abolish the ability of Cdc5 to functionally complement the defect associated with a cdc5-1 temperature-sensitive mutation, to localize to the spindle poles and cytokinetic neck filaments, and to induce elongated cells with ectopic septin ring structures . Consistent with the polo box-dependent subcellular localization, the C-terminal domain of Cdc5, but not its polo box mutant, is sufficient for subcellular localization, and its overexpression appears to inhibit cytokinesis . These data provide evidence that the polo box is required to direct Cdc5 to specific subcellular locations and induce or organize cytokinetic structures.

Mol Cell Biol, 2000 Jan, 20(1), 242 - 8
Dbf4p, an essential S phase-promoting factor, is targeted for degradation by the anaphase-promoting complex; Ferreira MF et al.; The Dbf4p/Cdc7p protein kinase is essential for the activation of replication origins during S phase . The catalytic subunit, Cdc7p, is present at constant levels throughout the cell cycle . In contrast, we show here that the levels of the regulatory subunit, Dbf4p, oscillate during the cell cycle . Dbf4p is absent from cells during G(1) and accumulates during the S and G(2) phases . Dbf4p is rapidly degraded at the time of chromosome segregation and remains highly unstable during pre-Start G(1) phase . The rapid degradation of Dbf4p during G(1) requires a functional anaphase-promoting complex (APC) . Mutation of a sequence in the N terminus of Dbf4p which resembles the cyclin destruction box eliminates this APC-dependent degradation of Dbf4p . We suggest that the coupling of Dbf4p degradation to chromosome separation may play a redundant role in ensuring that prereplicative complexes, which assemble after chromosome segregation, do not immediately refire.

Mol Cell Biol, 2000 Jan, 20(1), 104 - 12
Kin28, the TFIIH-associated carboxy-terminal domain kinase, facilitates the recruitment of mRNA processing machinery to RNA polymerase II; Rodriguez CR et al.; The cotranscriptional placement of the 7-methylguanosine cap on pre-mRNA is mediated by recruitment of capping enzyme to the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II . Immunoblotting suggests that the capping enzyme guanylyltransferase (Ceg1) is stabilized in vivo by its interaction with the CTD and that serine 5, the major site of phosphorylation within the CTD heptamer consensus YSPTSPS, is particularly important . We sought to identify the CTD kinase responsible for capping enzyme targeting . The candidate kinases Kin28-Ccl1, CTDK1, and Srb10-Srb11 can each phosphorylate a glutathione S-transferase-CTD fusion protein such that capping enzyme can bind in vitro . However, kin28 mutant alleles cause reduced Ceg1 levels in vivo and exhibit genetic interactions with a mutant ceg1 allele, while srb10 or ctk1 deletions do not . Therefore, only the TFIIH-associated CTD kinase Kin28 appears necessary for proper capping enzyme targeting in vivo . Interestingly, levels of the polyadenylation factor Pta1 are also reduced in kin28 mutants, while several other polyadenylation factors remain stable . Pta1 in yeast extracts binds specifically to the phosphorylated CTD, suggesting that this interaction may mediate coupling of polyadenylation and transcription.

Mol Cell Biol, 2000 Jan, 20(1), 1 - 11
Scanning mutagenesis of Mcm1: residues required for DNA binding, DNA bending, and transcriptional activation by a MADS-box protein; Acton TB et al.; MCM1 is an essential gene in the yeast Saccharomyces cerevisiae and is a member of the MADS-box family of transcriptional regulatory factors . To understand the nature of the protein-DNA interactions of this class of proteins, we have made a series of alanine substitutions in the DNA-binding domain of Mcm1 and examined the effects of these mutations in vivo and in vitro . Our results indicate which residues of Mcm1 are important for viability, transcriptional activation, and DNA binding and bending . Substitution of residues in Mcm1 which are highly conserved among the MADS-box proteins are lethal to the cell and abolish DNA binding in vitro . These positions have almost identical interactions with DNA in both the serum response factor-DNA and alpha2-Mcm1-DNA crystal structures, suggesting that these residues make up a conserved core of protein-DNA interactions responsible for docking MADS-box proteins to DNA . Substitution of residues which are not as well conserved among members of the MADS-box family play important roles in contributing to the specificity of DNA binding . These results suggest a general model of how MADS-box proteins recognize and bind DNA . We also provide evidence that the N-terminal extension of Mcm1 may have considerable conformational freedom, possibly to allow binding to different DNA sites . Finally, we have identified two mutants at positions which are critical for Mcm1-mediated DNA bending that have a slow-growth phenotype . This finding is consistent with our earlier results, indicating that DNA bending may have a role in Mcm1 function in the cell.

J Biol Chem, 1999 Dec 17, 274(51), 36428 - 38
An SC35-like protein and a novel serine/arginine-rich protein interact with Arabidopsis U1-70K protein; Golovkin M et al.; The U1 small nuclear ribonucleoprotein 70-kDa protein, a U1 small nuclear ribonucleoprotein-specific protein, has been shown to have multiple roles in nuclear precursor mRNA processing in animals . By using the C-terminal arginine-rich region of Arabidopsis U1-70K protein in the yeast two-hybrid system, we have identified an SC35-like (SR33) and a novel plant serine/arginine-rich (SR) protein (SR45) that interact with the plant U1-70K . The SR33 and SR45 proteins share several features with SR proteins including modular domains typical of splicing factors in the SR family of proteins . However, both plant SR proteins are rich in proline, and SR45, unlike most animal SR proteins, has two distinct arginine/serine-rich domains separated by an RNA recognition motif . By using coprecipitation assays we confirmed the interaction of plant U1-70K with SR33 and SR45 proteins . Furthermore, in vivo and in vitro protein-protein interaction experiments have shown that SR33 protein interacts with itself and with SR45 protein but not with two other members (SRZ21 and SRZ22) of the SR family that are known to interact with the Arabidopsis full-length U-70K only . A Clk/Sty protein kinase (AFC-2) from Arabidopsis phosphorylated four SR proteins (SR33, SR45, SRZ21, and SRZ22) . Coprecipitation studies have confirmed the interaction of SR proteins with AFC2 kinase, and the interaction between AFC2 and SR33 is modulated by the phosphorylation status of these proteins . These and our previous results suggest that the plant U1-70K interacts with at least four distinct members of the SR family including SR45 with its two arginine/serine-rich domains, and the interaction between the SR proteins and AFC2 is modulated by phosphorylation . The interaction of plant U1-70K with a novel set of proteins suggests the early stages of spliceosome assembly, and intron recognition in plants is likely to be different from animals.

Curr Genet, 1999 Oct, 36(4), 222 - 31
Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes; Poggeler S; To gain a deeper insight into the evolution of mating-type genes from filamentous ascomycetes, a comprehensive sequence analysis of PCR-amplified sequences corresponding to A- and a-specific mating-type sequences was undertaken . The study included nine homothallic (compatible) and eight heterothallic (incompatible) members of the genera Neurospora and Sordaria . Distance and parsimony trees based on gene fragments from the mat a-1 and mat A-1 genes were compared with trees derived from partial DNA sequences of the gpd glyceraldehyde-3-phosphate dehydrogenase gene . In contrast to the sequences from the gpd gene, mating-type genes show striking sequence differences, suggesting that these genes evolve very rapidly . Strong inter-relationships were found among homothallic, as well as among heterothallic, members of both genera, indicating that in each genus a change from one reproductive strategy to another might result from one single event.

Nucleic Acids Res, 2000 Jan 1, 28(1), 148 - 52
MitBASE : a comprehensive and integrated mitochondrial DNA database . The present status; Attimonelli M et al.; MitBASE is an integrated and comprehensive database of mitochondrial DNA data which collects, under a single interface, databases for Plant, Vertebrate, Invertebrate, Human, Protist and Fungal mtDNA and a Pilot database on nuclear genes involved in mitochondrial biogenesis in Saccharomyces cerevisiae . MitBASE reports all available information from different organisms and from intraspecies variants and mutants . Data have been drawn from the primary databases and from the literature; value adding information has been structured, e.g., editing information on protist mtDNA genomes, pathological information for human mtDNA variants, etc . The different databases, some of which are structured using commercial packages (Microsoft Access, File Maker Pro) while others use a flat-file format, have been integrated under ORACLE . Ad hoc retrieval systems have been devised for some of the above listed databases keeping into account their peculiarities . The database is resident at the EBI and is available at the following site: e.pl . The impact of this project is intended for both basic and applied research . The study of mitochondrial genetic diseases and mitochondrial DNA intraspecies diversity are key topics in several biotechnological fields . The database has been funded within the EU Biotechnology programme.

Nucleic Acids Res, 2000 Jan 1, 28(1), 120 - 2
XREFdb: cross-referencing the genetics and genes of mammals and model organisms; Ploger R et al.; XREFdb supports the investigation of protein function in the context of information available through work in multiple organisms . In addition to facilitating the association of functional data among known genes from multiple organisms, XREFdb has developed strategies that provide access to information associated with as-yet unstudied genes . The database organizes protein similarity and genetic map positional information from diverse sources in the public domain to facilitate investigator evaluation of potential functional significance . XREFdb is found at URL www.ncbi.nlm.nih.gov/XREFdb

Nucleic Acids Res, 2000 Jan 1, 28(1), 37 - 40
MIPS: a database for genomes and protein sequences; Mewes HW et al.; The Munich Information Center for Protein Sequences (MIPS-GSF), Martinsried, near Munich, Germany, continues its longstanding tradition to develop and maintain high quality curated genome databases . In addition, efforts have been intensified to cover the wealth of complete genome sequences in a systematic, comprehensive form . Bioinformatics, supporting national as well as European sequencing and functional analysis projects, has resulted in several up-to-date genome-oriented databases . This report describes growing databases reflecting the progress of sequencing the Arabidopsis thaliana (MATDB) and Neurospora crassa genomes (MNCDB), the yeast genome database (MYGD) extended by functional analysis data, the database of annotated human EST-clusters (HIB) and the database of the complete cDNA sequences from the DHGP (German Human Genome Project) . It also contains information on the up-to-date database of complete genomes (PEDANT), the classification of protein sequences (ProtFam) and the collection of protein sequence data within the framework of the PIR-International Protein Sequence Database . These databases can be accessed through the MIPS WWW server .

Nature, 1999 Dec 2, 402(6761), 555 - 60
The RCAF complex mediates chromatin assembly during DNA replication and repair; Tyler JK et al.; Chromatin assembly is a fundamental biological process that is essential for the replication and maintenance of the eukaryotic genome . In dividing cells, newly synthesized DNA is rapidly assembled into chromatin by the deposition of a tetramer of the histone proteins H3 and H4, followed by the deposition of two dimers of histones H2A and H2B to complete the nucleosome-the fundamental repeating unit of chromatin . Here we describe the identification, purification, cloning, and characterization of replication-coupling assembly factor (RCAF), a novel protein complex that facilitates the assembly of nucleosomes onto newly replicated DNA in vitro . RCAF comprises the Drosophila homologue of anti-silencing function 1 protein ASF1 and histones H3 and H4 . The specific acetylation pattern of H3 and H4 in RCAF is identical to that of newly synthesized histones . Genetic analyses in Saccharomyces cerevisiae demonstrate that ASF1 is essential for normal cell cycle progression, and suggest that RCAF mediates chromatin assembly after DNA replication and the repair of double-strand DNA damage in vivo.

Yakugaku Zasshi, 1999 Nov, 119(11), 822 - 34
{Analysis of xenobiotic detoxification system mediated by efflux transporters}; Suzuki H; The excretion of drugs mediated by transporters plays an important role in the detoxification of xenobiotics . In this article, I will summarize recent progress we have made in this field, particularly focusing on the roles of transporters responsible for exporting drugs . As far as the biliary excretion of xenobiotics is concerned, it has been suggested that canalicular multispecific organic anion transporter/multidrug resistance associated protein 2 (cMOAT/MRP2) is involved in the ATP-dependent export of organic anions across the bile canalicular membrane . By comparing the transport across this membrane between normal rats and Eisai hyperbilirubinemic rats whose cMOAT/MRP2 function is hereditarily defective, we were able to demonstrate the substrate specificity of cMOAT/MRP2 . This includes non-conjugated anionic drugs, and glutathione- and glucuronide-conjugates of xenobiotics . The role of cMOAT/MRP2 in drug disposition has also been clarified . Moreover, the cDNA of cMOAT/MRP2 has been cloned and its functional analysis has been completed . Thus, it may be possible to predict in vivo transport across the bile canalicular membrane from in vitro data using the recombinant transporter . We also cloned MRP3 as an inducible transporter in the liver under the cholestatic conditions . Although MRP3 mediates the cellular export of non-conjugated organic anions and glucuronide-conjugates, the substrate specificity of MRP3 is different from that of cMOAT/MRP2 in that glutathione-conjugates are poor substrates for MRP3 . It is possible that MRP3 plays an important role under certain pathological conditions in the liver . Since it has been shown that cMOAT/MRP2 and MRP 3 are expressed in the small intestine under physiological conditions, it seems reasonable that these transporters are responsible for the previously reported cellular extrusion of organic anions . We also found that there was MRP activity in the blood-brain and blood-cerebrospinal fluid barriers . RT-PCR resulted in the amplification of MRP1, 5 and 6 from freshly isolated rat cerebral endothelial cells . It has been suggested that there is basolateral localization of MRP1 in the choroid plexus . In conjunction with the P-glycoprotein located on the luminal membrane of cerebral endothelial cells, these transporters play significant roles in restricting the entry of xenobiotics from the circulating blood into the central nervous system . Regulation of the activity of these efflux transporters allows the disposition of drugs to be altered.

Adv Biochem Eng Biotechnol, 2000, 66, 209 - 31
Metabolic network analysis . A powerful tool in metabolic engineering; Christensen B et al.; Metabolic network analysis is a tool for investigating the features that identify the topology of a metabolic network and the relative activities of its individual branches . The pillars of metabolic network analysis are mathematical modeling, allowing for a quantitative analysis, biochemical knowledge of, for example, reaction stoichiometry, and the experimental techniques, providing input for the modeling part . The modeling part includes metabolite balancing, usually the basis for metabolic flux analysis, and isotope balancing . Isotope balancing can be used for both identification of active pathways and for estimation of the relative fluxes through two pathways leading to the same metabolite, aspects that are difficult to investigate using metabolite balancing . The combination of metabolite and isotope balancing is very powerful and constitutes the basis of metabolic network analysis . With the main focus being on investigating the metabolic network structure, this review describes how central metabolic features, for example, pathway identification, flux distribution, and compartmentation, can be addressed using a combination of metabolite balancing and labeling experiments.

Mutat Res, 1999 Nov 29, 430(1), 131 - 44
Codon 249 of the human TP53 tumor suppressor gene is no hot spot for aflatoxin B1 in a heterologous background; Sengstag C et al.; Mutations in the TP53 tumor suppressor gene are the most common alteration in cancer, and human primary liver cancers related to previous dietary exposure to the mycotoxin aflatoxin B1 (AFB1) exhibit a specific hot spot mutation at TP53 codon 249 . We have asked whether the 249 hot spot is related to a particular susceptibility to AFB1 of this TP53 region or whether it is related to a phenotype of the 249S p53 mutant protein . This was addressed by constructing a metabolically competent variant of Saccharomyces cerevisiae strain yIG397 expressing human cytochrome P450 1A2 and P450-reductase and isolating AFB1-induced mutants that failed to express the genomic ADE2 reporter gene . Molecular analysis revealed that only 8/40 mutants had a mutation in the TP53 target gene, whereas 32/40 mutants were due to a recombination event eliminating the ADE2 reporter gene . None of 19 mutations identified in the eight mutant TP53 plasmids altered codon 249, thus this codon was no hot spot if the TP53 gene was in the heterologous background yeast . The genotoxic action of AFB1 was completely different from that of the alkylating agent ethyl-methane-sulfonate, where 28/30 induced mutations were linked to the TP53 target gene.

Yeast, 1999 Dec, 15(16), 1761 - 8
New tools for protein linkage mapping and general two-hybrid screening; Durfee T et al.; The two-hybrid system has proved to be a facile method for detecting and analyzing protein-protein interactions . An expanded application of this system, protein linkage mapping, provides a means of identifying interactions on a global scale and should prove a powerful tool in analyzing whole genomes as their sequences become available . To overcome some of the inherent difficulties in such a large-scale approach, we have constructed a set of new strains and vectors that will allow for more efficient screening . The strains contain a GAL1-URA3 reporter for positive and negative selection, as well as a UAS(G)-lacZ reporter . The strains are of opposite mating types, permitting libraries present in one strain to be easily screened against a second library in the companion strain . We also constructed a family of CEN-based vectors for expression of both Gal4 DNA-binding and activation domain fusions . These plasmids include a hemagglutinin epitope tag and different polylinkers to increase the ease of subcloning . CEN-based vectors are maintained at 1-2 copies per cell, limiting the number of individual cells containing multiple plasmids that can confuse further analyses, and ensuring that fusions are not expressed at toxic levels . Using these vectors, both homo- and heterodimeric interactions resulted in up to 10-fold higher reporter gene transcription than obtained with 2micro;-based plasmids, despite significantly lower protein levels . In addition to protein linkage mapping, these reagents should be generally useful in standard two-hybrid applications .

Mol Biol Cell, 1999 Dec, 10(12), 4149 - 61
Promiscuity in Rab-SNARE interactions; Grote E et al.; Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins . We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles . Assembly of Sncp and Ssop into a SNARE complex is defective in a sec4-8 mutant strain . However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8 cells . In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles . In an alternate approach to look for selective Rab-SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop . However, Rab-SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp . We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.

Mol Biol Cell, 1999 Dec, 10(12), 4121 - 33
The Rho GTPase Rho3 has a direct role in exocytosis that is distinct from its role in actin polarity; Adamo JE et al.; Budding yeast grow asymmetrically by the polarized delivery of proteins and lipids to specific sites on the plasma membrane . This requires the coordinated polarization of the actin cytoskeleton and the secretory apparatus . We identified Rho3 on the basis of its genetic interactions with several late-acting secretory genes . Mutational analysis of the Rho3 effector domain reveals three distinct functions in cell polarity: regulation of actin polarity, transport of exocytic vesicles from the mother cell to the bud, and docking and fusion of vesicles with the plasma membrane . We provide evidence that the vesicle delivery function of Rho3 is mediated by the unconventional myosin Myo2 and that the docking and fusion function is mediated by the exocyst component Exo70 . These data suggest that Rho3 acts as a key regulator of cell polarity and exocytosis, coordinating several distinct events for delivery of proteins to specific sites on the cell surface.

Nat Cell Biol, 1999 Dec, 1(8), 514 - 21
Components of the spindle-assembly checkpoint are essential in Caenorhabditis elegans; Kitagawa R et al.; The spindle-assembly checkpoint ensures that, during mitosis and meiosis, chromosomes do not segregate until they are properly attached to the microtubules of the spindle . Here we show that mdf-1 and mdf-2 are components of the spindle-assembly checkpoint in Caenorhabditis elegans, and are essential for the long-term survival and fertility of this organism . Loss of function of either of these genes leads to the accumulation of a variety of defects, including chromosome abnormalities, X-chromosome non-disjunction or loss, problems in gonad development, and embryonic lethality . Antibodies that recognize the MDF-2 protein localize to nuclei of the cleaving embryo in a cell-cycle-dependent manner . mdf-1, a gene encoding a product that interacts with MDF-2, is required for cell-cycle arrest and proper chromosome segregation in premeiotic germ cells treated with nocodazole, a microtubule-depolymerizing agent . In the absence of mdf gene products, errors in chromosome segregation arise and accumulate, ultimately leading to genetic lethality.

Biophys J, 1999 Dec, 77(6), 3293 - 304
Sampling field heterogeneity at the heme of c-type cytochromes by spectral hole burning spectroscopy and electrostatic calculations; Laberge M et al.; We report on a comparative investigation of the heme pocket fields of two Zn-substituted c-type cytochromes-namely yeast and horse heart cytochromes c-using a combination of hole burning Stark spectroscopy and electrostatic calculations . The spectral hole burning experiments are consistent with different pocket fields experienced at the hemes of the respective cytochromes . In the case of horse heart Zn-cytochrome c, two distinguishable electronic origins with different electrostatic properties are observed . The yeast species, on the other hand, displays a single electronic origin . Electrostatic calculations and graphics modeling using the linearized finite-difference Poisson-Boltzmann equation performed at selected time intervals on nanosecond-molecular dynamics trajectories show that the hemes of the respective cytochromes sample different potentials as they explore conformational space . The electrostatic potentials generated by the protein matrix at the heme show different patterns in both cytochromes, and we suggest that the cytochromes differ by the number of "electrostatic substates" that they can sample, thus accounting for the different spectral populations observed in the two cytochromes.

Biochem J, 1999 Dec 15, 344 Pt 3, 903 - 14
Function of human mitochondrial 2,4-dienoyl-CoA reductase and rat monofunctional Delta3-Delta2-enoyl-CoA isomerase in beta-oxidation of unsaturated fatty acids; Gurvitz A et al.; Human 2,4-dienoyl-CoA reductase (2,4-reductase; DECR) and rat monofunctional Delta(3)-Delta(2)-enoyl-CoA isomerase (rat 3, 2-isomerase; ECI) are thought to be mitochondrial auxiliary enzymes involved in the beta-oxidation of unsaturated fatty acids . However, their function during this process has not been demonstrated . Although they lack obvious peroxisomal targeting signals (PTSs), both proteins have been suggested previously to also occur in the mammalian peroxisomal compartment . The putative function and peroxisomal location of the two mammalian proteins can be examined in yeast, since beta-oxidation of unsaturated fatty acids is a compartmentalized process in Saccharomyces cerevisiae requiring peroxisomal 2,4-dienoyl-CoA reductase (Sps19p) and peroxisomal 3, 2-isomerase (Eci1p) . A yeast sps19Delta mutant expressing human 2, 4-reductase ending with the native C-terminus could not grow on petroselinic acid {cis-C(18:1(6))} medium but could grow when the protein was extended with a PTS tripeptide, SKL (Ser-Lys-Leu) . We therefore reason that the human protein is a physiological 2, 4-reductase but that it is probably not peroxisomal . Rat 3, 2-isomerase expressed in a yeast eci1Delta strain was able to re-establish growth on oleic acid {cis-C(18:1(9))} medium irrespective of an SKL extension . Since we had shown that Delta(2,4) double bonds could not be metabolized extra-peroxisomally to restore growth of the sps19Delta strain, we postulate that rat 3,2-isomerase acted on the Delta(3) unsaturated metabolite of oleic acid by replacing the mutant's missing activity from within the peroxisomes . Immunoblotting of fractionated yeast cells expressing rat 3, 2-isomerase in combination with electron microscopy supported our proposal that the protein functioned in peroxisomes . The results presented here shed new light on the function and location of human mitochondrial 2,4-reductase and rat monofunctional 3,2-isomerase.

Anal Biochem, 1999 Dec 1, 276(1), 18 - 26
Usefulness of statistic experimental designs in enzymology: example with recombinant hCYP3A4 and 1A2; Bournique B et al.; First, the effects of 10 incubation factors were screened altogether on nifedipine dehydrogenase (NIF) and methoxyresorufin O-deethylase (MROD) activities catalyzed by recombinant human CYP3A4 and 1A2, respectively . Using a statistic experimental design, only 36 assays were needed to be exhaustive . Eight factors influenced CYP3A4-mediated NIF activity: buffer type, pH, temperature, Mg/EDTA, cytochrome b5, NADPH-P450 reductase, NADH, and solvent . Two factors had no significant effect: total ionic concentration and catalase/reduced glutathione . Six factors influenced CYP1A2-mediated MROD rates: buffer type, pH, temperature, Mg/EDTA, NADH, and glycerol . Four factors had no significant effect: total ionic concentration, cytochrome b5, reductase, and NAD+ . Secondly, the combined effects of ionic strength and Mg concentration on NIF/CYP3A4 were studied and easily modeled using another statistic experimental design . The effect of Mg was strong at a constant ionic strength of 100 mM and negligible at a constant ionic strength of 500 mM . Thirdly, the effects of influencing factors and their interactions on MROD/CYP1A2 were modeled after 40 assays using a third statistic experimental design . Later experiments confirmed the predictivity of the models and the efficiency of optimized conditions . This approach can be applied to other biochemistry areas .

Mol Gen Genet, 1999 Oct, 262(3), 426 - 36
A mutation in a mitochondrial ABC transporter results in mitochondrial dysfunction through oxidative damage of mitochondrial DNA; Senbongi H et al.; We have isolated a Saccharomyces cerevisiae mutant that shows an increased tendency to form cytoplasmic petites (respiration-deficient rho- or rho0 mutants) in response to treatment of cells growing on a solid medium with the DNA-damaging agent methyl methane-sulfonate or ultraviolet light . The mutation in this strain, atm1-1, was found to cause a single amino acid substitution in ATM1, a nuclear gene that encodes the mitochondrial ATP-binding cassette (ABC) transporter . When the mutant cells were grown in liquid glucose medium, they accumulated free iron within the mitochondria and at the same time gave rise to spontaneous cytoplasmic petite mutants, as seen previously in cells carrying a mutation in a gene homologous to the human gene responsible for Friedreich's ataxia . Analysis of the effects of free iron and malonic acid (an inhibitor of oxidative respiration in mitochondria) on the incidence of petites among the mutant cells indicated that spontaneous induction of petites was a consequence of oxidative stress rather than a direct effect of either a defect in the ATM1 gene or the accumulation of free iron . We observed an increase in the incidence of strand breaks in the mitochondrial DNA of the atm1-1 mutant cells . Furthermore, we found that rates of induction of petites and accumulation of strand breaks in mitochondrial DNA were enhanced in the atm1-1 mutant by the introduction of another mutation, mhr1-1, which results in a deficiency in mitochondrial DNA repair . These observations indicate that spontaneous induction of petites in the atm1-1 mutant is a consequence of oxidative damage to mitochondrial DNA mediated by enhanced accumulation of mitochondrial iron.

J Lipid Res, 1999 Dec, 40(12), 2244 - 54
Phytanoyl-CoA hydroxylase from rat liver . Protein purification and cDNA cloning with implications for the subcellular localization of phytanic acid alpha-oxidation; Jansen GA et al.; Phytanoyl-CoA hydroxylase (PhyH) catalyzes the conversion of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA, which is the first step in the phytanic acid alpha-oxidation pathway . Recently, several studies have shown that in humans, phytanic acid alpha-oxidation is localized in peroxisomes . In rat, however, the alpha-oxidation pathway has been reported to be mitochondrial . In order to clarify this differential subcellular distribution, we have studied the rat PhyH protein . We have purified PhyH from rat liver to apparent homogeneity as judged by SDS-PAGE . Sequence analysis of two PhyH peptide fragments allowed cloning of the rat PHYH cDNA encoding a 38 . 6 kDa protein . The deduced amino acid sequence revealed strong homology to human PhyH including the presence of a peroxisome targeting signal type 2 (PTS2) . Heterologous expression of rat PHYH in Saccharomyces cerevisiae yielded a 38.6 kDa protein whereas the PhyH purified from rat liver had a molecular mass of 35 kDa . This indicates that PhyH is probably processed in rat by proteolytic removal of a leader sequence containing the PTS2 . This type of processing has been reported in several other peroxisomal proteins that contain a PTS2 . Subcellular localization studies using equilibrium density centrifugation showed that PhyH is indeed a peroxisomal protein in rat . The finding that PhyH is peroxisomal in both rat and humans provides strong evidence against the concept of a differential subcellular localization of phytanic acid alpha-oxidation in rat and human.

J Lipid Res, 1999 Dec, 40(12), 2204 - 11
Family of human oxysterol binding protein (OSBP) homologues . A novel member implicated in brain sterol metabolism; Laitinen S et al.; Oxysterol binding protein (OSBP) is a cytosolic protein that undergoes ligand-induced binding to the Golgi apparatus and has been implicated in the regulation of cellular cholesterol metabolism . In the yeast Saccharomyces cerevisiae an OSBP homologue is involved in membrane trafficking through the Golgi complex . Prompted by the multitude of OSBP-related genes in the yeast genome, we carried out a search for human expressed sequence tags (ESTs) displaying homology to the sterol-binding domain of OSBP . This revealed a minimum of six novel OSBP-related proteins, designated ORP-1 to ORP-6 . ORP cDNA probes were generated by reverse transcription-PCR from human liver mRNA, and used for Northern blot analysis of human tissue transcript panels . This verified that each of them represents a different gene product and showed that they display distinct tissue-specific expression patterns . The ORP-1 and -2 mRNA expression levels were similar to or higher than that of OSBP while the ORP-3 to -6 mRNAs were detected at lower levels in specific tissues . The most abundantly expressed new gene, ORP-1, was transcribed at strikingly high levels in the cortical areas of human brain and displayed sterol-regulated expression in a cultured human neuroblastoma cell line . This indicates that ORP-1 may play an important role in maintaining the sterol balance in cells of the central nervous system . Together with OSBP, the identified gene products constitute a novel human protein family that may provide a link between organellar sterol status and membrane dynamics.

J Mol Biol, 1999 Dec 10, 294(4), 1041 - 9
Structure and function of a new phosphopeptide-binding domain containing the FHA2 of Rad53; Liao H et al.; The forkhead-associated (FHA) domain is a 55-75 amino acid residue module found in >20 proteins from yeast to human . It has been suggested to participate in signal transduction pathways, perhaps via protein-protein interactions involving recognition of phosphopeptides . Neither the structure nor the ligand of FHA is known . Yeast Rad53, a checkpoint protein involved in DNA damage response, contains two FHA domains, FHA1 (residues 66-116) and FHA2 (residues 601-664), the second of which recognizes phosphorylated Rad9 . We herein report the solution structure of an "FHA2-containing domain" of Rad53 (residues 573-730) . The structure consists of a beta-sandwich containing two antiparallel beta-sheets and a short, C-terminal alpha-helix . Binding experiments suggested that the FHA2-containing domain specifically recognizes pTyr and a pTyr-containing peptide from Rad9, and that the binding site involves residues highly conserved across FHA domains . The results, along with other recent reports, suggest that FHA domains could have pTyr and pSer/Thr dual specificity .

Dev Biol, 1999 Dec 1, 216(1), 210 - 29
Wingless modulates the effects of dominant negative notch molecules in the developing wing of Drosophila; Brennan K et al.; The development and patterning of the wing in Drosophila relies on a sequence of cell interactions molecularly driven by a number of ligands and receptors . Genetic analysis indicates that a receptor encoded by the Notch gene and a signal encoded by the wingless gene play a number of interdependent roles in this process and display very strong functional interactions . At certain times and places, during wing development, the expression of wingless requires Notch activity and that of its ligands Delta and Serrate . This has led to the proposal that all the interactions between Notch and wingless can be understood in terms of this regulatory relationship . Here we have tested this proposal by analysing interactions between Delta- and Serrate-activated Notch signalling and Wingless signalling during wing development and patterning . We find that the cell death caused by expressing dominant negative Notch molecules during wing development cannot be rescued by coexpressing Nintra . This suggests that the dominant negative Notch molecules cannot only disrupt Delta and Serrate signalling but can also disrupt signalling through another pathway . One possibility is the Wingless signalling pathway as the cell death caused by expressing dominant negative Notch molecules can be rescued by activating Wingless signalling . Furthermore, we observe that the outcome of the interactions between Notch and Wingless signalling differs when we activate Wingless signalling by expressing either Wingless itself or an activated form of the Armadillo . For example, the effect of expressing the activated form of Armadillo with a dominant negative Notch on the patterning of sense organ precursors in the wing resembles the effects of expressing Wingless alone . This result suggests that signalling activated by Wingless leads to two effects, a reduction of Notch signalling and an activation of Armadillo .

Proc Natl Acad Sci U S A, 1999 Dec 7, 96(25), 14647 - 51
Herbicide sensitivity determinant of wheat plastid acetyl-CoA carboxylase is located in a 400-amino acid fragment of the carboxyltransferase domain; Nikolskaya T et al.; A series of chimeral genes, consisting of the yeast GAL10 promoter, yeast ACC1 leader, wheat acetyl-CoA carboxylase (ACCase; EC 6.4.1.2) cDNA, and yeast ACC1 3'-tail, was used to complement a yeast ACC1 mutation . These genes encode a full-length plastid enzyme, with and without the putative chloroplast transit peptide, as well as five chimeric cytosolic/plastid proteins . Four of the genes, all containing at least half of the wheat cytosolic ACCase coding region at the 5'-end, complement the yeast mutation . Aryloxyphenoxypropionate and cyclohexanedione herbicides, at concentrations below 10 microM, inhibit the growth of haploid yeast strains that express two of the chimeric ACCases . This inhibition resembles the inhibition of wheat plastid ACCase observed in vitro and in vivo . The differential response to herbicides localizes the sensitivity determinant to the third quarter of the multidomain plastid ACCase . Sequence comparisons of different multidomain and multisubunit ACCases suggest that this region includes part of the carboxyltransferase domain, and therefore that the carboxyltransferase activity of ACCase (second half-reaction) is the target of the inhibitors . The highly sensitive yeast gene-replacement strains described here provide a convenient system to study herbicide interaction with the enzyme and a powerful screening system for new inhibitors.

FEMS Microbiol Lett, 1999 Dec 15, 181(2), 225 - 8
Isolation and transformation of uracil auxotrophs of the edible basidiomycete Pleurotus ostreatus; Kim BG et al.; Uracil auxotrophs of Pleurotus ostreatus were isolated using the selectable marker, resistance to 5'-fluoro-orotic acid (5'-FOA) . Two of the nine uracil auxotrophs obtained were transformed to prototrophy using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from Trichoderma reesei . Southern blot analyses of the transformants showed that the transforming DNA had integrated into the genome of the protoplasts . Using 2 x 10(7) protoplasts, this system gave a transformation efficiency of about 30 transformants per microg of DNA . Normal fruiting bodies were induced in the transformants by crossing them with wild-type monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to prototrophy . These results indicate the integrated DNA was stably inherited.

J Biol Chem, 1999 Dec 10, 274(50), 36009 - 14
The assembly of the CAAT-box binding complex at a photosynthesis gene promoter is regulated by light, cytokinin, and the stage of the plastids; Kusnetsov V et al.; A functionally important region in the promoter of the spinach photosynthesis gene AtpC, which encodes the subunit gamma of the chloroplast ATP synthase, is located immediately upstream of the CAAT-box . A single nucleotide exchange in this region (AAAATTCAAT --> AAGATCAAT) uncouples the expression of an AtpC promoter::uidA gene fusion from the regulation by light, cytokinin, and functional plastids and results in a high constitutive expression of the reporter gene . By screening an Arabidopsis thaliana expression library with a double-stranded wild-type oligonucleotide from this promoter region, we have isolated cDNAs from Arabidopsis libraries that code for plant homologs of the CAAT-box binding factor (CBF)-C . Binding occurs only in the presence of nuclear extracts, consistent with reports from metazoa CBFs that the subunits A and B in addition to C are required for the formation of the CBF-DNA complex . At least eight genes with homologies to CBF-C are present in the Arabidopsis genome; one of them exhibits striking similarities to the gene for the human global transcriptional repressor Drap1 . In gel mobility shift assays, low binding activity of CBF to the wild-type AtpC promoter sequence was observed with nuclear extracts from tissue with low AtpC expression levels, i.e . extracts from etiolated and photobleached seedlings, whereas high binding activity was detectable with extracts from tissues with high AtpC expression levels, i.e . extracts from light-grown seedlings and etiolated seedlings treated with cytokinin . Binding to the mutant sequence, which directs constitutive high level uidA expression in vivo, is significantly stronger than to the wild-type sequence . The data are consistent with the idea that the assembly of CBF at the AtpC promoter is regulated in response to light and cytokinin and that the low level of expression in etiolated and photobleached material is caused by an inhibitory effect . The structure/function relationships of the Arabidopsis CBFs are discussed in relation to their regulatory function in AtpC gene expression.

J Biol Chem, 1999 Dec 10, 274(50), 35999 - 6008
Involvement of a cellular glycolytic enzyme, phosphoglycerate kinase, in Sendai virus transcription; Ogino T et al.; In vitro mRNA synthesis of Sendai virus is almost entirely dependent on the addition of cellular proteins (host factors) . Previous studies indicated that the host factor activity from bovine brain was resolved into at least two complementary fractions, one of which may be tubulin . In this study, the host factor activity that stimulates the transcription in the presence of tubulin was further purified from bovine brain . This fraction was found to contain at least two complementary factors, and one of them was purified to a single polypeptide chain with an apparent M(r) of 46,000 (p46) . From the amino acid sequence, biochemical, and immunological analyses, p46 was identified as a glycolytic enzyme, phosphoglycerate kinase (PGK) . Purified native PGK from rabbit and yeast, and a recombinant human PGK substituted for p46 . Although, as previously suggested, tubulin was involved in the transcription initiation complex formation by being integrated into the complex, p46 and its complementary factor had little effect on the complex formation . On the other hand, when p46 and the complementary factor were added to the RNA chain elongation reaction from the isolated initiation complex formed with tubulin, mRNA synthesis was dramatically stimulated . The enzymatic activity per se of PGK did not seem to be required for its activity . West-Western blot analysis showed that PGK could directly interact with tubulin . These data suggest that PGK stimulates Sendai virus mRNA synthesis at the elongation step, probably through its interaction with tubulin in the initiation complex.

J Biol Chem, 1999 Dec 10, 274(50), 35991 - 8
Translational regulation of ribonucleotide reductase by eukaryotic initiation factor 4E links protein synthesis to the control of DNA replication; Abid MR et al.; Ribonucleotide reductase synthesizes dNDPs, a specific and limiting step in DNA synthesis, and can participate in neoplastic transformation when overexpressed . The small subunit (ribonucleotide reductase 2 (RNR2)) was cloned as a major product in a subtraction library from eukaryotic initiation factor 4E (eIF4E)-transformed cells (Chinese hamster ovary-4E (CHO-4E)) . CHO-4E cells have 20-40-fold elevated RNR2 protein, reflecting an increased distribution of RNR2 mRNA to the heavy polysomes . CHO-4E cells display an altered cell cycle with shortened S phase, similar to cells selected for RNR2 overexpression with hydroxyurea . The function of ribonucleotide reductase as a checkpoint component of S progression was studied in yeast in which elevated eIF4E rescued S-arrested rnr2-68(ts) cells, by increasing recruitment of its mRNA to polysomes . Crosses between rnr2-68(ts) and mutant eIF4E (cdc33-1(ts)) engendered conditional synthetic lethality, with extreme sensitivity to hydroxyurea and the microtubule depolymerizing agent, benomyl . The double mutant (cdc33-1 rnr2-68) also identified a unique terminal phenotype, arrested with small bud and a randomly distributed single nucleus, which is distinct from those of both parental single mutants . This phenotype defines eIF4E and RNR2 as determinants in an important cell cycle checkpoint, in early/mid-S phase . These results also provide a link between protein and DNA synthesis and provide an explanation for cell cycle alterations induced by elevated eIF4E.

J Biol Chem, 1999 Dec 10, 274(50), 35734 - 40
How an inhibitor of the HIV-I protease modulates proteasome activity; Schmidtke G et al.; The human immunodeficiency virus, type I protease inhibitor Ritonavir has been used successfully in AIDS therapy for 4 years . Clinical observations suggested that Ritonavir may exert a direct effect on the immune system unrelated to inhibition of the human immunodeficiency virus, type I protease . In fact, Ritonavir inhibited the major histocompatibility complex class I restricted presentation of several viral antigens at therapeutically relevant concentrations (5 microM) . In search of a molecular target we found that Ritonavir inhibited the chymotrypsin-like activity of the proteasome whereas the tryptic activity was enhanced . In this study we kinetically analyzed how Ritonavir modulates proteasome activity and what consequences this has on cellular functions of the proteasome . Ritonavir is a reversible effector of proteasome activity that protected the subunits MB-1 (X) and/or LMP7 from covalent active site modification with the vinyl sulfone inhibitor(125)I-NLVS, suggesting that they are the prime targets for competitive inhibition by Ritonavir . At low concentrations of Ritonavir (5 microM) cells were more sensitive to canavanine but proliferated normally whereas at higher concentrations (50 microM) protein degradation was affected, and the cell cycle was arrested in the G(1)/S phase . Ritonavir thus modulates antigen processing at concentrations at which vital cellular functions of the proteasome are not yet severely impeded . Proteasome modulators may hence qualify as therapeutics for the control of the cytotoxic immune response.

J Biol Chem, 1999 Dec 10, 274(50), 35583 - 90
Self-association of the alpha subunit of phosphorylase kinase as determined by two-hybrid screening; Ayers NA et al.; The structural organization of the (alphabetagammadelta)(4) phosphorylase kinase complex has been studied using the yeast two-hybrid screen for the purpose of elucidating regions of alpha subunit interactions . By screening a rabbit skeletal muscle cDNA library with residues 1-1059 of the alpha subunit of phosphorylase kinase, we have isolated 16 interacting, independent, yet overlapping transcripts of the alpha subunit containing its C-terminal region . Domain mapping of binary interactions between alpha constructs revealed two regions involved in the self-association of the alpha subunit: residues 833-854, a previously unrecognized leucine zipper, and an unspecified region within residues 1015-1237 . The cognate binding partner for the latter domain has been inferred to lie within the stretch from residues 864-1059 . Indirect evidence from the literature suggests that the interacting domains contained within the latter two, overlapping regions may be further narrowed to the stretches from 1057 to 1237 and from 864 to 971 . Cross-linking of the nonactivated holoenzyme with N-(gamma-maleimidobutyroxy)sulfosuccin-imide ester produced intramolecularly cross-linked alpha-alpha dimers, consistent with portions of two alpha subunits in the holoenyzme being in sufficient proximity to associate . This is the first report to identify potential areas of contact between the alpha subunits of phosphorylase kinase . Additionally, issues regarding the general utility of two-hybrid screening as a method for studying homodimeric interactions are discussed.

J Biol Chem, 1999 Dec 10, 274(50), 35434 - 40
Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates; Yang X et al.; Diphosphoinositol polyphosphate phosphohydrolase (DIPP) hydrolyzes diadenosine 5',5"'-P(1),P(6)-hexaphosphate (Ap(6)A), a Nudix (nucleoside diphosphate attached-moiety "x") substrate, and two non-Nudix compounds: diphosphoinositol pentakisphosphate (PP-InsP(5)) and bis-diphosphoinositol tetrakisphosphate ((PP)(2)-InsP(4)) . Guided by multiple sequence alignments, we used site-directed mutagenesis to obtain new information concerning catalytically essential amino acid residues in DIPP . Mutagenesis of either of two conserved glutamate residues (Glu(66) and Glu(70)) within the Nudt (Nudix-type) catalytic motif impaired hydrolysis of Ap(6)A, PP-InsP(5), and (PP)(2)-InsP(4) >95%; thus, all three substrates are hydrolyzed at the same active site . Two Gly-rich domains (glycine-rich regions 1 and 2 (GR1 and GR2)) flank the Nudt motif with potential sites for cation coordination and substrate binding . GR1 comprises a GGG tripeptide, while GR2 is identified as a new functional motif (GX(2)GX(6)G) that is conserved in yeast homologues of DIPP . Mutagenesis of any of these Gly residues in GR1 and GR2 reduced catalytic activity toward all three substrates by up to 95% . More distal to the Nudt motif, H91L and F84Y mutations substantially decreased the rate of Ap(6)A and (PP)(2)-InsP(4) metabolism (by 71 and 96%), yet PP-InsP(5) hydrolysis was only mildly reduced (by 30%); these results indicate substrate-specific roles for His(91) and Phe(84) . This new information helps define DIPP's structural, functional, and evolutionary relationships to Nudix hydrolases.

J Biol Chem, 1999 Dec 10, 274(50), 35393 - 9
Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A; Snoek GT et al.; In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein alpha (PI-TPalpha), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPalpha . Two stable cell lines, i.e . SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPalpha . Overexpression of PI-TPalpha resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13-14 h for SPI6 and SPI8 cells . Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G(1) phase is mainly responsible for this decrease . The saturation density of the cells increased from 0.20 x 10(5) cells/cm(2) for wtNIH3T3 cells to 0.53 x 10(5) cells/cm(2) for SPI6 and SPI8 cells . However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar . Upon equilibrium labeling of the cells with myo-{(3)H} inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2-3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells . A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4, 5-bisphosphate were the same as those in control cells . The addition of PI-TPalpha to a total lysate of myo-{(3)H}inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns . The addition of Ca(2+) further increased this formation . Based on these observations, we propose that PI-TPalpha is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns . The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells . The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.

J Biol Chem, 1999 Dec 10, 274(50), 35337 - 42
Binding of nucleotides to guanylate kinase, p21(ras), and nucleoside-diphosphate kinase studied by nano-electrospray mass spectrometry; Prinz H et al.; The binding of nucleotides to three different nucleotide-binding proteins and to a control protein was studied by means of nano-electrospray mass spectrometry applied to aqueous nondenaturing solutions . The method leads to unambiguous identification of enzyme complexes with substrates and products but does not allow the determination of dissociation constants or even stoichiometries relevant to the binding in solution . For guanylate kinase (EC 2.7.4 . 8), the transfer of HPO(3) between nucleotides was observed whenever a ternary complex with adenylate or guanylate nucleotides was formed . Guanosine 5'-tetraphosphate was generated after prolonged incubation with GDP or GTP . Mg(2+) binding was considerably enhanced in functional high affinity complexes, such as observed between guanylate kinase and its bisubstrate inhibitor P(1)-(5'-guanosyl)-P(5)-(5'-adenosyl) pentaphosphate or with the tight nucleotide-binding protein p21(ras) and GDP . Nucleoside-diphosphate kinase (EC 2.7.4.6) itself was phosphorylated in accordance to its known ping-pong mechanism . All nucleotide-binding proteins were shown to bind sulfate (SO(4)(2-)) with presumably high affinity and slow exchange rate . The binding of phosphate (PO(4)(3-)) could be inferred indirectly from competition with SO(4)(2-).

Exp Cell Res, 1999 Dec 15, 253(2), 637 - 48
Transient overexpression of murine dishevelled genes results in apoptotic cell death; Strovel ET et al.; The Dishevelled (Dvl) gene family encodes cytoplasmic proteins that are implicated in Wnt signal transduction . In mammals, the manner in which Wnt signals are transduced remains unclear . The biochemical and molecular mechanisms defining the Wnt-1 pathway are of great interest because of its important role in development and its activation in murine breast tumors . In order to elucidate Dvl's role in Wnt signaling, we attempted to overexpress Dvl in cells, but were unable to obtain stable cell lines . We show here that the overexpression of Dvl genes alters nuclear and cellular morphology of COS-1 and C57MG cells and causes cell death due to the induction of apoptosis . Deletion studies demonstrate that all three conserved domains of Dvl (DIX, PDZ, and DEP) are required for Dvl-mediated cell death . Coexpression of protein phosphatase 2Calpha, a Dvl-interacting protein identified in yeast two-hybrid studies, protects cells from the cell death observed in cells overexpressing Dvl alone . Furthermore, the adenomatous polyposis coli (APC) gene product appears to be required for Dvl-mediated cell death . The relevance of these findings to Wnt signal transduction, as well as to developmental processes and disease, are discussed .

Philos Trans R Soc Lond B Biol Sci, 1999 Sep 29, 354(1389), 1533 - 50
SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis; Willems AR et al.; Many key activators and inhibitors of cell division are targeted for degradation by a recently described family of E3 ubiquitin protein ligases termed Skp1-Cdc53-F-box protein (SCF) complexes . SCF complexes physically link substrate proteins to the E2 ubiquitin-conjugating enzyme Cdc34, which catalyses substrate ubiquitination, leading to subsequent degradation by the 26S proteasome . SCF complexes contain a variable subunit called an F-box protein that confers substrate specificity on an invariant core complex composed of the subunits Cdc34, Skp1 and Cdc53 . Here, we review the substrates and pathways regulated by the yeast F-box proteins Cdc4, Grr1 and Met30 . The concepts of SCF ubiquitin ligase function are illustrated by analysis of the degradation pathway for the G1 cyclin Cln2 . Through mass spectrometric analysis of Cdc53 associated proteins, we have identified three novel F-box proteins that appear to participate in SCF-like complexes . As many F-box proteins can be found in sequence databases, it appears that a host of cellular pathways will be regulated by SCF-dependent proteolysis.

Nat Struct Biol, 1999 Dec, 6(12), 1081 - 3
If the loop fits..; Frankel AD; The structure of the yeast L30 ribosomal protein bound to its autoregulatory RNA site has been determined by NMR spectroscopy . The intricate architecture of the RNA internal loop and the structure of the binding region of the protein both are stabilized in the complex, highlighting the importance of mutually-induced fit in RNA-protein interactions.

Appl Environ Microbiol, 1999 Dec, 65(12), 5546 - 53
The bglA gene of Aspergillus kawachii encodes both extracellular and cell wall-bound beta-glucosidases; Iwashita K et al.; We cloned the genomic DNA and cDNA of bglA, which encodes beta-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound beta-glucosidase CB-1 . The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein . Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound beta-glucosidase CB-1 . The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal beta-glucosidases classified in subfamily B . We expressed the bglA cDNA in Saccharomyces cerevisiae and detected the recombinant beta-glucosidase in the periplasm fraction of the recombinant yeast . A . kawachii can produce two extracellular beta-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound beta-glucosidase . A . kawachii in which the bglA gene was disrupted produced none of the three beta-glucosidases, as determined by enzyme assays and a Western blot analysis . Thus, we concluded that the bglA gene encodes both extracellular and cell wall-bound beta-glucosidases in A . kawachii.

Appl Environ Microbiol, 1999 Dec, 65(12), 5252 - 6
Disruption of TRI101, the gene encoding trichothecene 3-O-acetyltransferase, from Fusarium sporotrichioides; McCormick SP et al.; We screened a Fusarium sporotrichioides NRRL 3299 cDNA expression library in a toxin-sensitive Saccharomyces cerevisiae strain lacking a functional PDR5 gene . Fourteen yeast transformants were identified as resistant to the trichothecene 4,15-diacetoxyscirpenol, and each carried a cDNA encoding the trichothecene 3-O-acetyltransferase that is the F . sporotrichioides homolog of the Fusarium graminearum TRI101 gene . Mutants of F . sporotrichioides NRRL 3299 produced by disruption of TRI101 were altered in their abilities to synthesize T-2 toxin and accumulated isotrichodermol and small amounts of 3, 15-didecalonectrin and 3-decalonectrin, trichothecenes that are not observed in cultures of the parent strain . Our results indicate that TRI101 converts isotrichodermol to isotrichodermin and is required for the biosynthesis of T-2 toxin.

J Neurochem, 1999 Dec, 73(6), 2240 - 9
Identification of a mammalian homologue of the fungal Tom70 mitochondrial precursor protein import receptor as a thyroid hormone-regulated gene in specific brain regions; Alvarez-Dolado M et al.; Thyroid hormone is an important regulator of mammalian brain maturation . By differential display PCR, we isolated a cDNA clone (S2) that is specifically up-regulated in the striatum of neonatal hypothyroid rats . S2 was identified as KIAA0719, the first human gene distantly homologous to the fungal Tom70, which encodes a member of the translocase mitochondrial outer membrane complex involved in the import of preproteins into the mitochondria . By northern and in situ hybridization studies, KIAA0719 was found to be up-regulated in the striatum, nucleus accumbens, and discrete cortical layers of 15-day-old hypothyroid rats . In contrast, lower expression was found in the olfactory tubercle, whereas no differences were detected in other brain regions . Significantly, treatment of hypothyroid animals with single injections of thyroxine restored the normal levels of KIAA0719 expression . Moreover, treatment of control animals with thyroxine led to a reduced expression, demonstrating a negative hormonal regulation in vivo . Thus, KIAA0719 gene expression is regulated by thyroid hormone in the neonatal rat brain in a region-specific fashion . Given the role of the homologous Tom70 gene, the alteration of KIAA0719 expression may contribute to the changes in mitochondrial morphology and physiology caused by hypothyroidism in the developing rat brain.

EMBO J, 1999 Dec 1, 18(23), 6832 - 44
The E2-E3 interaction in the N-end rule pathway: the RING-H2 finger of E3 is required for the synthesis of multiubiquitin chain; Xie Y et al.; We dissected physical and functional interactions between the ubiquitin-conjugating (E2) enzyme Ubc2p and Ubr1p, the E3 component of the N-end rule pathway in Saccharomyces cerevisiae . The binding of the 20 kDa Ubc2p by the 225 kDa Ubr1p is shown to be mediated largely by the basic residue-rich (BRR) region of Ubr1p . However, mutations of the BRR domain that strongly decrease the interaction between Ubr1p and Ubc2p do not prevent the degradation of N-end rule substrates . In contrast, this degradation is completely dependent on the RING-H2 finger of Ubr1p adjacent to the BRR domain . Specifically, the first cysteine of RING-H2 is required for the ubiquitylation activity of the Ubr1p-Ubc2p complex, although this cysteine plays no detectable role in either the binding of N-end rule substrates by Ubr1p or the physical affinity between Ubr1p and Ubc2p . These results defined the topography of the Ubc2p-Ubr1p interaction and revealed the essential function of the RING-H2 finger, a domain that is present in many otherwise dissimilar E3 proteins of the ubiquitin system.

EMBO J, 1999 Dec 1, 18(23), 6816 - 22
Carbohydrate deficient glycoprotein syndrome type IV: deficiency of dolichyl-P-Man:Man(5)GlcNAc(2)-PP-dolichyl mannosyltransferase; Korner C et al.; Type IV of the carbohydrate deficient glycoprotein syndromes (CDGS) is characterized by microcephaly, severe epilepsy, minimal psychomotor development and partial deficiency of sialic acids in serum glycoproteins . Here we show that the molecular defect in the index patient is a missense mutation in the gene encoding the mannosyltransferase that transfers mannose from dolichyl-phosphate mannose on to the lipid-linked oligosaccharide (LLO) intermediate Man(5)GlcNAc(2)-PP-dolichol . The defect results in the accumulation of the LLO intermediate and, due to its leaky nature, a residual formation of full-length LLOs . N-glycosylation is abnormal because of the transfer of truncated oligosaccharides in addition to that of full-length oligosaccharides and because of the incomplete utilization of N-glycosylation sites . The mannosyltransferase is the structural and functional orthologue of the Saccharomyces cerevisiae ALG3 gene.

EMBO J, 1999 Dec 1, 18(23), 6744 - 51
Hsp26: a temperature-regulated chaperone; Haslbeck M et al.; Small heat shock proteins (sHsps) are a conserved protein family, with members found in all organisms analysed so far . Several sHsps have been shown to exhibit chaperone activity and protect proteins from irreversible aggregation in vitro . Here we show that Hsp26, an sHsp from Saccharomyces cerevisiae, is a temperature-regulated molecular chaperone . Like other sHsps, Hsp26 forms large oligomeric complexes . At heat shock temperatures, however, the 24mer chaperone complex dissociates . Interestingly, chaperone assays performed at different temperatures show that the dissociation of the Hsp26 complex at heat shock temperatures is a prerequisite for efficient chaperone activity . Binding of non-native proteins to dissociated Hsp26 produces large globular assemblies with a structure that appears to be completely reorganized relative to the original Hsp26 oligomers . In this complex one monomer of substrate is bound per Hsp26 dimer . The temperature-dependent dissociation of the large storage form of Hsp26 into a smaller, active species and the subsequent re-association to a defined large chaperone-substrate complex represents a novel mechanism for the functional activation of a molecular chaperone.

EMBO J, 1999 Dec 1, 18(23), 6672 - 81
Gal83 mediates the interaction of the Snf1 kinase complex with the transcription activator Sip4; Vincent O et al.; The Snf1/AMPK protein kinase family is widely conserved in eukaryotes . In Saccharomyces cerevisiae, the Snf1 kinase is an essential element of the glucose response pathway and has diverse regulatory roles . The Snf1 complex contains one of the related proteins Sip1, Sip2 and Gal83, which are also conserved in higher eukaryotes . Previous studies showed that the Sip1/Sip2/Gal83 component plays a structural role in the complex . We present evidence that this component also mediates the interaction of the Snf1 kinase complex with specific targets . We show that Gal83 mediates the association of the kinase with Sip4, a Snf1-regulated transcription activator of gluconeogenic genes . Gal83 interacts with Sip4 in two-hybrid assays in vivo, and bacterially expressed proteins bind in vitro . Moreover, Gal83 is required for the two-hybrid interaction of Sip4 with the Snf1 kinase . Gal83 also facilitates the rapid Snf1-dependent phosphorylation and activation of Sip4 in response to glucose limitation, indicating that Gal83 mediates the functional interaction of Snf1 with Sip4 . Evidence indicates that Sip1 and Sip2 do not interact with Sip4 . We propose that members of the Sip1/Sip2/Gal83 family confer specificity to the kinase complex in its interactions with target proteins.

EMBO J, 1999 Dec 1, 18(23), 6662 - 71
A new regulatory domain on the TATA-binding protein; Cang Y et al.; Recognition of the TATA box by the TATA-binding protein (TBP) is a highly regulated step in RNA polymerase II-dependent transcription . Several proteins have been proposed to regulate TBP activity, yet the TBP domains responsive to all these regulators have not been defined . Here we describe a new class of TBP mutants that increase transcription from core promoters in vivo . The majority of these mutations alter amino acids that cluster tightly on the TBP surface, defining a new TBP regulatory domain . The mutant TBP proteins are defective for binding the transcriptional regulator yNC2, are resistant to inhibition by yNC2 in vitro and exhibit allele-specific genetic interactions with yNC2 . These results provide strong biochemical and genetic evidence that TBP is directly repressed in vivo, and define a new TBP domain important for transcriptional regulation.

EMBO J, 1999 Dec 1, 18(23), 6619 - 29
Mre11 is essential for the maintenance of chromosomal DNA in vertebrate cells; Yamaguchi-Iwai Y et al.; Yeast Mre11 functions with Rad50 and Xrs2 in a complex that has pivotal roles in homologous recombination (HR) and non-homologous end-joining (NHEJ) DNA double-strand break (DSB) repair pathways . Vertebrate Mre11 is essential . Conditionally, MRE11 null chicken DT40 cells accumulate chromosome breaks and die upon Mre11 repression, showing frequent centrosome amplification . Mre11 deficiency also causes increased radiosensitivity and strongly reduced targeted integration frequencies . Mre11 is, therefore, crucial for HR and essential in mitosis through its role in chromosome maintenance by recombinational repair . Surprisingly perhaps, given the role of Mre11 in yeast NHEJ, disruption of NHEJ by deletion of KU70 greatly exacerbates the effects of MRE11 deficiency, revealing a significant Mre11-independent component of metazoan NHEJ.

Biochem Biophys Res Commun, 1999 Dec 9, 266(1), 147 - 51
MIDA1, an Id-associating protein, has two distinct DNA binding activities that are converted by the association with Id1: a novel function of Id protein; Inoue T et al.; Id proteins not only regulate cell differentiation negatively, but they also promote growth, immortalization, and apoptosis . To know the mechanism of how Id regulates cell fate, we previously isolated an Id-associating protein, MIDA1, which positively regulates cell growth (1) . Its predicted amino acid sequence consists of a Zuotin (a Z-DNA binding protein in yeast) homology region and tryptophan-mediated repeats (Tryp-med repeats) . MIDA1 exhibits a sequence-specific DNA binding activity through the Tryp-med repeats (manuscript in preparation) . In this study, we revealed that, like Zuotin, MIDA1 can specifically bind to Z-DNA . This suggested that MIDA is a novel DNA binding protein that has two different DNA binding activities . Furthermore, association of Id1 with MIDA1 stimulated the sequence-specific DNA binding activity, while it inhibited the Z-DNA binding activity . Therefore, we concluded that MIDA1 may act as a mediator of the growth-promoting function of Id, by switching the two DNA binding activities of MIDA1 .

Nature, 1999 Nov 18, 402(6759), 320 - 2
Sustained oscillations in living cells; Dano S et al.; Glycolytic oscillations in yeast have been studied for many years simply by adding a glucose pulse to a suspension of cells and measuring the resulting transient oscillations of NADH . Here we show, using a suspension of yeast cells, that living cells can be kept in a well defined oscillating state indefinitely when starved cells, glucose and cyanide are pumped into a cuvette with outflow of surplus liquid . Our results show that the transitions between stationary and oscillatory behaviour are uniquely described mathematically by the Hopf bifurcation . This result characterizes the dynamical properties close to the transition point . Our perturbation experiments show that the cells remain strongly coupled very close to the transition . Therefore, the transition takes place in each of the cells and is not a desynchronization phenomenon . With these two observations, a study of the kinetic details of glycolysis, as it actually takes place in a living cell, is possible using experiments designed in the framework of nonlinear dynamics . Acetaldehyde is known to synchronize the oscillations . Our results show that glucose is another messenger substance, as long as the glucose transporter is not saturated.

J Hepatol, 1999 Nov, 31(5), 783 - 90
Involvement of phosphatidylserine and non-phospholipid components of the hepatitis B virus envelope in human Annexin V binding and in HBV infection in vitro; De Meyer S et al.; BACKGROUND/AIMS: We have previously demonstrated that human liver Annexin V (hAV), a Ca2+-dependent phospholipid binding protein, binds specifically to small HBsAg (SHBsAg) . Because of the propensity of AV to bind phospholipids, we here examine the role of phospholipids, as component of the HBV envelope, in binding to hAV and in HBV infection . METHODS: The influence of phospholipids (phosphatidylserine and phosphatidylcholine) on the binding of hAV to SHBsAg or to anti-hAV monoclonals was determined by ELISA . Their influence on HBV infection was investigated using an in vitro HBV infection assay . RESULTS: Two monoclonals, specific against hAV, were able to block the binding of hAV to SHBsAg and recognized different epitopes of hAV . The binding of one of these monoclonals to hAV could be inhibited by phosphatidylserine, but not by phosphatidylcholine . Further experiments revealed that phosphatidylserine could also inhibit the binding of hAV to SHBsAg and could even prevent HBV infection in vitro . Phosphatidylcholine had no effect on the binding of hAV to SHBsAg and could not prevent HBV infection in vitro . However, since phosphatidylserine was not able to abolish the binding of the other blocking monoclonal to hAV, a non-phospholipid component of the HBV envelope must also be involved in hAV binding . CONCLUSIONS: These results indicate that phosphatidylserine and a non-phospholipid component of the HBV envelope are involved in hAV binding and in HBV infection.

Genes Dev, 1999 Nov 15, 13(22), 3027 - 33
Regulation of CDK4 activity by a novel CDK4-binding protein, p34(SEI-1); Sugimoto M et al.; The p16(INK4a) tumor suppressor inhibits cyclin-dependent kinases (CDK4 and CDK6) . Here we report the isolation of a novel gene, SEI-1, whose product (p34(SEI-1)) appears to antagonize the function of p16(INK4a) . Addition of p34(SEI-1) to cyclin D1-CDK4 renders the complex resistant to inhibition by p16(INK4a) . Expression of SEI-1 is rapidly induced on addition of serum to quiescent fibroblasts, and ectopic expression of p34(SEI-1) enables fibroblasts to proliferate even in low serum concentrations . p34(SEI-1) seems to act as a growth factor sensor and may facilitate the formation and activation of cyclin D-CDK complexes in the face of inhibitory levels of INK4 proteins.

Genes Dev, 1999 Nov 15, 13(22), 2946 - 57
Dephosphorylation of cyclin-dependent kinases by type 2C protein phosphatases; Cheng A et al.; Activating phosphorylation of cyclin-dependent protein kinases (CDKs) is necessary for their kinase activity and cell cycle progression . This phosphorylation is carried out by the Cdk-activating kinase (CAK); in contrast, little is known about the corresponding protein phosphatase . We show that type 2C protein phosphatases (PP2Cs) are responsible for this dephosphorylation of Cdc28p, the major budding yeast CDK . Two yeast PP2Cs, Ptc2p and Ptc3p, display Cdc28p phosphatase activity in vitro and in vivo, and account for approximately 90% of Cdc28p phosphatase activity in yeast extracts . Overexpression of PTC2 or PTC3 results in synthetic lethality in strains temperature-sensitive for yeast CAK1, and disruptions of PTC2 and PTC3 suppress the growth defect of a cak1 mutant . Furthermore, PP2C-like enzymes are the predominant phosphatases toward human Cdk2 in HeLa cell extracts, indicating that the substrate specificity of PP2Cs toward CDKs is evolutionarily conserved.

Genes Dev, 1999 Nov 15, 13(22), 2940 - 5
The Spt components of SAGA facilitate TBP binding to a promoter at a post-activator-binding step in vivo; Dudley AM et al.; The SAGA complex of Saccharomyces cerevisiae is required for the transcription of many RNA polymerase II-dependent genes . Previous studies have demonstrated that SAGA possesses histone acetyltransferase activity, catalyzed by the SAGA component Gcn5 . However, the transcription of many genes, although SAGA dependent, is Gcn5 independent, suggesting the existence of distinct SAGA activities . We have studied the in vivo role of two other SAGA components, Spt3 and Spt20, at the well-characterized GAL1 promoter . Our results demonstrate that both Spt3 and Spt20 are required for the binding of TATA-binding protein but not of the activator Gal4 and that this role is Gcn5 independent . These results suggest a coactivator role for Spt3 and Spt20 in the recruitment of TBP.

Genes Dev, 1999 Nov 15, 13(22), 2934 - 9
Different upstream transcriptional activators have distinct coactivator requirements; Lee DK et al.; Activated transcription by RNA polymerase II (Pol II) requires coactivators, one of which is the SRB/mediator . Whereas Srb4, an essential subunit of the SRB/mediator, is broadly required for Pol II transcription in yeast, we have shown that it is dispensable for the transcriptional activation of some genes . Here, we show that transcriptional activation by different natural activators, and by artificial recruitment of various transcription factors, have very different degrees of Srb4 independence . These data, and the analysis of an rgr1 mutant, point to an Rgr1 subcomplex of the SRB/mediator as the mechanistic route of activation by Srb4-independent activators in vivo.

Genes Dev, 1999 Nov 15, 13(22), 2928 - 33
The Rbx1 subunit of SCF and VHL E3 ubiquitin ligase activates Rub1 modification of cullins Cdc53 and Cul2; Kamura T et al.; The RING-H2 finger protein Rbx1 is a subunit of the related SCF (Skp1-Cdc53/Cul1-F-box protein) and von Hippel-Lindau (VHL) tumor suppressor (elongin BC-Cul2-VHL) E3 ubiquitin ligase complexes, where it functions as a component of Cdc53/Rbx1 and Cul2/Rbx1 modules that activate ubiquitination of target proteins by the E2 ubiquitin-conjugating enzymes Cdc34 and Ubc5 . Here we demonstrate that the Cdc53/Rbx1 and Cul2/Rbx1 modules also activate conjugation of the ubiquitin-like protein Rub1 to Cdc53 and Cul2 by the dedicated E2 Rub1 conjugating enzyme Ubc12 . Our findings identify Rbx1 as a common component of enzyme systems responsible for ubiquitin and Rub1 modification of target proteins.

Gastroenterology, 1999 Dec, 117(6), 1408 - 15
Differential expression of basolateral and canalicular organic anion transporters during regeneration of rat liver; Gerloff T et al.; BACKGROUND & AIMS: Liver regeneration in response to various forms of injury or surgical resection is a complex process resulting in restoration of the original liver mass and maintenance of liver-specific functions such as bile formation . However, liver regeneration is frequently associated with cholestasis, whose molecular pathogenesis remains unknown . METHODS: To study the molecular mechanisms leading to cholestasis, expression of all major hepatic organic anion transporters contributing to bile formation was determined for up to 2 weeks in rats after 70% partial hepatectomy . RESULTS: Inversely related to serum bile acid levels, basolateral transporters including the sodium-taurocholate cotransporter (Ntcp) and the organic anion transporting polypeptides Oatp1 and Oatp2 were markedly down-regulated at both protein and steady-state mRNA levels by 50%-60% of controls (P < 0.05) during early replicative stages of regeneration (12 hours to 2 days) with a slightly delayed time course for Oatp2 . Expression of all basolateral transporters returned to control values between 4 and 4 days after partial hepatectomy . In contrast, protein and mRNA expression of both the canalicular ATP-dependent bile salt export pump (Bsep) and the multiorganic anion transporter Mrp2 remained unchanged or were slightly increased during liver regeneration, but also returned to control values 7-14 days after partial hepatectomy . CONCLUSIONS: The data suggest a differential regulation of basolateral and canalicular organic anion transporters in the regenerating liver . Unaltered expression of Bsep and Mrp2 provides a potential molecular mechanism for regenerating liver cells to maintain or even increase bile secretion expressed per weight of remaining liver . However, down-regulation of basolateral organic anion transporters might protect replicating liver cells by diminishing uptake of potentially hepatotoxic bile salts, because the remaining liver initially cannot cope with the original bile acid pool size.

Methods, 1999 Nov, 19(3), 410 - 6
Transcriptional analysis of purified histone acetyltransferase complexes; Steger DJ et al.; Acetylation of lysine residues within the amino-terminal tails of the core histone proteins is strongly correlated to the regulation of gene transcription in vivo . To directly study the effects of histone acetylation on transcription, we have developed a biochemical system examining the regulation of RNA polymerase II-directed transcription by native histone acetyltransferases (HATs) . For the promoter sequences investigated, it has been demonstrated that HATs facilitate transcription from nucleosomal DNA templates in an acetyl-CoA-dependent fashion but do not affect transcription from histone-free templates . Here, protocols are presented describing the in vitro assembly of evenly spaced nucleosomal arrays on DNA fragments harboring gene regulatory sequences and the use of these templates with purified HAT complexes in transcription assays .

Dev Comp Immunol, 1999 Oct-Dec, 23(7-8), 545 - 52
Maternal transmission of immunity to white spot syndrome associated virus (WSSV) in shrimp (Penaeus monodon); Huang CC et al.; Beta-1,3-1,6-glucan, derived from bakers' yeast Saccharomyces cerevisiae, was used in the present study to investigate the extent to which glucan is able to protect spawners from white spot syndrome associated virus (WSSV), and whether this protection (if any) can be passed on to hatchlings via maternal transmission of immunity . Results showed that fewer spawners in the glucan-injected groups showed the clinical symptoms of red body coloration and white spots on the shell during the 15 days between eyestalk ablation and the end of repeated spawning . This suggests that the application of glucan might lead to a slight enhancement of disease resistance in spawners, although the differences were not statistically significant within the confidence limit chosen . Challenge results showed a significant increase in relative percent survival for larvae derived from groups of glucan-injected spawners compared to those derived from groups of untreated spawners . It therefore seems that a maternally transmitted disease resistance induced by glucan, protected the larvae against a WSSV infection . Glucan immersion was not only shown to be effective for nauplii derived from spawners that were not injected with glucan, it also provided additional, cumulative protection for nauplii which already had a maternally transmitted resistance to WSSV . This is the first documented demonstration of a maternal transmission of immunity in invertebrates.

Mol Cell Biol Res Commun, 1999 May, 1(2), 158 - 61
GAL4 is a substrate for caspases: implications for two-hybrid screening and other GAL4-based assays; van Criekinge W et al.; Yeast two-hybrid technology as well as mammalian reporter assays use fusions between a protein of interest and the GAL4 DNA-binding domain (GAL4DB) . We demonstrate that expression of a GAL4DB/caspase-1 chimeric protein in yeast leads to autoproteolytic cleavage of GAL4DB . Moreover, recombinant GAL4DB is a good in vitro substrate for recombinant caspase-1 and several other caspases . Cleavage sites map at the C-terminus of GAL4DB and result in release of the fused protein . The finding that GAL4DB can be cleaved by caspases has important implications for the use of caspases in two-hybrid analysis and in the interpretation of mammalian assays based on GAL4-dependent reporter gene expression.

Front Biosci, 1999 Dec 01, 4, D805 - 15
The metazoan origin recognition complex; Quintana DG et al.; Regulated initiation of DNA replication relies on the firing of initiator proteins that bind specifically to origin DNA . The discovery of the first eukaryotic initiator, the Saccharomyces cerevisiae Origin Recognition Complex (ORC) has allowed us to discern some aspects of how the onset of replication is regulated . However, understanding the specifics of replication in metazoan organisms can only be achieved by directly addressing these questions in animal cells . This review deals with the current state of knowledge on the metazoan Origin Recognition Complex, its composition and regulation in higher eukaryotes, its role in the initiation of replication and beyond replication, and its possible connection with human pathology.

Front Biosci, 1999 Dec 01, 4, D824 - 33
Transcription factors in DNA replication; Murakami Y et al.; Accumulating evidence suggests the involvement of transcription factors in the regulation of DNA replication in eukaryotic cells . Almost all eukaryotic DNA viruses contain binding sites for transcription factors which function as auxiliary elements for DNA replication initiation at replication origins, and, indeed, the binding of transcription factors to these elements has been shown to stimulate DNA replication . Transcription factors also regulate some of the chromosome DNA replication origins of budding yeast, indicating that transcription factor involvement in DNA replication is not restricted to viruses . Consistent with this notion, recently determined replication origins of higher eukaryotes have been found occasionally to associate with transcription factor binding sites, although there is no direct evidence for the involvement of the factors that bind to these sequences in DNA replication . Analyses using viral and yeast systems have suggested that transcription factors stimulate the formation of the replication initiation complex by engaging in specific interactions with proteins of the initiation complex and/or by modulating the repressive chromatin structure around origins of replication . These mechanisms are analogous to those advanced to explain stimulation of transcription by transcription factors . The accumulated data suggests that transcription factors play a general role in the formation of functional complexes on chromosomes.

Front Biosci, 1999 Dec 01, 4, D834 - 40
CDC7 kinase complex as a molecular switch for DNA replication; Masai H et al.; Cdc7 kinase and its activator Dbf4 protein, originally identified in budding yeast Saccharomyces cerevisiae, are widely conserved in eukaryotes including fission yeast and human . Dbf4-related activators bind and stimulate kinase activity of Cdc7-like catalytic subunit . Its kinase activity is cell cycle-regulated, mainly through availability of the activation subunit whose level increases at G1/S boundary and is maintained at a high level throughout S phase . MCM2 protein is among physiologically important substrates . Genetic studies in fission yeast indicate that Cdc7-related kinase complex also functions in meiosis, uninduced mutagenesis, DNA replication checkpoint signaling and maintenance of chromatin structures during S phase.

Front Biosci, 1999 Dec 01, 4, D816 - 23
Cell cycle regulation of DNA replication initiation proteins in mammalian cells; Fujita M; Genomic DNA has to be replicated completely and only once during a single cell cycle in order to maintain integrity . Eukaryotes have developed highly regulated machinery for precisely replicating genomic DNA that is fragmented into multiple chromosomes . Our knowledge of such mechanisms largely depends on findings with budding yeast, since identification of specific DNA sequences acting as replication origins, autonomously replicating sequences, has allowed extensive analyses of the initiation of DNA replication . Several factors essential for regulation of initiation have been identified, including ORC, CDC6 and MCM . Subsequent work has suggested that the fundamental machinery for DNA replication may be conserved in metazoan embryonic cells in which replication occurs sequence-independently, and also in mammalian nonembryonic cells, where replication origins are more specific . However, there are specific differences . In this review, information on function and regulation of mammalian initiation factors, ORC, CDC6 and MCM, is summarized, and yeast and embryonic systems are compared . A hypothetical model for the state of prereplication chromatin in mammalian cell nuclei and regulation during the cell cycle is also proposed.

Mol Cell Neurosci, 1999 Sep, 14(3), 213 - 24
A novel neuron-specific DNA end-binding factor in the murine brain; Hurd YL et al.; To characterize the distribution of transcription factor AP-1 and YY1 DNA-binding activities in the rat brain, the labeled target oligonucleotides were loaded on brain sections and after incubation and washing, the residual signal was registered by autoradiography . The binding was predominantly associated with neurons and was regionally specific with highest levels in the cerebellum, hippocampus, and piriform cortex . The identified binding factor was not, however, sequence-specific, but apparently recognized DNA ends and was activated by long double-stranded DNA . UV cross-linking identified the molecular mass of the factor to be about 80 kDa . The factor was not found in soluble brain extracts, suggesting its association with membranes or the nuclear matrix . Despite apparent similarities with Ku protein, which targets DNA-ends, the DNA end-binding activity was present in brains of Ku86- and Ku70-deficient mice . Since DNA end-binding factors are generally involved in DNA repair, the same function may be suggested for the novel factor identified in the present study.

Curr Biol, 1999 Nov 18, 9(22), 1323 - 6
Synthesis of diphosphoinositol pentakisphosphate by a newly identified family of higher inositol polyphosphate kinases; Saiardi A et al.; Inositol (1,4,5) trisphosphate (Ins(1,4,5)P(3)) is a well-known messenger molecule that releases calcium from intracellular stores . Homologues with up to six phosphates have been characterized and recently, homologues with seven or eight phosphate groups, including pyrophosphates, have been identified . These homologues are diphosphoinositol pentakisphosphate (PP-InsP(5)/InsP(7)) and bis(diphospho)inositol tetrakisphosphate (bis-PP-InsP(4)/InsP(8)) {1}, the rapid turnover of which {2} is regulated by calcium {2} and adrenergic receptor activity {3} . It has been proposed that the high-energy pyrophosphates might participate in protein phosphorylation {4} . We have purified InsP(6) kinase {5} and PP-InsP(5) kinase {6}, both of which display ATP synthase activity, transferring phosphate to ADP . Here, we report the cloning of two mammalian InsP(6) kinases and a yeast InsP(6) kinase . Furthermore, we show that the yeast protein, ArgRIII, is an inositol-polyphosphate kinase that can convert InsP(3) to InsP(4), InsP(5) and InsP(6) . We have identified a new family of highly conserved inositol-polyphosphate kinases that contain a newly identified, unique consensus sequence.

Curr Biol, 1999 Nov 18, 9(22), 1297 - 303
A class VII unconventional myosin is required for phagocytosis; Titus MA; BACKGROUND: Phagocytosis, the process by which cells internalize particles, is essential for the defense of multicellular organisms against invading pathogens and is the major means by which many unicellular organisms obtain nutrients . The actin cytoskeleton plays a critical role in phagocytosis and the observation that a significant amount of force (10-20 nN) is generated during internalization, suggests that a myosin participates in the process . Although more than 15 distinct classes of myosin have been identified, their roles in phagocytosis are unknown . RESULTS: The identification of a class VII unconventional myosin (DdMVII) in the Dictyostelium discoideum amoeba, which is a model for phagocytosis, is reported here . Mutant cells lacking DdMVII exhibited an 80% decrease in the uptake of particles whereas all other actin-based behaviors that were tested, including pinocytosis, exocytosis, cytokinesis and morphogenesis, proceeded normally . The defect in phagocytosis was neither because of altered particle binding nor inability to form actin-filled phagocytic cups . CONCLUSIONS: Molecular genetic analysis of Dictyostelium myosin VII reveals that this motor protein plays a specific and significant role in phagocytosis.

J Cell Sci, 1999 Dec, 112 ( Pt 24), 4547 - 56
Identification of the putative mammalian orthologue of Sec31P, a component of the COPII coat; Shugrue CA et al.; The regulation of intracellular vesicular trafficking is mediated by specific families of proteins that are involved in vesicular budding, translocation, and fusion with target membranes . We purified a vesicle-associated protein from hepatic microsomes using sequential column chromatography and partially sequenced it . Oliogonucleotides based on these sequences were used to clone the protein from a rat liver cDNA library . The clone encoded a novel protein with a predicted mass of 137 kDa (p137) . The protein had an N terminus WD repeat motif with significant homology to Sec31p, a member of the yeast COPII coat that complexes with Sec13p . We found that p137 interacted with mammalian Sec13p using several approaches: co-elution through sequential column chromatography, co-immunoprecipitation from intact cells, and yeast two-hybrid analysis . Morphologically, the p137 protein was localized to small punctate structures in the cytoplasm of multiple cultured cell lines . When Sec13p was transfected into these cells, it demonstrated considerable overlap with p137 . This overlap was maintained through several pharmacological manipulations . The p137 compartment also demonstrated partial overlap with ts045-VSVG protein when infected cells were incubated at 15 degrees C . These findings suggest that p137 is the mammalian orthologue of Sec31p.

Science, 1999 Nov 26, 286(5445), 1700 - 5
Molecular architecture of the rotary motor in ATP synthase; Stock D et al.; Adenosine triphosphate (ATP) synthase contains a rotary motor involved in biological energy conversion . Its membrane-embedded F0 sector has a rotation generator fueled by the proton-motive force, which provides the energy required for the synthesis of ATP by the F1 domain . An electron density map obtained from crystals of a subcomplex of yeast mitochondrial ATP synthase shows a ring of 10 c subunits . Each c subunit forms an alpha-helical hairpin . The interhelical loops of six to seven of the c subunits are in close contact with the gamma and delta subunits of the central stalk . The extensive contact between the c ring and the stalk suggests that they may rotate as an ensemble during catalysis.

J Biol Chem, 1999 Dec 3, 274(49), 35269 - 77
c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads; Akiyoshi S et al.; Smads are intracellular signaling mediators of the transforming growth factor-beta (TGF-beta) superfamily that regulates a wide variety of biological processes . Among them, Smads 2 and 3 are activated specifically by TGF-beta . We identified c-Ski as a Smad2 interacting protein . c-Ski is the cellular homologue of the v-ski oncogene product and has been shown to repress transcription by recruiting histone deacetylase (HDAC) . Smad2/3 interacts with c-Ski through its C-terminal MH2 domain in a TGF-beta-dependent manner . c-Ski contains two distinct Smad-binding sites with different binding properties . c-Ski strongly inhibits transactivation of various reporter genes by TGF-beta . c-Ski is incorporated in the Smad DNA binding complex, interferes with the interaction of Smad3 with a transcriptional co-activator, p300, and in turn recruits HDAC . c-Ski is thus a transcriptional co-repressor that links Smads to HDAC in TGF-beta signaling.

Nature, 1999 Nov 4, 402(6757), 83 - 6
A combined algorithm for genome-wide prediction of protein function; Marcotte EM et al.; The availability of over 20 fully sequenced genomes has driven the development of new methods to find protein function and interactions . Here we group proteins by correlated evolution, correlated messenger RNA expression patterns and patterns of domain fusion to determine functional relationships among the 6,217 proteins of the yeast Saccharomyces cerevisiae . Using these methods, we discover over 93,000 pairwise links between functionally related yeast proteins . Links between characterized and uncharacterized proteins allow a general function to be assigned to more than half of the 2,557 previously uncharacterized yeast proteins . Examples of functional links are given for a protein family of previously unknown function, a protein whose human homologues are implicated in colon cancer and the yeast prion Sup35.

RNA, 1999 Oct, 5(10), 1299 - 307
Aberrant mRNAs with extended 3' UTRs are substrates for rapid degradation by mRNA surveillance; Muhlrad D et al.; The mRNA surveillance system is known to rapidly degrade aberrant mRNAs that contain premature termination codons in a process referred to as nonsense-mediated decay . A second class of aberrant mRNAs are those wherein the 3' UTR is abnormally extended due to a mutation in the polyadenylation site . We provide several observations that these abnormally 3'-extended mRNAs are degraded by the same machinery that degrades mRNAs with premature nonsense codons . First, the decay of the 3'-extended mRNAs is dependent on the same decapping enzyme and 5'-to-3' exonuclease . Second, the decay is also dependent on the proteins encoded by the UPF1, UPF2, and UPF3 genes, which are known to be specifically required for the rapid decay of mRNAs containing nonsense codons . Third, the ability of an extended 3' UTR to trigger decay is prevented by stabilizing sequences within the PGK1 coding region that are known to protect mRNAs from the rapid decay induced by premature nonsense codons . These results indicate that the mRNA surveillance system plays a role in degrading abnormally extended 3' UTRs . Based on these results, we propose a model in which the mRNA surveillance machinery degrades aberrant mRNAs due to the absence of the proper spatial arrangement of the translation-termination codon with respect to the 3' UTR element as defined by the utilization of a polyadenylation site.

Nucleic Acids Res, 1999 Dec 15, 27(24), 4679 - 86
Geometry of a complex formed by double strand break repair proteins at a single DNA end: recruitment of DNA-PKcs induces inward translocation of Ku protein; Yoo S et al.; Ku protein and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) are essential components of the double-strand break repair machinery in higher eukaryotic cells . Ku protein binds to broken DNA ends and recruits DNA-PKcs to form an enzymatically active complex . To characterize the arrangement of proteins in this complex, we developed a set of photocross-linking probes, each with a single free end . We have previously used this approach to characterize the contacts in an initial Ku-DNA complex, and we have now applied the same technology to define the events that occur when Ku recruits DNA-PKcs . The new probes allow the binding of one molecule of Ku protein and one molecule of DNA-PKcs in a defined position and orientation . Photocross-linking reveals that DNA-PKcs makes direct contact with the DNA termini, occupying an approximately 10 bp region proximal to the free end . Characterization of the Ku protein cross-linking pattern in the presence and absence of DNA-PKcs suggests that Ku binds to form an initial complex at the DNA ends, and that recruitment of DNA-PKcs induces an inward translocation of this Ku molecule by about one helical turn . The presence of ATP had no effect on protein-DNA contacts, suggesting that neither DNA-PK-mediated phosphorylation nor a putative Ku helicase activity plays a role in modulating protein conformation under the conditions tested.

Plant J, 1999 Nov, 20(3), 343 - 8
Short communication: unsaturated fatty acids inhibit MP2C, a protein phosphatase 2C involved in the wound-induced MAP kinase pathway regulation; Baudouin E et al.; When mechanically injured, plants develop multiple defense systems including the activation of specific genes . These responses are triggered by a complex network of signalling events that include Ca2+ fluxes, the production of free fatty acids from membrane lipids, as well as the activation of mitogen-activated protein kinases (MAPK) . In the present paper, we address the question of the regulation of the MAPK pathway by wound-induced Ca2+ and fatty acid signals . We report that MP2C, a serine/threonine protein phosphatase 2C from alfalfa involved in MAPK pathway inactivation, is inhibited specifically in vitro by long-carbon-chain polyunsaturated fatty acids, and alpha-linolenic acid, the primary product of the octadecanoid pathway, was found to be the most potent inhibitor . Ca2+ also inhibits MP2C, but only at high concentrations, and other divalent cations show similar inhibitory effect, making it unlikely that Ca2+ is involved in the regulation of MP2C in vivo . Overall, our data suggest that cross-talk between wound-induced MAPK and octadecanoid pathways may occur at the level of protein phosphatase 2C and linolenic acid.

Int J Radiat Oncol Biol Phys, 1999 Nov 1, 45(4), 1005 - 10
The immunohistochemical expression of DNA-PKCS and Ku (p70/p80) in head and neck cancers: relationships with radiosensitivity; Bjork-Eriksson T et al.; PURPOSE: The DNA-PK complex is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation . This study evaluated the relationship between the immunohistochemical expression of the individual components of DNA-PK and cellular radiosensitivity in head and neck cancers . METHODS AND MATERIALS: Biopsies from patients with previously untreated squamous cell carcinomas of the head and neck were assessed for inherent tumor radiosensitivity measured as the surviving fraction at 2 Gy (SF2) using a soft agar clonogenic assay . Paraffin-embedded tumor material from 64 successfully grown specimens was immunohistochemically stained for expression of DNA-PKcs and Ku (p70/p80) . The same tumor material was previously analyzed for the immunohistochemical expression of p53 . RESULTS: A significant correlation was found between the degree of expression of DNA-PKcs and Ku (p70/p80) (r = 0.55, p<0.001) . There were no overall significant differences in the levels of expression of DNA-PKcs and Ku (p70/p80) in tumors from patients of either sex, different sites, histologies, and stages . No relationship was found between SF2 and the expression of either DNA-PKcs (r = 0.22, p = 0.081) or Ku (p70/p80) (r = 0.064, p = 0.62) . Comparison with previous immunohistochemical characterization showed no significant correlations between the expression levels of p53 and either DNA-PKcs (r = 0.093, p = 0.46) or Ku (p70/p80) (r = -0.17, p = 0.17) . CONCLUSIONS: This study suggests that determining the immunohistochemical expression of DNA-PK in head and neck cancers from multiple sites does not have a role as a predictive assay of tumor in vitro radiosensitivity.

Dev Genet, 1999, 25(4), 312 - 20
Genetic analysis of the role of the drosophila fat facets gene in the ubiquitin pathway; Wu Z et al.; The Drosophila fat facets gene encodes a deubiquitinating enzyme required during eye development to limit the number of photoreceptors in each facet to eight . Ubiquitin is a small polypeptide that targets proteins for degradation by the proteasome . Deubiquitinating enzymes cleave ubiquitin-protein bonds . In order to investigate the role of FAT FACETS in the ubiquitin pathway, genetic interactions between fat facets and the Drosophila UbcD1 gene were assessed . In addition, three yeast deubiquitinating enzyme genes were tested for their ability to substitute for fat facets in the developing Drosophila eye and for their effects on eye morphology . The results of these experiments support the hypothesis that FAT FACETS activity antagonizes that of the proteolytic machinery . The implications of these results for the specificity of FAF and yeast UBPs are discussed as well .

Biochim Biophys Acta, 1999 Nov 23, 1441(2-3), 268 - 77
PI transfer protein: the specific recognition of phospholipids and its functions; Sha B et al.; Phosphatidylinositol transfer proteins (PITPs) can bind specifically and transfer a single phosphatidylinositol (PI) molecule between phospholipid membranes in an ATP-independent manner in vitro . PITPs exist in all the eukaryotic systems from yeast to human . PITP plays an essential role in intracellular vesicle flow and inositol lipid signaling . The crystal structure of yeast PITP Sec14p reveals a large hydrophobic pocket to accommodate the acyl chains of phospholipid molecules . At the opening of the pocket, a hydrogen bond network may render Sec14p the binding specificity to PI molecules . The structure suggests that the PI-binding ability may play an important role in the in vivo function of PITPs.

Biochim Biophys Acta, 1999 Nov 23, 1441(2-3), 162 - 72
The structure of myristoyl-CoA:protein N-myristoyltransferase; Bhatnagar RS et al.; Protein N-myristoylation is a covalent modification that occurs co-translationally in eukaryotes . Myristate, a rare 14 carbon saturated fatty acid (C14:0), is attached, via an amide linkage, to the N-terminal glycine of a subset of eukaryotic and viral proteins by myristoyl-CoA:protein N-myristoyltransferase (Nmt) . Genetic and biochemical studies have established that Nmt is a target for development of a new class of fungicidal drugs . The enzyme is also a potential target for development of antiviral and antineoplastic agents . The structure of Saccharomyces cerevisiae Nmt1p has been determined recently with bound substrate analogs . The Nmt fold resembles the fold of members of the GCN5-related N-acetyltransferase superfamily . The structure reveals how Nmt's myristoyl-CoA and peptide substrates are recognized and bound, and what elements control the enzyme's ordered kinetic mechanism . Acyl transfer occurs through the nucleophilic addition-elimination reaction: an oxyanion hole formed by main chain atoms polarizes the thioester carbonyl and stabilizes the transition state while deprotonation of the ammonium of the Gly acceptor appears to be mediated by Nmt's C-terminal carboxylate . The use of main chain carboxylate atoms as general base catalyst is a novel feature.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 13926 - 31
Association of terminal deoxynucleotidyl transferase with Ku; Mahajan KN et al.; Terminal deoxynucleotidyl transferase (TdT) catalyzes the addition of nucleotides at the junctions of rearranging Ig and T cell receptor gene segments, thereby generating antigen receptor diversity . Ku is a heterodimeric protein composed of 70- and 86-kDa subunits that binds DNA ends and is required for V(D)J recombination and DNA double-strand break (DSB) repair . We provide evidence for a direct interaction between TdT and Ku proteins . Studies with a baculovirus expression system show that TdT can interact specifically with each of the Ku subunits and with the heterodimer . The interaction between Ku and TdT is also observed in pre-T cells with endogenously expressed proteins . The protein-protein interaction is DNA independent and occurs at physiological salt concentrations . Deletion mutagenesis experiments reveal that the N-terminal region of TdT (131 amino acids) is essential for interaction with the Ku heterodimer . This region, although not important for TdT polymerization activity, contains a BRCA1 C-terminal domain that has been shown to mediate interactions of proteins involved in DNA repair . The induction of DSBs in Cos-7 cells transfected with a human TdT expression construct resulted in the appearance of discrete nuclear foci in which TdT and Ku colocalize . The physical association of TdT with Ku suggests a possible mechanism by which TdT is recruited to the sites of DSBs such as V(D)J recombination intermediates.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 13789 - 94
Evidence that Myb-related CDC5 proteins are required for pre-mRNA splicing; Burns CG et al.; The conserved CDC5 family of Myb-related proteins performs an essential function in cell cycle control at G(2)/M . Although c-Myb and many Myb-related proteins act as transcription factors, herein, we implicate CDC5 proteins in pre-mRNA splicing . Mammalian CDC5 colocalizes with pre-mRNA splicing factors in the nuclei of mammalian cells, associates with core components of the splicing machinery in nuclear extracts, and interacts with the spliceosome throughout the splicing reaction in vitro . Furthermore, genetic depletion of the homolog of CDC5 in Saccharomyces cerevisiae, CEF1, blocks the first step of pre-mRNA processing in vivo . These data provide evidence that eukaryotic cells require CDC5 proteins for pre-mRNA splicing.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 13691 - 6
Neural restrictive silencer factor recruits mSin3 and histone deacetylase complex to repress neuron-specific target genes; Naruse Y et al.; Accumulative evidence suggests that more than 20 neuron-specific genes are regulated by a transcriptional cis-regulatory element known as the neural restrictive silencer (NRS) . A trans-acting repressor that binds the NRS, NRSF {also designated RE1-silencing transcription factor (REST)} has been cloned, but the mechanism by which it represses transcription is unknown . Here we show evidence that NRSF represses transcription of its target genes by recruiting mSin3 and histone deacetylase . Transfection experiments using a series of NRSF deletion constructs revealed the presence of two repression domains, RD-1 and RD-2, within the N- and C-terminal regions, respectively . A yeast two-hybrid screen using the RD-1 region as a bait identified a short form of mSin3B . In vitro pull-down assays and in vivo immunoprecipitation-Western analyses revealed a specific interaction between NRSF-RD1 and mSin3 PAH1-PAH2 domains . Furthermore, NRSF and mSin3 formed a complex with histone deacetylase 1, suggesting that NRSF-mediated repression involves histone deacetylation . When the deacetylation of histones was inhibited by tricostatin A in non-neuronal cells, mRNAs encoding several neuronal-specific genes such as SCG10, NMDAR1, and choline acetyltransferase became detectable . These results indicate that NRSF recruits mSin3 and histone deacetylase 1 to silence neural-specific genes and suggest further that repression of histone deacetylation is crucial for transcriptional activation of neural-specific genes during neuronal terminal differentiation.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 13685 - 90
Topoisomerase II drives DNA transport by hydrolyzing one ATP; Baird CL et al.; DNA topoisomerase II is a homodimeric molecular machine that couples ATP usage to the transport of one DNA segment through a transient break in another segment . In the presence of a nonhydrolyzable ATP analog, the enzyme is known to promote a single turnover of DNA transport . Current models for the enzyme's mechanism based on this result have hydrolysis of two ATPs as the last step, used only to reset the enzyme for another round of reaction . Using rapid-quench techniques, topoisomerase II recently was shown to hydrolyze its two bound ATPs in a strictly sequential manner . This result is incongruous with the models based on the nonhydrolyzable ATP analog data . Here we present evidence that hydrolysis of one ATP by topoisomerase II precedes, and accelerates, DNA transport . These results indicate that important features of this enzyme's mechanism previously have been overlooked because of the reliance on nonhydrolyzable analogs for studying a single reaction turnover . A model for the mechanism of topoisomerase II is presented to show how hydrolysis of one ATP could drive DNA transport.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 13650 - 5
RNA-controlled polymorphism in the in vivo assembly of 180-subunit and 120-subunit virions from a single capsid protein; Krol MA et al.; Repeated, specific interactions between capsid protein (CP) subunits direct virus capsid assembly and exemplify regulated protein-protein interactions . The results presented here reveal a striking in vivo switch in CP assembly . Using cryoelectron microscopy, three-dimensional image reconstruction, and molecular modeling, we show that brome mosaic virus (BMV) CP can assemble in vivo two remarkably distinct capsids that selectively package BMV-derived RNAs in the absence of BMV RNA replication: a 180-subunit capsid indistinguishable from virions produced in natural infections and a previously unobserved BMV capsid type with 120 subunits arranged as 60 CP dimers . Each such dimer contains two CPs in distinct, nonequivalent environments, in contrast to the quasi-equivalent CP environments throughout the 180-subunit capsid . This 120-subunit capsid utilizes most of the CP interactions of the 180-subunit capsid plus nonequivalent CP-CP interactions . Thus, the CP of BMV, and perhaps other viruses, can encode CP-CP interactions that are not apparent from mature virions and may function in assembly or disassembly . Shared structural features suggest that the 120- and 180-subunit capsids share assembly steps and that a common pentamer of CP dimers may be an important assembly intermediate . The ability of a single CP to switch between distinct capsids by means of alternate interactions also implies reduced evolutionary barriers between different capsid structures . The in vivo switch between alternate BMV capsids is controlled by the RNA packaged: a natural BMV genomic RNA was packaged in 180-subunit capsids, whereas an engineered mRNA containing only the BMV CP gene was packaged in 120-subunit capsids . RNA features can thus direct the assembly of a ribonucleoprotein complex between alternate structural pathways.

Proc Natl Acad Sci U S A, 1999 Nov 23, 96(24), 13603 - 10
The protein kinases of Caenorhabditis elegans: a model for signal transduction in multicellular organisms; Plowman GD et al.; Caenorhabditis elegans should soon be the first multicellular organism whose complete genomic sequence has been determined . This achievement provides a unique opportunity for a comprehensive assessment of the signal transduction molecules required for the existence of a multicellular animal . Although the worm C . elegans may not much resemble humans, the molecules that regulate signal transduction in these two organisms prove to be quite similar . We focus here on the content and diversity of protein kinases present in worms, together with an assessment of other classes of proteins that regulate protein phosphorylation . By systematic analysis of the 19,099 predicted C . elegans proteins, and thorough analysis of the finished and unfinished genomic sequences, we have identified 411 full length protein kinases and 21 partial kinase fragments . We also describe 82 additional proteins that are predicted to be structurally similar to conventional protein kinases even though they share minimal primary sequence identity . Finally, the richness of phosphorylation-dependent signaling pathways in worms is further supported with the identification of 185 protein phosphatases and 128 phosphoprotein-binding domains (SH2, PTB, STYX, SBF, 14-3-3, FHA, and WW) in the worm genome.

Mol Pharmacol, 1999 Dec, 56(6), 1105 - 15
Domain interactions affecting human DNA topoisomerase I catalysis and camptothecin sensitivity; Fiorani P et al.; DNA topoisomerase I (Top1p) relaxes supercoiled DNA by the formation of a covalent intermediate in which the active site tyrosine is transiently bound to the severed DNA strand . The antineoplastic agent camptothecin (Cpt) specifically targets Top1p and several mutations have been isolated that render the enzyme Cpt resistant . The mutated residues, although located in different regions of the enzyme, may constitute part of the Cpt binding site . To begin identifying the structural features of DNA Top1p important for Cpt-induced cytotoxicity, we developed a novel yeast genetic screen to isolate catalytically active, yet Cpt-resistant enzymes from a pool of human top1 mutants . Among the mutations isolated were substitutions of Ser or Val for Gly363, which like the Gly363 to Cys mutation previously reported by us, suppressed the Cpt sensitivity of Top1p . In contrast, each amino-acid substitution differed in its ability to suppress the lethal phenotype and catalytic activity of a human top1 mutant top1T718A that resembles Cpt by stabilizing the covalent intermediate . Biochemical analyses and molecular modeling support a model where interactions between two conserved domains, a central "lip" region containing residue Gly363 and the residues around the active site tyrosine (Tyr723), directly affect the formation of the Cpt-binding site and enzyme catalysis.

Biochemistry, 1999 Nov 23, 38(47), 15666 - 72
Streptomyces lividans potassium channel contains poly-(R)-3-hydroxybutyrate and inorganic polyphosphate; Reusch RN; The Streptomyces lividans KcsA potassium channel, a homotetramer of 17.6 kDa subunits, was found to contain two nonproteinaceous polymers, namely, poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) . PHB and polyP are ubiquitous cellular constituents with a demonstrated capacity for cation selection and transport . PHB was detected in both tetramer and monomer species of KcsA by reaction to anti-PHB IgG on Western blots, and estimated as 28 monomer units of PHB per KcsA tetramer by a chemical assay in which PHB is converted to its unique degradation product, crotonic acid . PolyP was detected in KcsA tetramers, but not in monomers, by metachromatic reaction to o-toluidine blue stain on SDS-PAGE gels . A band of free polyP was also visible, suggesting that polyP is released when tetramers dissociate . The exopolyphosphatase of Saccharomyces cerevisiae degraded the free polyP, but tetramer-associated polyP was not affected, indicating it was inaccessible to the enzyme . PolyP in KcsA was estimated as 15 monomer units per tetramer by an enzymatic assay in which polyphosphate kinase is used to transfer phosphates from polyP to {(14)C}ADP, yielding {(14)C}ATP . The experimentally determined isoelectric point of KcsA tetramer was 6.5-7.5, substantially more acidic than the theoretical pI of 10.3, and consistent with the inclusion of a polyanion . The results suggest that PHB is covalently bound to KcsA subunits while polyP is held within tetramers by ionic forces . It is posited that KcsA protein creates an environment in which PHB/polyP is selective for K(+) . The basic amino acids attenuate the negative charge density of polyP, thereby transforming the cation binding preference from multivalent to monovalent, and discrimination between K(+) and Na(+) is accomplished by adjusting the ligand geometry in cation binding cavities formed by PHB and polyP.

Biochemistry, 1999 Nov 23, 38(47), 15653 - 8
Oxidation of the His-52 --> Leu mutant of cytochrome c peroxidase by p-nitroperoxybenzoic acid: role of the distal histidine in hydroperoxide activation; Palamakumbura AH et al.; Both cytochrome c peroxidase (CcP) and a mutant cytochrome c peroxidase in which the distal histidine has been replaced by leucine, CcP(H52L), are converted to hydroxy-ligated derivatives at alkaline pH . In CcP, the hydroxy-ligated derivative is subsequently converted to a bis-imidazole species prior to protein denaturation while the initial hydroxy-ligated CcP(H52L) is converted to a second, spectroscopically distinct hydroxy-ligated species prior to denaturation . The spectra of the alkaline forms of CcP and CcP(H52L) have been determined between 310 and 700 nm . The pH dependence of the rate of reaction between CcP(H52L) and hydrogen peroxide has been extended to pH 10 . The hydroxy-ligated form of CcP(H52L) reacts with hydrogen peroxide 4 times more rapidly than the pentacoordinate, high-spin form of CcP(H52L) that exists at neutral pH . The rate of the reaction between p-nitroperoxybenzoic acid and CcP(H52L) has been measured between pH 4 and pH 8 . Neutral p-nitroperoxybenzoic acid reacts with CcP(H52L) 10(5) times more slowly than with CcP while the negatively charged p-nitroperoxybenzoate reacts with CcP(H52L) 10(3) times more slowly than with CcP . These data indicate that the role of the distal histidine during the initial formation of the peroxy anion/heme iron complex is not simply base catalysis.

Biochemistry, 1999 Nov 23, 38(47), 15647 - 52
Oxidation of cytochrome c peroxidase to compound I by peroxyacids: evidence for rate-limiting diffusion through the protein matrix; Palamakumbura AH et al.; The rate of the reaction between p-nitroperoxybenzoic acid and cytochrome c peroxidase (CcP) has been investigated as a function of pH and ionic strength . The pH dependence of the reaction between CcP and peracetic acid has also been determined . The rate of the reactions are influenced by two heme-linked ionizations in the protein . The enzyme is active when His-52 (pK(a) 3.8 +/- 0.1) is unprotonated and an unknown group with a pK(a) of 9.8 +/- 0.1 is protonated . The bimolecular rate constant for the reaction between peracetic acid and CcP and between p-nitroperoxybenzoic acid and CcP are (1.8 +/- 0.1) x 10(7) and (1.6 +/- 0.2) x 10(7) M(-)(1) s(-)(1), respectively . These rates are about 60% slower than the reaction between hydrogen peroxide and CcP . A critical comparison of the pH dependence of the reactions of hydrogen peroxide, peracetic acid, and p-nitroperoxybenzoic acid with CcP provides evidence that both the neutral and anionic forms of the two peroxyacids react directly with the enzyme . The peracetate and p-nitroperoxybenzoate anions react with CcP with rates of (1.5 +/- 0.1) x 10(6) and (1.6 +/- 0.2) x 10(6) M(-)(1) s(-)(1), respectively, about 10 times slower than the neutral peroxyacids . These data indicate that CcP discriminates between the neutral peroxyacids and their negatively charged ions . However, the apparent bimolecular rate constant for reaction between p-nitroperoxybenzoate and CcP is independent of ionic strength in the range of 0.01-1.0 M, suggesting that electrostatic repulsion between the anion and CcP is not the cause of the lower reactivity for the peroxybenzoate anion . The data are consistent with the hypothesis that the rate-limiting step for the oxidation of CcP to compound I by both neutral peroxyacid and the negatively charged peroxide ion is diffusion of the reactants through the protein matrix, from the surface of the protein to the distal heme pocket.

Biochemistry, 1999 Nov 23, 38(47), 15580 - 6
DNA topoisomerases as targets for the anticancer drug TAS-103: DNA interactions and topoisomerase catalytic inhibition; Fortune JM et al.; TAS-103 is a novel anticancer drug that kills cells by increasing levels of DNA cleavage mediated by topoisomerase II . While most drugs that stimulate topoisomerase II-mediated DNA scission (i.e., topoisomerase II poisons) also inhibit the catalytic activity of the enzyme, they typically do so only at concentrations above the clinical range . TAS-103 is unusual in that it reportedly inhibits the catalytic activity of both topoisomerase I and II and does so at physiologically relevant concentrations {Utsugi, T., et al . (1997) Jpn . J . Cancer Res . 88, 992-1002} . Without a topoisomerase activity to relieve accumulating torsional stress, the DNA tracking systems that promote the action of TAS-103 as a topoisomerase II poison would be undermined . Therefore, the effects of TAS-103 on the catalytic activity of topoisomerase I and II were characterized . DNA binding and unwinding assays indicate that the drug intercalates into DNA with an apparent dissociation constant of approximately 2.2 microM . Furthermore, DNA strand passage assays with mammalian topoisomerase I indicate that TAS-103 does not inhibit the catalytic activity of the type I enzyme . Rather, the previously reported inhibition of topoisomerase I-catalyzed DNA relaxation results from a drug-induced alteration in the apparent topology of the nucleic acid substrate . TAS-103 does inhibit the catalytic activity of human topoisomerase IIalpha, apparently by blocking the DNA religation reaction of the enzyme . The lack of inhibition of topoisomerase I catalytic activity by TAS-103 explains how the drug is able to function as a topoisomerase II poison in treated cells.

Biochemistry, 1999 Nov 23, 38(47), 15573 - 9
DNA topoisomerases as targets for the anticancer drug TAS-103: primary cellular target and DNA cleavage enhancement; Byl JA et al.; TAS-103 is a novel antineoplastic agent that is active against in vivo tumor models {Utsugi, T., et al . (1997) Jpn . J . Cancer Res . 88, 992-1002} . This drug is believed to be a dual topoisomerase I/II-targeted agent, because it enhances both topoisomerase I- and topoisomerase II-mediated DNA cleavage in treated cells . However, the relative importance of these two enzymes for the cytotoxic actions of TAS-103 is not known . Therefore, the primary cellular target of the drug and its mode of action were determined . TAS-103 stimulated DNA cleavage mediated by mammalian topoisomerase I and human topoisomerase IIalpha and beta in vitro . The drug was less active than camptothecin against the type I enzyme but was equipotent to etoposide against topoisomerase IIalpha . A yeast genetic system that allowed manipulation of topoisomerase activity and drug sensitivity was used to determine the contributions of topoisomerase I and II to drug cytotoxicity . Results indicate that topoisomerase II is the primary cellular target of TAS-103 . In addition, TAS-103 binds to human topoisomerase IIalpha in the absence of DNA, suggesting that enzyme-drug interactions play a role in formation of the ternary topoisomerase II.drug.DNA complex . TAS-103 induced topoisomerase II-mediated DNA cleavage at sites similar to those observed in the presence of etoposide . Like etoposide, it enhanced cleavage primarily by inhibiting the religation reaction of the enzyme . Based on these findings, it is suggested that TAS-103 be classified as a topoisomerase II-targeted drug.

Mol Cell Biol, 1999 Dec, 19(12), 8422 - 32
Hsp90 binds and regulates Gcn2, the ligand-inducible kinase of the alpha subunit of eukaryotic translation initiation factor 2 {corrected}; Donze O et al.; The protein kinase Gcn2 stimulates translation of the yeast transcription factor Gcn4 upon amino acid starvation . Using genetic and biochemical approaches, we show that Gcn2 is regulated by the molecular chaperone Hsp90 in budding yeast Saccharomyces cerevisiae . Specifically, we found that (i) several Hsp90 mutant strains exhibit constitutive expression of a GCN4-lacZ reporter plasmid; (ii) Gcn2 and Hsp90 form a complex in vitro as well as in vivo; (iii) the specific inhibitors of Hsp90, geldanamycin and macbecin I, enhance the association of Gcn2 with Hsp90 and inhibit its kinase activity in vitro; (iv) in vivo, macbecin I strongly reduces the levels of Gcn2; (v) in a strain expressing the temperature-sensitive Hsp90 mutant G170D, both the accumulation and activity of Gcn2 are abolished at the restrictive temperature; and (vi) the Hsp90 cochaperones Cdc37, Sti1, and Sba1 are required for the response to amino acid starvation . Taken together, these data identify Gcn2 as a novel target for Hsp90, which plays a crucial role for the maturation and regulation of Gcn2.

Mol Cell Biol, 1999 Dec, 19(12), 8136 - 45
PCAF interacts with tax and stimulates tax transactivation in a histone acetyltransferase-independent manner; Jiang H et al.; Recent studies have shown that the p300/CREB binding protein (CBP)-associated factor (PCAF) is involved in transcriptional activation . PCAF activity has been shown strongly associated with histone acetyltransferase (HAT) activity . In this report, we present evidence for a HAT-independent transcription function that is activated in the presence of the human T-cell leukemia virus type 1 (HTLV-1) Tax protein . In vitro and in vivo GST-Tax pull-down and coimmunoprecipitation experiments demonstrate that there is a direct interaction between Tax and PCAF, independent of p300/CBP . PCAF can be recruited to the HTLV-1 Tax responsive element in the presence of Tax, and PCAF cooperates with Tax in vivo to activate transcription from the HTLV-1 LTR over 10-fold . Point mutations at Tax amino acid 318 (TaxS318A) or 319 to 320 (Tax M47), which have decreased or no activity on the HTLV-1 promoter, are defective for PCAF binding . Strikingly, the ability of PCAF to stimulate Tax transactivation is not solely dependent on the PCAF HAT domain . Two independent PCAF HAT mutants, which knock out acetyltransferase enzyme activity, activate Tax transactivation to approximately the same level as wild-type PCAF . In contrast, p300 stimulation of Tax transactivation is HAT dependent . These studies provide experimental evidence that PCAF contains a coactivator transcription function independent of the HAT activity on the viral long terminal repeat.

Nat Cell Biol, 1999 Nov, 1(7), 423 - 30
Evidence for a COP-I-independent transport route from the Golgi complex to the endoplasmic reticulum; Girod A et al.; The cytosolic coat-protein complex COP-I interacts with cytoplasmic 'retrieval' signals present in membrane proteins that cycle between the endoplasmic reticulum (ER) and the Golgi complex, and is required for both anterograde and retrograde transport in the secretory pathway . Here we study the role of COP-I in Golgi-to-ER transport of several distinct marker molecules . Microinjection of anti-COP-I antibodies inhibits retrieval of the lectin-like molecule ERGIC-53 and of the KDEL receptor from the Golgi to the ER . Transport to the ER of protein toxins, which contain a sequence that is recognized by the KDEL receptor, is also inhibited . In contrast, microinjection of anti-COP-I antibodies or expression of a GTP-restricted Arf-1 mutant does not interfere with Golgi-to-ER transport of Shiga toxin/Shiga-like toxin-1 or with the apparent recycling to the ER of Golgi-resident glycosylation enzymes . Overexpression of a GDP-restricted mutant of Rab6 blocks transport to the ER of Shiga toxin/Shiga-like toxin-1 and glycosylation enzymes, but not of ERGIC-53, the KDEL receptor or KDEL-containing toxins . These data indicate the existence of at least two distinct pathways for Golgi-to-ER transport, one COP-I dependent and the other COP-I independent . The COP-I-independent pathway is specifically regulated by Rab6 and is used by Golgi glycosylation enzymes and Shiga toxin/Shiga-like toxin-1.

Nat Cell Biol, 1999 Nov, 1(7), 415 - 22
G1-phase and B-type cyclins exclude the DNA-replication factor Mcm4 from the nucleus; Labib K et al.; Cyclin-dependent kinases (CDKs) activate the firing of replication origins during the S phase of the cell cycle . They also block re-initiation of DNA replication within a single cell cycle, by preventing the assembly of prereplicative complexes at origins . We show here that, in budding yeast, CDKs exclude the essential prereplicative-complex component Mcm4 from the nucleus . Although origin firing can be triggered by the B-type cyclins only, both G1-phase and B-type cyclins cause exit of Mcm4 from the nucleus . These results suggest that G1 cyclins may diminish the cell's capacity to assemble prereplicative complexes before B-type cyclins trigger origin firing during S phase.

Nat Cell Biol, 1999 Oct, 1(6), 358 - 61
De novo generation of a PrPSc-like conformation in living cells; Ma J et al.; Conformational conversion of the cellular PrPC protein to PrPSc is a central aspect of the prion diseases, but how PrP initially converts to this conformation remains a mystery . Here we show that PrP expressed in the yeast cytoplasm, instead of the endoplasmic reticulum, acquires the characteristics of PrPSc, namely detergent insolubility and a distinct pattern of protease resistance . Neuroblastoma cells cultured under reducing, glycosylation-inhibiting conditions produce PrP with the same characteristics . We therefore describe what is, to our knowledge, the first conversion of full-length PrP in a heterologous system, show the importance of reducing and deglycosylation conditions in PrP conformational transitions, and suggest a model for initiating events in sporadic and inherited prion diseases.

Nat Cell Biol, 1999 Oct, 1(6), 346 - 53
Formation of AP-3 transport intermediates requires Vps41 function; Rehling P et al.; Transport of a subset of membrane proteins to the yeast vacuole requires the function of the AP-3 adaptor protein complex . To define the molecular requirements of vesicular transport in this pathway, we used a biochemical approach to analyse the formation and content of the AP-3 transport intermediate . A vam3tsf (vacuolar t-SNARE) mutant blocks vesicle docking and fusion with the vacuole and causes the accumulation of 50-130-nanometre membrane vesicles, which we isolated and showed by biochemical analysis and immunocytochemistry to contain both AP-3 adaptors and alkaline phosphatase (ALP) pathway cargoes . Inactivation of AP-3 or the protein Vps41 blocks formation of this vesicular intermediate . Vps41 binds to the AP-3 delta-adaptin subunit, suggesting that they function together in the formation of ALP pathway transport intermediates at the late Golgi.

J Biol Chem, 1999 Nov 19, 274(47), 33202 - 5
Receptor-independent activators of heterotrimeric G-protein signaling pathways; Takesono A et al.; Heterotrimeric G-protein signaling systems are activated via cell surface receptors possessing the seven-membrane span motif . Several observations suggest the existence of other modes of stimulus input to heterotrimeric G-proteins . As part of an overall effort to identify such proteins we developed a functional screen based upon the pheromone response pathway in Saccharomyces cerevisiae . We identified two mammalian proteins, AGS2 and AGS3 (activators of G-protein signaling), that activated the pheromone response pathway at the level of heterotrimeric G-proteins in the absence of a typical receptor . beta-galactosidase reporter assays in yeast strains expressing different Galpha subunits (Gpa1, G(s)alpha, G(i)alpha(2(Gpa1(1-41))), G(i)alpha(3(Gpa1(1-41))), Galpha(16(Gpa1(1-41)))) indicated that AGS proteins selectively activated G-protein heterotrimers . AGS3 was only active in the G(i)alpha(2) and G(i)alpha(3) genetic backgrounds, whereas AGS2 was active in each of the genetic backgrounds except Gpa1 . In protein interaction studies, AGS2 selectively associated with Gbetagamma, whereas AGS3 bound Galpha and exhibited a preference for GalphaGDP versus GalphaGTPgammaS . Subsequent studies indicated that the mechanisms of G-protein activation by AGS2 and AGS3 were distinct from that of a typical G-protein-coupled receptor . AGS proteins provide unexpected mechanisms for input to heterotrimeric G-protein signaling pathways . AGS2 and AGS3 may also serve as novel binding partners for Galpha and Gbetagamma that allow the subunits to subserve functions that do not require initial heterotrimer formation.

FEBS Lett, 1999 Nov 19, 461(3), 253 - 7
Interactions between the full complement of human RNA polymerase II subunits; Schaller S et al.; As an approach to elucidating the rules governing the assembly of human RNA polymerase II (hRPB), interactions between its subunits have been systematically analyzed . Eleven of the 12 expected hRPB subunits have previously been tested for reciprocal interactions (J . Biol . Chem . 272 (1997) 16815-16821) . We now report the results obtained for the last subunit (hRPB4; Mol . Cell . Biol . 18 (1998) 1935-1945) and propose an essentially complete picture of the potential interactions occurring within hRPB . Finally, complementation experiments in yeast indicated that hRPB4 expression efficiently cured both heat and cold-sensitivity of RPB4-lacking strains, supporting the existence of conserved functional subunit interactions.

Mol Cell Biol, 1999 Dec, 19(12), 8673 - 85
Analysis of TFIIA function In vivo: evidence for a role in TATA-binding protein recruitment and gene-specific activation; Liu Q et al.; Activation of transcription can occur by the facilitated recruitment of TFIID to promoters by gene-specific activators . To investigate the role of TFIIA in TFIID recruitment in vivo, we exploited a class of yeast TATA-binding protein (TBP) mutants that is activation and DNA binding defective . We found that co-overexpression of TOA1 and TOA2, the genes that encode yeast TFIIA, overcomes the activation defects caused by the TBP mutants . Using a genetic screen, we isolated a new class of TFIIA mutants and identified three regions on TFIIA that are likely to be involved in TBP recruitment or stabilization of the TBP-TATA complex in vivo . Amino acid replacements in only one of these regions enhance TFIIA-TBP-DNA complex formation in vitro, suggesting that the other regions are involved in regulatory interactions . To determine the relative importance of TFIIA in the regulation of different genes, we constructed yeast strains to conditionally deplete TFIIA levels prior to gene activation . While the activation of certain genes, such as INO1, was dramatically impaired by TFIIA depletion, activation of other genes, such as CUP1, was unaffected . These data suggest that TFIIA facilitates DNA binding by TBP in vivo, that TFIIA may be regulated by factors that target distinct regions of the protein, and that promoters vary significantly in the degree to which they require TFIIA for activation.

Mol Cell Biol, 1999 Dec, 19(12), 8633 - 45
Synthetic lethality with conditional dbp6 alleles identifies rsa1p, a nucleoplasmic protein involved in the assembly of 60S ribosomal subunits; Kressler D et al.; Dbp6p is an essential putative ATP-dependent RNA helicase that is required for 60S-ribosomal-subunit assembly in the yeast Saccharomyces cerevisiae (D . Kressler, J . de la Cruz, M . Rojo, and P . Linder, Mol . Cell . Biol . 18:1855-1865, 1998) . To identify factors that are functionally interacting with Dbp6p, we have performed a synthetic lethal screen with conditional dbp6 mutants . Here, we describe the cloning and the phenotypic analysis of the previously uncharacterized open reading frame YPL193W, which we renamed RSA1 (ribosome assembly 1) . Rsa1p is not essential for cell viability; however, rsa1 null mutant strains display a slow-growth phenotype, which is exacerbated at elevated temperatures . The rsa1 null allele synthetically enhances the mild growth defect of weak dbp6 alleles and confers synthetic lethality when combined with stronger dbp6 alleles . Polysome profile analysis shows that the absence of Rsa1p results in the accumulation of half-mer polysomes . However, the pool of free 60S ribosomal subunits is only moderately decreased; this is reminiscent of polysome profiles from mutants defective in 60S-to-40S subunit joining . Pulse-chase labeling of pre-rRNA in the rsa1 null mutant strain indicates that formation of the mature 25S rRNA is decreased at the nonpermissive temperature . Interestingly, free 60S ribosomal subunits of a rsa1 null mutant strain that was grown for two generations at 37 degrees C are practically devoid of the 60S-ribosomal-subunit protein Qsr1p/Rpl10p, which is required for joining of 60S and 40S subunits (D . P . Eisinger, F . A . Dick, and B . L . Trumpower, Mol . Cell . Biol . 17:5136-5145, 1997) . Moreover, the combination of the Deltarsa1 and qsr1-1 mutations leads to a strong synthetic growth inhibition . Finally, a hemagglutinin epitope-tagged Rsa1p localizes predominantly to the nucleoplasm . Together, these results point towards a function for Rsa1p in a late nucleoplasmic step of 60S-ribosomal-subunit assembly.

Mol Cell Biol, 1999 Dec, 19(12), 8314 - 25
Characterization of a novel member of the DOK family that binds and modulates Abl signaling; Cong F et al.; A novel member of the p62(dok) family of proteins, termed DOKL, is described . DOKL contains features of intracellular signaling molecules, including an N-terminal PH (pleckstrin homology) domain, a central PTB (phosphotyrosine binding) domain, and a C-terminal domain with multiple potential tyrosine phosphorylation sites and proline-rich regions, which might serve as docking sites for SH2- and SH3-containing proteins . The DOKL gene is predominantly expressed in bone marrow, spleen, and lung, although low-level expression of the RNA can also be detected in other tissues . DOKL and p62(dok) bind through their PTB domains to the Abelson tyrosine kinase in a kinase-dependent manner in both yeast and mammalian cells . DOKL is phosphorylated by the Abl tyrosine kinase in vivo . In contrast to p62(dok), DOKL lacks YxxP motifs in the C terminus and does not bind to Ras GTPase-activating protein (RasGAP) upon phosphorylation . Overexpression of DOKL, but not p62(dok), suppresses v-Abl-induced mitogen-activated protein (MAP) kinase activation but has no effect on constitutively activated Ras- and epidermal growth factor-induced MAP kinase activation . The inhibitory effect requires the PTB domain of DOKL . Finally, overexpression of DOKL in NIH 3T3 cells inhibits the transforming activity of v-Abl . These results suggest that DOKL may modulate Abl function.

Mol Cell Biol, 1999 Dec, 19(12), 8302 - 13
Yap1p activates gene transcription in an oxidant-specific fashion; Coleman ST et al.; Positive regulation of gene expression by the yeast Saccharomyces cerevisiae transcription factor Yap1p is required for normal tolerance of oxidative stress elicited by the redox-active agents diamide and H(2)O(2) . Several groups have provided evidence that a cluster of cysteine residues in the extreme C terminus of the factor are required for normal modulation of Yap1p by oxidant challenge . Deletion of this C-terminal cysteine-rich domain (c-CRD) produces a protein that is highly active under both stressed and nonstressed conditions and is constitutively located in the nucleus . We have found that a variety of different c-CRD mutant proteins are hyperactive in terms of their ability to confer diamide tolerance to cells but fail to provide even normal levels of H(2)O(2) resistance . Although the c-CRD mutant forms of Yap1p activate an artificial Yap1p-responsive gene to the same high level in the presence of either diamide or H(2)O(2), these mutant factors confer hyperresistance to diamide but hypersensitivity to H(2)O(2) . To address this discrepancy, we have examined the ability of c-CRD mutant forms of Yap1p to activate expression of an authentic target gene required for H(2)O(2) tolerance, TRX2 . When assayed in the presence of c-CRD mutant forms of Yap1p, a TRX2-lacZ fusion gene fails to induce in response to H(2)O(2) . We have also identified a second cysteine-rich domain, in the N terminus (n-CRD), that is required for H(2)O(2) but not diamide resistance and influences the localization of the protein . These data are consistent with the idea that the function of Yap1p is different at promoters of loci involved in H(2)O(2) tolerance from promoters of genes involved in diamide resistance.

Mol Cell Biol, 1999 Dec, 19(12), 8123 - 35
Biochemical analysis of distinct activation functions in p300 that enhance transcription initiation with chromatin templates; Kraus WL et al.; To investigate the mechanisms of transcriptional enhancement by the p300 coactivator, we analyzed wild-type and mutant versions of p300 with a chromatin transcription system in vitro . Estrogen receptor, NF-kappaB p65 plus Sp1, and Gal4-VP16 were used as different sequence-specific activators . The CH3 domain (or E1A-binding region) was found to be essential for the function of each of the activators tested . The bromodomain was also observed to be generally important for p300 coactivator activity, though to a lesser extent than the CH3 domain/E1A-binding region . The acetyltransferase activity and the C-terminal region (containing the steroid receptor coactivator/p160-binding region and the glutamine-rich region) were each found to be important for activation by estrogen receptor but not for that by Gal4-VP16 . The N-terminal region of p300, which had been previously found to interact with nuclear hormone receptors, was not seen to be required for any of the activators, including estrogen receptor . Single-round transcription experiments revealed that the functionally important subregions of p300 contribute to its ability to promote the assembly of transcription initiation complexes . In addition, the acetyltransferase activity of p300 was observed to be distinct from the broadly essential activation function of the CH3 domain/E1A-binding region . These results indicate that specific regions of p300 possess distinct activation functions that are differentially required to enhance the assembly of transcription initiation complexes . Interestingly, with the estrogen receptor, four distinct regions of p300 each have an essential role in the transcription activation process . These data exemplify a situation in which a network of multiple activation functions is required to achieve gene transcription.

Mol Cell Biol, 1999 Dec, 19(12), 8003 - 15
Biochemical analysis of the intrinsic Mcm4-Mcm6-mcm7 DNA helicase activity; You Z et al.; Mcm proteins play an essential role in eukaryotic DNA replication, but their biochemical functions are poorly understood . Recently, we reported that a DNA helicase activity is associated with an Mcm4-Mcm6-Mcm7 (Mcm4,6,7) complex, suggesting that this complex is involved in the initiation of DNA replication as a DNA-unwinding enzyme . In this study, we have expressed and isolated the mouse Mcm2, 4,6,7 proteins from insect cells and characterized various mutant Mcm4,6,7 complexes in which the conserved ATPase motifs of the Mcm4 and Mcm6 proteins were mutated . The activities associated with such preparations demonstrated that the DNA helicase activity is intrinsically associated with the Mcm4,6,7 complex . Biochemical analyses of these mutant Mcm4,6,7 complexes indicated that the ATP binding activity of the Mcm6 protein in the complex is critical for DNA helicase activity and that the Mcm4 protein may play a role in the single-stranded DNA binding activity of the complex . The results also indicated that the two activities of DNA helicase and single-stranded DNA binding can be separated.

Mol Cell Biol, 1999 Dec, 19(12), 7933 - 43
Splicing of the meiosis-specific HOP2 transcript utilizes a unique 5' splice site; Leu JY et al.; The Saccharomyces cerevisiae HOP2 gene is required to prevent formation of synaptonemal complex between nonhomologous chromosomes during meiosis . The HOP2 gene is expressed specifically in meiotic cells, with the transcript reaching maximum abundance early in meiotic prophase . The HOP2 coding region is interrupted by an intron located near the 5' end of the gene . This intron contains a nonconsensus 5' splice site (GUUAAGU) that differs from the consensus 5' splice signal (GUAPyGU) by the insertion of a nucleotide and by a single nucleotide substitution . Bases flanking the HOP2 5' splice site have the potential to pair with sequences in U1 small nuclear RNA, and mutations disrupting this pairing reduce splicing efficiency . HOP2 pre-mRNA is spliced efficiently in the absence of the Mer1 and Nam8 proteins, which are required for splicing the transcripts of two other meiosis-specific genes.

J Biol Chem, 1999 Nov 26, 274(48), 34045 - 52
Domain requirements of DnaJ-like (Hsp40) molecular chaperones in the activation of a steroid hormone receptor; Fliss AE et al.; DnaJ-like proteins function in association with Hsp70 molecular chaperones to facilitate protein folding . We previously demonstrated that a yeast DnaJ-like protein, Ydj1p, was important for activation of heterologously expressed steroid hormone receptors (Caplan, A . J., Langley, E., Wilson, E . M., and Vidal, J . (1995) J . Biol . Chem . 270, 5251-5257) . In the present study, we analyzed Ydj1p function by assaying hormone binding to the human androgen receptor (AR) heterologously expressed in yeast . We analyzed hormone binding in strains that were wild type or deleted for the YDJ1 gene . In the deletion mutant, the AR did not bind hormone to the same extent as the wild type . Introduction of mutant forms of Ydj1p to the deletion strain revealed that the J-domain is necessary but not sufficient for Ydj1p action, and that other domains of the protein are also functionally important . Of three human DnaJ-like proteins introduced into the deletion mutant, only Hdj2, which displays full domain conservation with Ydj1p, suppressed the hormone binding defect of the deletion mutant . By comparison of the domains shared by these three human proteins, and with mutants of Ydj1p that were functional, it was deduced that the cysteine-rich zinc binding domain is important for Hdj2/Ydj1p action in hormone receptor function . A model for the mechanism of DnaJ-like protein action is discussed.

J Biol Chem, 1999 Nov 26, 274(48), 33913 - 20
The prosegments of furin and PC7 as potent inhibitors of proprotein convertases . In vitro and ex vivo assessment of their efficacy and selectivity; Zhong M et al.; All proprotein convertases (PCs) of the subtilisin/kexin family contain an N-terminal prosegment that is presumed to act both as an intramolecular chaperone and an inhibitor of its parent enzyme . In this work, we examined inhibition by purified, recombinant bacterial prosegments of furin and PC7 on the in vitro processing of either the fluorogenic peptide pERTKR-MCA or the human immunodeficiency virus envelope glycoprotein gp160 . These propeptides are potent inhibitors that display measurable selectivity toward specific proprotein convertases . Small, synthetic decapeptides derived from the C termini of the prosegments are also potent inhibitors, albeit less so than the full-length proteins, and the C-terminal P1 arginine is essential for inhibition . The bacterial, recombinant prosegments were also used to generate specific antisera, allowing us to study the intracellular metabolic fate of the prosegments of furin and PC7 expressed via vaccinia virus constructs . These vaccinia virus recombinants, along with transient transfectants of the preprosegments of furin and PC7, efficiently inhibited the ex vivo processing of the neurotrophins nerve growth factor and brain-derived neurotrophic factor . Thus, we have demonstrated for the first time that PC prosegments, expressed ex vivo as independent domains, can act in trans to inhibit precursor maturation by intracellular PCs.

Crit Rev Biochem Mol Biol, 1999, 34(5), 285 - 314
Biochemistry of iron uptake; Wessling-Resnick M; Recent information gained from genetic and biochemical studies of iron transport in yeast, coupled with the identification of specific mutations causing iron uptake disorders in mice and man, has provided new clues about the mechanisms involved in iron uptake . This article summarizes these discoveries and discusses their impact on our current understanding of the biochemistry of iron uptake.

Curr Opin Clin Nutr Metab Care, 1998 Jul, 1(4), 341 - 6
Vitamin A and regulation of gene expression; Nagpal S et al.; The biologically active form of vitamin A, retinoic acid, and its synthetic analogs exhibit potent anti-proliferative normalization of differentiation and anti-inflammatory activities, which appear to account for their therapeutic effects in hyperproliferative and inflammatory diseases, such as acne, psoriasis, photoaging and neoplasias . These therapeutic effects are achieved by their ability to regulate complex programs of gene expression in target cells by binding to nuclear receptors, which are ligand-dependent transcription factors . This article reviews retinoid regulated genes and recently identified mechanisms which play important roles in the regulation of transcription by retinoids.

Gene, 1999 Nov 15, 240(1), 115 - 23
Identification, characterization and comparative analysis of a novel chorismate mutase gene in Arabidopsis thaliana; Mobley EM et al.; Phenylalanine, tyrosine, and tryptophan have a dual biosynthetic role in plants; they are required for protein synthesis and are also precursors to a number of aromatic secondary metabolites critical to normal development and stress responses . Whereas much has been learned in recent years about the genetic control of tryptophan biosynthesis in Arabidopsis and other plants, relatively little is known about the genetic regulation of phenylalanine and tyrosine synthesis . We have isolated, characterized and determined the expression of Arabidopsis thaliana genes encoding chorismate mutase, the enzyme catalyzing the first committed step in phenylalanine and tyrosine synthesis . Three independent Arabidopsis chorismate mutase cDNAs were isolated by functional complementation of a Saccharomyces cerevisiae mutation . Two of these cDNAs have been reported independently (Eberhard et al., 1993 . FEBS 334, 233-236; Eberhard et al., 1996 . Plant J . 10, 815-821), but the third (designated CM-3) represents a novel gene . The different organ-specific expression patterns of these cDNAs, their regulation in response to pathogen infiltration, as well as the different enzymatic characteristics of the proteins they encode are also described . Together, these data suggest that each isoform may play a distinct physiological role in coordinating chorismate mutase activity with developmental and environmental signals.

Biochim Biophys Acta, 1999 Nov 16, 1472(3), 498 - 508
Isolation and characterization of Cox17p from porcine heart by determining its survival-promoting activity in NIH3T3 cells; Takenouchi T et al.; We have found that the gel filtration fraction of porcine heart extract clearly promoted the survival of NIH3T3 fibroblast cells in the serum-free medium condition . A structural analysis showed that the active fraction contained a novel peptide, porcine Cox17p (p-Cox17p), which was recently reported by Chen et al . as dopuin (Z . W . Chen et al., Eur . J . Biochem . 249 (1997) 518-522) . Porcine Cox17p/dopuin possesses high sequence homology to the product of human COX17 gene (h-Cox17p) . Although Cox17p has been implied to be involved in copper recruitment to mitochondria and in the functional assembly of cytochrome oxidase in yeast, its role in mammalian cells is unknown . In this study, we chemically synthesized p-Cox17p to investigate its biological effects . Refolding experiments of synthesized linear p-Cox17p revealed the existence of mostly one pattern of three intrachain disulfide bridges similar to that of native p-Cox17p, because the main oxidized p-Cox17p was completely co-eluted with the natural product . The addition of heavy metal ions such as copper, zinc and cadmium significantly inhibited the formation of the oxidized form, suggesting that reduced p-Cox17p may interact directly with these metal ions . The reduced and oxidized forms of p-Cox17p were also confirmed to promote the survival of NIH3T3 cells in serum-free medium as observed with the natural product, indicating that Cox17p may be a bioactive peptide.

J Cell Sci, 1999 Dec, 112 ( Pt 23), 4185 - 91
Surfing the Sec61 channel: bidirectional protein translocation across the ER membrane; Romisch K; Misfolded secretory and transmembrane proteins are retained in the endoplasmic reticulum (ER) and subsequently degraded . Degradation is primarily mediated by cytosolic proteasomes and thus requires retrograde transport out of the ER back to the cytosol . The available evidence suggests that the protein-conducting channel formed by the Sec61 complex is responsible for both forward and retrograde transport of proteins across the ER membrane . For transmembrane proteins, retrograde transport can be viewed as a reversal of integration of membrane proteins into the ER membrane . Retrograde transport of soluble proteins through the Sec61 channel after signal-peptide cleavage, however, must be mechanistically distinct from signal-peptide-mediated import into the ER through the same channel.

J Biomol Struct Dyn, 1999 Oct, 17(2), 281 - 8
Statistical analysis of the exon-intron structure of higher and lower eukaryote genes; Kriventseva EV et al.; Statistics of the exon-intron structure and splicing sites of several diverse eukaryotes was studied . The yeast exon-intron structures have a number of unique features . A yeast gene usually have at most one intron . The branch site is strongly conserved, whereas the polypirimidine tract is short . Long yeast introns tend to have stronger acceptor sites . In other species the branch site is less conserved and often cannot be determined . In non-yeast samples there is an almost universal correlation between lengths of neighboring exons (all samples excluding protists) and correlation between lengths of neighboring introns (human, drosophila, protists) . On the average first introns are longer, and anomalously long introns are usually first introns in a gene . There is a universal preference for exons and exon pairs with the (total) length divisible by 3 . Introns positioned between codons are preferred, whereas those positioned between the first and second positions in codon are avoided . The choice of A or G at the third position of intron (the donor splice sites generally prefer purines at this position) is correlated with the overall GC-composition of the gene . In all samples dinucleotide AG is avoided in the region preceding the acceptor site.

EMBO J, 1999 Nov 15, 18(22), 6561 - 72
Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase; Pellicioli A et al.; The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle . We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins . Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect . Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells . Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis . We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.

EMBO J, 1999 Nov 15, 18(22), 6439 - 47
Proteasome-mediated degradation of transcriptional activators correlates with activation domain potency in vivo; Molinari E et al.; We show that the intracellular concentration of transcriptional activator proteins is regulated by the proteasome-mediated protein degradation pathway . The rate of degradation of activators by proteasomes correlates with activation domain potency in vivo . Mutations either in the activation domain residues involved in target protein interaction or in the DNA-binding domain residues essential for DNA binding abolish the transcriptional activation function in vivo and render the activator resistant to degradation by proteasomes . Finally, using a rapamycin-regulated gene expression system, we show that recruiting activation domains to DNA-bound receptor proteins greatly enhanced the rate of degradation of reconstituted activators . These observations suggest that in mammalian cells efficient recruitment of activator-target protein complexes to the promoter means that they are subjected to rapid degradation by proteasomes . We propose that proteasome-mediated control of the intracellular levels of transcriptional activators could play an important role in the regulation of gene expression.

EMBO J, 1999 Nov 15, 18(22), 6407 - 14
Chromatin remodelling at the PHO8 promoter requires SWI-SNF and SAGA at a step subsequent to activator binding; Gregory PD et al.; The SWI-SNF and SAGA complexes possess ATP-dependent nucleosome remodelling activity and histone acetyltransferase (HAT) activity, respectively . Mutations that eliminate the ATPase activity of the SWI-SNF complex, or the HAT activity of SAGA, abolish proper chromatin remodelling at the PHO8 promoter in vivo . These effects are mechanistically distinct, since the absence of SWI-SNF freezes chromatin in the repressed state, while the absence of Gcn5 permits a localized perturbation of chromatin structure immediately adjacent to the upstream transactivator binding site . However, this remodelling is not propagated to the proximal promoter, and no activation is observed under all conditions . Furthermore, Pho4 is bound to the PHO8 promoter in the absence of Snf2 or Gcn5, confirming a role for SWI-SNF and SAGA in chromatin remodelling independent of activator binding . These data provide new insights into the roles of the SWI-SNF and SAGA complexes in chromatin remodelling in vivo.

J Cell Biol, 1999 Nov 15, 147(4), 699 - 706
Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape; Sesaki H et al.; In yeast, mitochondrial division and fusion are highly regulated during growth, mating and sporulation, yet the mechanisms controlling these activities are unknown . Using a novel screen, we isolated mutants in which mitochondria lose their normal structure, and instead form a large network of interconnected tubules . These mutants, which appear defective in mitochondrial division, all carried mutations in DNM1, a dynamin-related protein that localizes to mitochondria . We also isolated mutants containing numerous mitochondrial fragments . These mutants were defective in FZO1, a gene previously shown to be required for mitochondrial fusion . Surprisingly, we found that in dnm1 fzo1 double mutants, normal mitochondrial shape is restored . Induction of Dnm1p expression in dnm1 fzo1 cells caused rapid fragmentation of mitochondria . We propose that dnm1 mutants are defective in the mitochondrial division, an activity antagonistic to fusion . Our results thus suggest that mitochondrial shape is normally controlled by a balance between division and fusion which requires Dnm1p and Fzo1p, respectively.

Glycobiology, 1999 Dec, 9(12), 1287 - 93
Characterization of an alg2 mutant of the zygomycete fungus Rhizomucor pusillus; Takeuchi K et al.; The zygomycete fungus Rhizomucor pusillus secretes an aspartic proteinase (MPP) that contains asparagine ( N )-linked oligosaccharides at two sites . Mutant strain 1116 defective in N -glycosylation secretes MPP with truncated oligo-saccharide chains . Lipid-linked oligosaccharides in mutant 1116 were labeled with {6-(3)H}glucosamine and {2-(3)H}mannose, prepared by cycles of solvent extraction, and analyzed by gel filtration chromatography on a Bio-Gel P-4 column after mild acid-hydrolysis . Mutant 1116 accumulated an intermediate, Man(1)GlcNAc(2)-dolichol pyrophosphate (PP-Dol), whereas wild-type strain F27 synthesized the fully assembled oligosaccharide precursor Glc(3)Man(9)GlcNAc(2)-PP-Dol . Consistent with this, alg2 encoding a mannosyltransferase in the lipid-linked oligosaccharide biosynthetic pathway in mutant 1116 had a 5 bp insertion that generated a stop codon in the middle of the coding sequence . Transformation of mutant 1116 with the intact alg2 gene on a pUC19-derived plasmid generated transformants that contained multicopies of alg2 at the alg2 locus . Glycosylation of the total proteins in the transformants was recovered to the same level as in strain F27, as determined with peroxidase-concanavalin A . These transformants produced MPP mainly with the same N -linked oligosaccharides as that produced by strain F27, but still with truncated oligosaccharides in small amounts . All of these data show that Alg2 is an alpha-1,3 or alpha-1,6 mannosyltransferase that elongates Man(1)GlcNAc(2)-PP-Dol to Man(2)GlcNAc(2)-PP-Dol . The slower growth of mutant 1116 was significantly recovered on introduction of alg2 . The viability of the alg2 mutants of the zygomycete R.pusillus makes a contrast with the lethal effect of ALG2 mutations in the yeast Saccharomyces cerevisiae.

Mech Dev, 1999 Dec, 89(1-2), 155 - 9
Nrk: a murine X-linked NIK (Nck-interacting kinase)-related kinase gene expressed in skeletal muscle; Kanai-Azuma M et al.; We report the cloning and expression pattern of a novel Ste20-type kinase gene, NIK-related kinase (Nrk), located on the mouse X chromosome . The full-length Nrk cDNA encodes a 1455-amino-acid polypeptide characterized by a N-terminal Ste20-type catalytic domain and a C-terminal regulatory domain characteristic of the group I GCK subfamily . The overall structure of the NRK protein is closely related to that of Nck-interacting kinase (Nik) . In situ hybridization revealed that Nrk was predominantly expressed in skeletal muscle during mouse embryogenesis . Nrk gene expression was detected in the myotome at 10.5 dpc and, thereafter, was observed in developing skeletal musculature from 11.5 to 13.5 dpc . However, expression in skeletal muscle was not observed in adults.

J Biol Chem, 1999 Nov 19, 274(47), 33785 - 9
An unexpected link between the secretory path and the organization of the nucleus; Nanduri J et al.; Yeast sec mutations define the machinery of vesicular traffic . Surprisingly, many of these mutations also inhibit ribosome biogenesis by reducing transcription of rRNA and genes encoding ribosomal proteins . We observe that these mutants reversibly inhibit protein import into the nucleus, with import cargo accumulating at the nucleoplasmic face of nuclear pore complexes, as when Ran-GTP cannot bind importins . They also rapidly and reversibly relocate multiple nucleolar and nucleoplasmic proteins to the cytoplasm . The import block and relocation are antagonized by overexpression of yeast Ran, Hog1p kinase, or Ssa/Hsp70 proteins or by inhibition of protein synthesis . These nucleocytoplasmic signaling events document an extraordinary plasticity of nuclear organization.

J Biol Chem, 1999 Nov 19, 274(47), 33778 - 84
Genetic dissection of hTAF(II)130 defines a hydrophobic surface required for interaction with glutamine-rich activators; Rojo-Niersbach E et al.; The general transcription factor TFIID is a multiprotein complex consisting of the TATA box-binding protein and multiple TATA box-binding protein-associated factors (TAF(II)s) . The central domain of human TAF(II)130 contains four glutamine-rich regions Q1-Q4 that interact with transcriptional activators such as Sp1 and CREB and mediate activation . We screened in yeast random point mutations introduced into Q1-Q4 against the Sp1 activation domain and obtained a distinct set of hTAF(II)130s with alterations in TAF(II)-activator interaction . Here we characterize functionally an hTAF(II)130 mutant containing a phenylalanine to serine change at position 311 (F311S) that is compromised in its ability to associate with Sp1B and CREB-N activation domains . Substitution of phenylalanine with tyrosine but not with isoleucine or tryptophan also reduced hTAF(II)130 interaction, suggesting that the hydrophobic character rather than the specific amino acid at this position is a key determinant of interaction . Deletion of nine amino acids (Delta9) surrounding Phe(311) abolished the interaction of hTAF(II)130 with Sp1 . Overexpression of hTAF(II)130Q1/Q2 and Q1-Q4 strongly inhibited Sp1-dependent transcriptional enhancement in transient transfection assays, whereas expression of either F311S or Delta9 only partially suppressed Sp1-mediated activation . Thus, a short hydrophobic sequence motif encompassing Phe(311) in hTAF(II)130 represents a critical surface with which Sp1B interacts to activate transcription.

J Biol Chem, 1999 Nov 19, 274(47), 33287 - 95
Human JIK, a novel member of the STE20 kinase family that inhibits JNK and is negatively regulated by epidermal growth factor; Tassi E et al.; Mammalian members related to Saccharomyces cerevisiae serine/threonine kinase STE20 can be divided into two subfamilies based on their structure and function . The PAK subfamily is characterized by an N-terminal p21-binding domain (also known as CRIB domain), a C-terminal kinase domain, and is regulated by the small GTP-binding proteins Rac1 and Cdc42Hs . The second group is represented by the GCK-like members, which contain an N-terminal catalytic domain and lack the p21-binding domain . Some of them have been demonstrated to induce c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) cascade, while others have been shown to be activated by a subset of stress conditions or apoptotic agents, although little is known about their specific function . Here, we have identified a novel human STE20-related serine/threonine kinase, belonging to the GCK-like subfamily . This kinase does not induce the JNK/SAPK pathway, but, instead, inhibits the basal activity of JNK/SAPK, and diminishes its activation in response to human epidermal growth factor (EGF) . Therefore, we designated this molecule JIK for JNK/SAPK-inhibitory kinase . The inhibition of JNK/SAPK signaling pathway by JIK was found to occur between the EGF receptor and the small GTP-binding proteins Rac1 and Cdc42Hs . In contrast, JIK does not activate nor does it inhibit ERK2, ERK6, p38, or ERK5 . Furthermore, JIK kinase activity is not modulated by any exogenous stimuli, but, interestingly, it is dramatically decreased upon EGF receptor activation . Thus, JIK might represent the first member of the STE20 kinase family whose activity can be negatively regulated by tyrosine kinase receptors, and whose downstream targets inhibit, rather than enhance, JNK/SAPK activation.

Biochem Biophys Res Commun, 1999 Nov 19, 265(2), 509 - 12
Molecular cloning of rat ATX1 homologue protein; Hiromura M et al.; An ATX1 homologue of 503 bp length was cloned from a rat cDNA library, and the deduced protein from the cDNA was found to contain 68 amino acids with a predicted molecular mass of 7.2 kDa . The rat ATX1 homologue protein (Rah1p), which shows 35%, 38%, and 89% identities with Atx1p, CUC-1, and HAH1, respectively, conserves both the MTCXXC copper-binding site in the N terminus and the KTGK lysine-rich region in the C terminus . In Northern blot analysis, rah1 mRNA was found to be expressed at high levels in the liver, small intestine, and testis . Expression of rah1 cDNA complemented a null atx1 mutant strain in yeast . Thus, Rah1p was concluded to be a functional copper chaperone .

Mol Biol Cell, 1999 Nov, 10(11), 3549 - 65
Shr3p mediates specific COPII coatomer-cargo interactions required for the packaging of amino acid permeases into ER-derived transport vesicles; Gilstring CF et al.; The SHR3 gene of Saccharomyces cerevisiae encodes an integral membrane component of the endoplasmic reticulum (ER) with four membrane-spanning segments and a hydrophilic, cytoplasmically oriented carboxyl-terminal domain . Mutations in SHR3 specifically impede the transport of all 18 members of the amino acid permease (aap) gene family away from the ER . Shr3p does not itself exit the ER . Aaps fully integrate into the ER membrane and fold properly independently of Shr3p . Shr3p physically associates with the general aap Gap1p but not Sec61p, Gal2p, or Pma1p in a complex that can be purified from N-dodecylmaltoside-solubilized membranes . Pulse-chase experiments indicate that the Shr3p-Gap1p association is transient, a reflection of the exit of Gap1p from the ER . The ER-derived vesicle COPII coatomer components Sec13p, Sec23p, Sec24p, and Sec31p but not Sar1p bind Shr3p via interactions with its carboxyl-terminal domain . The mutant shr3-23p, a nonfunctional membrane-associated protein, is unable to associate with aaps but retains the capacity to bind COPII components . The overexpression of either Shr3p or shr3-23p partially suppresses the temperature-sensitive sec12-1 allele . These results are consistent with a model in which Shr3p acts as a packaging chaperone that initiates ER-derived transport vesicle formation in the proximity of aaps by facilitating the membrane association and assembly of COPII coatomer components.

Nat Cell Biol, 1999 Aug, 1(4), 221 - 6
The base of the proteasome regulatory particle exhibits chaperone-like activity; Braun BC et al.; Protein substrates of the proteasome must apparently be unfolded and translocated through a narrow channel to gain access to the proteolytic active sites of the enzyme . Protein folding in vivo is mediated by molecular chaperones . Here, to test for chaperone activity of the proteasome, we assay the reactivation of denatured citrate synthase . Both human and yeast proteasomes stimulate the recovery of the native structure of citrate synthase . We map this chaperone-like activity to the base of the regulatory particle of the proteasome, that is, to the ATPase-containing assembly located at the substrate-entry ports of the channel . Denatured but not native citrate synthase is bound by the base complex . Ubiquitination of citrate synthase is not required for its binding or refolding by the base complex of the proteasome . These data suggest a model in which ubiquitin-protein conjugates are initially tethered to the proteasome by specific recognition of their ubiquitin chains; this step is followed by a nonspecific interaction between the base and the target protein, which promotes substrate unfolding and translocation.

Nat Cell Biol, 1999 Jul, 1(3), 130 - 5
Competition between glutathione and protein thiols for disulphide-bond formation; Cuozzo JW et al.; It has long been assumed that the oxidized form of glutathione, the tripeptide glutamate-cysteine-glycine, is a source of oxidizing equivalents needed for the formation of disulphide bonds in proteins within the endoplasmic reticulum (ER), although the in vivo function of glutathione in the ER has never been studied directly . Here we show that the major pathway for oxidation in the yeast ER, defined by the protein Ero1, is responsible for the oxidation of both glutathione and protein thiols . However, mutation and overexpression studies show that glutathione competes with protein thiols for the oxidizing machinery . Thus, contrary to expectation, cellular glutathione contributes net reducing equivalents to the ER; these reducing equivalents can buffer the ER against transient hyperoxidizing conditions.

Nat Cell Biol, 1999 Jun, 1(2), E47 - 53
Regulation of the APC and the exit from mitosis; Morgan DO; The events of late mitosis, from sister-chromatid separation to cytokinesis, are governed by the anaphase-promoting complex (APC), a multisubunit assembly that triggers the ubiquitin-dependent proteloysis of key regulatory proteins . An intricate regulatory network governs APC activity and helps to ensure that late mitotic events are properly timed and coordinated.

J Bacteriol, 1999 Nov, 181(22), 6876 - 81
CzcD is a heavy metal ion transporter involved in regulation of heavy metal resistance in Ralstonia sp . strain CH34; Anton A et al.; The Czc system of Ralstonia sp . strain CH34 mediates resistance to cobalt, zinc, and cadmium through ion efflux catalyzed by the CzcCB(2)A cation-proton antiporter . The CzcD protein is involved in the regulation of the Czc system . It is a membrane-bound protein with at least four transmembrane alpha-helices and is a member of a subfamily of the cation diffusion facilitator (CDF) protein family, which occurs in all three domains of life . The deletion of czcD in a Ralstonia sp . led to partially constitutive expression of the Czc system due to an increased transcription of the structural czcCBA genes, both in the absence and presence of inducers . The czcD deletion could be fully complemented in trans by CzcD and two other CDF proteins from Saccharomyces cerevisiae, ZRC1p and COT1p . All three proteins mediated a small but significant resistance to cobalt, zinc, and cadmium in Ralstonia, and this resistance was based on a reduced accumulation of the cations . Thus, CzcD appeared to repress the Czc system by an export of the inducing cations.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13542 - 7
A mitogen-activated protein kinase of the corn leaf pathogen Cochliobolus heterostrophus is involved in conidiation, appressorium formation, and pathogenicity: diverse roles for mitogen-activated protein kinase homologs in foliar pathogens; Lev S et al.; Fungal pathogens perceive and respond to molecules from the plant, triggering pathogenic development . Transduction of these signals may use heterotrimeric G proteins, and it is thought that protein phosphorylation cascades are also important . We have isolated a mitogen-activated protein kinase homolog from the corn pathogen Cochliobolus heterostrophus to test its role as a component of the transduction pathways . The new gene, CHK1, has a deduced amino acid sequence 90% identical to Pmk1 of the rice blast fungus Magnaporthe grisea and 59% identical to Fus3 of Saccharomyces cerevisiae . A series of chk1 deletion mutants has poorly developed aerial hyphae, autolysis, and no conidia . No pseudothecia are formed when a cross between two Deltachk1 mutants is attempted . The ability of Deltachk1 mutants to infect corn plants is reduced severely . The growth pattern of hyphae on a glass surface is strikingly altered from that of the wild type, forming coils or loops, but no appressoria . This set of phenotypes overlaps only partially with that of pmk1 mutants, the homologous gene of the rice blast fungus . In particular, sexual and asexual sporulation both require Chk1 function in Cochliobolus heterostrophus, in contrast to Pmk1, but perhaps more similar to yeast, where Fus3 transmits the mating signal . Chk1 is required for efficient colonization of leaf tissue, which can be compared with filamentous invasive growth of yeast, modulated through another closely related mitogen-activated protein kinase, Kss1 . Ubiquitous signaling elements thus are used in diverse ways in different plant pathogens, perhaps the result of coevolution of the transducers and their targets.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13363 - 8
Interaction of the copper chaperone HAH1 with the Wilson disease protein is essential for copper homeostasis; Hamza I et al.; The delivery of copper to specific sites within the cell is mediated by distinct intracellular carrier proteins termed copper chaperones . Previous studies in Saccharomyces cerevisiae suggested that the human copper chaperone HAH1 may play a role in copper trafficking to the secretory pathway of the cell . In this current study, HAH1 was detected in lysates from multiple human cell lines and tissues as a single-chain protein distributed throughout the cytoplasm and nucleus . Studies with a glutathione S-transferase-HAH1 fusion protein demonstrated direct protein-protein interaction between HAH1 and the Wilson disease protein, which required the cysteine copper ligands in the amino terminus of HAH1 . Consistent with these in vitro observations, coimmunoprecipitation experiments revealed that HAH1 interacts with both the Wilson and Menkes proteins in vivo and that this interaction depends on available copper . When these studies were repeated utilizing three disease-associated mutations in the amino terminus of the Wilson protein, a marked diminution in HAH1 interaction was observed, suggesting that impaired copper delivery by HAH1 constitutes the molecular basis of Wilson disease in patients harboring these mutations . Taken together, these data provide a mechanism for the function of HAH1 as a copper chaperone in mammalian cells and demonstrate that this protein is essential for copper homeostasis.

Proc Natl Acad Sci U S A, 1999 Nov 9, 96(23), 13086 - 90
The dimensions of the protein import channels in the outer and inner mitochondrial membranes; Schwartz MP et al.; Most mitochondrial proteins are imported into mitochondria through transmembrane channels composed largely, and perhaps exclusively, of proteins . We have determined the effective internal diameter of the protein import channel in the mitochondrial outer membrane to be between 20 A and 26 A during translocation . The diameter of the import channel in the inner membrane is smaller than the diameter of the outer membrane import channel . These results were obtained by measuring the effect of rigid steric bulk introduced into precursor proteins on import.

Genes Dev, 1999 Nov 1, 13(21), 2811 - 27
A specific protein-protein interaction accounts for the in vivo substrate selectivity of Ptp3 towards the Fus3 MAP kinase; Zhan XL et al.; The mitogen-activated protein kinases (MAPKs) play critical roles in many signal transduction processes . Several MAPKs have been found in Saccharomyces cerevisiae, including Fus3 in the mating pathway and Hog1 in the osmotic-stress response pathway . Cells lacking Fus3 or Hog1 activity are deficient in mating or adaptation to osmotic shock, respectively . However, constitutive activation of either Fus3 or Hog1 is lethal . Therefore, yeast cells have to tightly regulate both the activation and inactivation of Fus3 and Hog1 MAPKs, which are controlled mainly by phosphorylation and dephosphorylation . Previous studies have shown that Fus3 activity is negatively regulated by protein tyrosine phosphatase Ptp3 . In contrast, the Hog1 MAPK is mainly dephosphorylated by Ptp2 even though the two phosphatases share a high degree of sequence similarity . To understand the mechanisms of MAPK regulation, we examined the molecular basis underlying the in vivo substrate specificity between phosphatases and MAPKs . We observed that the amino-terminal noncatalytic domain of Ptp3 directly interacts with Fus3 via CH2 (Cdc25 homology) domain conserved among yeast PTPases and mammalian MAP kinase phosphatases and is responsible for the in vivo substrate selectivity of the phosphatase . Interaction between Ptp3 and Fus3 is required for dephosphorylation and inactivation of Fus3 under physiological conditions . Mutations in either Ptp3 or Fus3 that abolish this interaction cause a dysregulation of the Fus3 MAPK . Our data demonstrate that the specificity of MAP kinase inactivation in vivo by phosphatases is determined by specific protein-protein interactions outside of the phosphatase catalytic domain.

Biochim Biophys Acta, 1999 Sep 21, 1451(2-3), 242 - 54
Functional characterization of ARAKIN (ATMEKK1): a possible mediator in an osmotic stress response pathway in higher plants; Covic L et al.; The Arabidopsis thaliana ARAKIN (ATMEKK1) gene shows strong homology to members of the (MAP) mitogen-activated protein kinase family, and was previously shown to functionally complement a mating defect in Saccharomyces cerevisiae at the level of the MEKK kinase ste11 . The yeast STE11 is an integral component of two MAP kinase cascades: the mating pheromone pathway and the HOG (high osmolarity glycerol response) pathway . The HOG signal transduction pathway is activated by osmotic stress and causes increased glycerol synthesis . Here, we first demonstrate that ATMEKK1 encodes a protein with kinase activity, examine its properties in yeast MAP kinase cascades, then examine its expression under stress in A . thaliana . Yeast cells expressing the A . thaliana ATMEKK1 survive and grow under high salt (NaCl) stress, conditions that kill wild-type cells . Enhanced glycerol production, observed in non-stressed cells expressing ATMEKK1 is the probable cause of yeast survival . Downstream components of the HOG response pathway, HOG1 and PBS2, are required for ATMEKK1-mediated yeast survival . Because ATMEKK1 functionally complements the sho1/ssk2/ssk22 triple mutant, it appears to function at the level of the MEKK kinase step of the HOG response pathway . In A . thaliana, ATMEKK1 expression is rapidly (within 5 min) induced by osmotic (NaCl) stress . This is the same time frame for osmoticum-induced effects on the electrical properties of A . thaliana cells, both an immediate response and adaptation . Therefore, we propose that the A . thaliana ATMEKK1 may be a part of the signal transduction pathway involved in osmotic stress.

FEBS Lett, 1999 Nov 5, 460(3), 505 - 12
A thermostable vacuolar-type membrane pyrophosphatase from the archaeon Pyrobaculum aerophilum: implications for the origins of pyrophosphate-energized pumps; Drozdowicz YM et al.; Vacuolar-type H(+)-translocating pyrophosphatases (V-PPases) have been considered to be restricted to plants, a few species of phototrophic proteobacteria and protists . Here, we describe PVP, a thermostable, sequence-divergent V-PPase from the facultatively aerobic hyperthermophilic archaeon Pyrobaculum aerophilum . PVP shares only 38% sequence identity with both the prototypical V-PPase from Arabidopsis thaliana and the H(+)-PPi synthase from Rhodospirillum rubrum, yet possesses most of the structural features characteristic of V-PPases . Heterologous expression of PVP in Saccharomyces cerevisiae yields a M(r) 64 inverted question mark omitted inverted question mark000 membrane polypeptide that specifically catalyzes Mg(2+)-dependent PPi hydrolysis . The existence of PVP implies that PPi-energized H(+)-translocation is phylogenetically more deeply rooted than previously thought.

Hum Mol Genet, 1999 Dec, 8(13), 2533 - 40
Characterization of SURF-1 expression and Surf-1p function in normal and disease conditions; Tiranti V et al.; Loss-of-function mutations of the SURF-1 gene have been associated with Leigh syndrome with cytochrome c oxidase (COX) deficiency . Mature Surf-1 protein (Surf-1p) is a 30 kDa hydrophobic polypeptide whose function is still unknown . Using antibodies against a recombinant, hemagglutinin-tagged Surf-1p, we have demonstrated that this protein is imported into mitochondria as a larger precursor, which is then processed into the mature product by cleaving off an N-terminal leader polypeptide of approximately 40 amino acids . By using western blot analysis with specific antibodies, we showed that Surf-1p is localized in and tightly bound to the mitochondrial inner membrane . The same analysis revealed that no protein is present in cell lines harboring loss-of-function mutations of SURF-1, regardless of their type and position . Northern blot analysis showed the virtual absence of specific SURF-1 transcripts in different mutant cell lines . This result suggests that several mutations of SURF-1 are associated with severe mRNA instability . To understand better whether and which domains of the protein are essential for function, we generated several constructs with truncated or partially deleted SURF-1 cDNAs . None of these constructs, expressed into Surf-1p null mutant cells, were able to rescue the COX phenotype, suggesting that different regions of the protein are all essential for function . Finally, experiments based on blue native two-dimensional gel electrophoresis indicated that assembly of COX in Surf-1p null mutants is blocked at an early step, most likely before the incorporation of subunit II in the nascent intermediates composed of subunit I alone or subunit I plus subunit IV . However, detection of residual amounts of fully assembled complex suggests a certain degree of redundancy of this system.

Curr Biol, 1999 Nov 4, 9(21), R803 - 6
Nucleocytoplasmic transport: Inside out regulation; Hopper AK; Cells can respond to extracellular signals by redistributing transcription regulators between the nucleus and cytosol . Recent findings in budding yeast indicate that the nuclear transport receptor Msn5p mediates the nuclear export of several transcription regulators after their phosphorylation in the nucleus.

J Mol Evol, 1999 Nov, 49(5), 567 - 82
The evolution of the MAP kinase pathways: coduplication of interacting proteins leads to new signaling cascades; Caffrey DR et al.; The MAP-kinase pathways are intracellular signaling modules that are likely to exist in all eukaryotes . We provide an evolutionary model for these signaling pathways by focusing on the gene duplications that have occurred since the divergence of animals from yeast . Construction of evolutionary trees with confidence assessed by bootstrap clearly shows that the mammalian JNK and p38 pathways arose from an ancestral hyperosmolarity pathway after the split from yeast and before the split from C . elegans . These coduplications of interacting proteins at the MAPK and MEK levels have since evolved toward substrate specificity, thus giving distinct pathways . Mammalian duplications since the split from C . elegans are often associated with divergent tissue distribution but do not appear to confer detectable substrate specificity . The yeast kinase cascades have undergone similar fundamental functional changes since the split from mammals, with duplications giving rise to central signaling components of the filamentous and hypoosmolarity pathways . Experimentally defined cross-talk between yeast pheromone and hyperosmolarity pathways is mirrored with corresponding cross-talk in mammalian pathways, suggesting the existence of ancient orthologous cross-talk; our analysis of gene duplications at all levels of the cascade is consistent with this model but does not always provide significant bootstrap support . Our data also provide insights at different levels of the cascade where conflicting experimental evidence exists.

J Biol Chem, 1999 Nov 12, 274(46), 32565 - 73
Identification of a central phosphorylation site in p21-activated kinase regulating autoinhibition and kinase activity; Zenke FT et al.; p21-activated kinases (Pak)/Ste20 kinases are regulated in vitro and in vivo by the small GTP-binding proteins Rac and Cdc42 and lipids, such as sphingosine, which stimulate autophosphorylation and phosphorylation of exogenous substrates . The mechanism of Pak activation by these agents remains unclear . We investigated Pak kinase activation in more detail to gain insight into the interplay between the GTPase/sphingosine binding, an intramolecular inhibitory interaction, and autophosphorylation . We present biochemical evidence that an autoinhibitory domain (ID) contained within amino acid residues 67-150 of Pak1 interacts with the carboxyl-terminal kinase domain and that this interaction is regulated in a GTPase-dependent fashion . Cdc42- and sphingosine-stimulated Pak1 activity can be inhibited in trans by recombinant ID peptide, indicating similarities in their mode of activation . However, Pak1, which was autophosphorylated in response to either GTPase or sphingosine, is highly active and is insensitive to inhibition by the ID peptide . We identified phospho-acceptor site threonine 423 in the kinase activation loop as a critical determinant for the sensitivity to autoinhibition and enzymatic activity . Phosphorylation studies suggested that the stimulatory effect of both GTPase and sphingosine results in exposure of the activation loop, making it accessible for intermolecular phosphorylation.

Nat Genet, 1999 Nov, 23(3), 281 - 5
Diverse and dynamic functions of the Sir silencing complex; Guarente L; The yeast Sir protein complex has been implicated in transcriptional silencing and suppression of recombination . The Sir complex creates structured chromosomal domains at telomeres, silent mating-type loci and ribosomal DNA to invoke these functional states . Mechanistic insights into the function of Sir proteins implicate a range of activities in yeast, including repair of DNA double-strand breaks, regulation of the mitotic cell cycle, meiosis and ageing . I speculate that the Sir proteins may be capable of enzymatic modification of chromatin and other substrates, which enables them to carry out a broad range of cellular functions.

Science, 1999 Nov 5, 286(5442), 1166 - 71
Control of the DNA damage checkpoint by chk1 and rad53 protein kinases through distinct mechanisms; Sanchez Y et al.; In response to DNA damage, cells activate checkpoint pathways that prevent cell cycle progression . In fission yeast and mammals, mitotic arrest in response to DNA damage requires inhibitory Cdk phosphorylation regulated by Chk1 . This study indicates that Chk1 is required for function of the DNA damage checkpoint in Saccharomyces cerevisiae but acts through a distinct mechanism maintaining the abundance of Pds1, an anaphase inhibitor . Unlike other checkpoint mutants, chk1 mutants were only mildly sensitive to DNA damage, indicating that checkpoint functions besides cell cycle arrest influence damage sensitivity . Another kinase, Rad53, was required to both maintain active cyclin-dependent kinase 1, Cdk1(Cdc28), and prevent anaphase entry after checkpoint activation . Evidence suggests that Rad53 exerts its role in checkpoint control through regulation of the Polo kinase Cdc5 . These results support a model in which Chk1 and Rad53 function in parallel through Pds1 and Cdc5, respectively, to prevent anaphase entry and mitotic exit after DNA damage . This model provides a possible explanation for the role of Cdc5 in DNA damage checkpoint adaptation.

Science, 1999 Nov 5, 286(5442), 1153 - 5
A biochemical genomics approach for identifying genes by the activity of their products; Martzen MR et al.; For the identification of yeast genes specifying biochemical activities, a genomic strategy that is rapid, sensitive, and widely applicable was developed with an array of 6144 individual yeast strains, each containing a different yeast open reading frame (ORF) fused to glutathione S-transferase (GST) . For the identification of ORF-associated activities, strains were grown in defined pools, and GST-ORFs were purified . Then, pools were assayed for activities, and active pools were deconvoluted to identify the source strains . Three previously unknown ORF-associated activities were identified with this strategy: a cyclic phosphodiesterase that acts on adenosine diphosphate-ribose 1"-2" cyclic phosphate (Appr>p), an Appr-1"-p-processing activity, and a cytochrome c methyltransferase.

Science, 1999 Nov 5, 286(5442), 1146 - 9
An adenosine deaminase that generates inosine at the wobble position of tRNAs; Gerber AP et al.; Several transfer RNAs (tRNAs) contain inosine (I) at the first position of their anticodon (position 34); this modification is thought to enlarge the codon recognition capacity during protein synthesis . The tRNA-specific adenosine deaminase of Saccharomyces cerevisiae that forms I(34) in tRNAs is described . The heterodimeric enzyme consists of two sequence-related subunits (Tad2p/ADAT2 and Tad3p/ADAT3), both of which contain cytidine deaminase (CDA) motifs . Each subunit is encoded by an essential gene (TAD2 and TAD3), indicating that I(34) is an indispensable base modification in elongating tRNAs . These results provide an evolutionary link between the CDA superfamily and RNA-dependent adenosine deaminases (ADARs/ADATs).

Neuroreport, 1999 Sep 29, 10(14), 3037 - 43
A protein with the characters of a zinc-finger is implicated in the differentiation of Schwann cells; Labatut-Cazabat I et al.; During the development of the PNS, Schwann cells (SC) differentiate into myelinating and nonmyelinating cells, implying regulation by different transcription factors such as ZF proteins . Employing an original strategy using monoclonal antibodies specifically directed against the conserved ZF motif, we have identified a new ZF protein of 55 kDa present in rat sciatic nerve extract (SCp55) . We used polyclonal antibodies and cloned cDNA to characterize the expression of SCp55 by immunohistochemistry and in situ hybridization . This protein is specific for SC and shows differential expression both during development and between the two SC phenotypes . When they differentiate the protein is first induced in myelinating SC and then in nonmyelinating SC . The nature of this protein together with its differential expression suggests that it is a transcription factor that may have a role in the development of SC.

Mol Cell, 1999 Oct, 4(4), 657 - 64
Transcriptional activation by Gcn4p involves independent interactions with the SWI/SNF complex and the SRB/mediator; Natarajan K et al.; Mutations in three subunits of the SWI/SNF complex and in the Med2p subunit of the SRB/mediator of pol II holoenzyme impaired Gcn4p-activated transcription of HIS3 without reducing Gcn4p-independent transcription of this gene . Recombinant Gcn4p interacted with SWI/SNF and SRB/mediator subunits in cell extracts in a manner dependent on the same hydrophobic clusters in the Gcn4p activation domain; however, higher concentrations of Gcn4p were required for binding to SWI/SNF versus SRB/mediator subunits . In addition, SRB/mediator and SWI/SNF subunits did not coimmunopreciptate from the extracts . These findings, together with the fact that Gcn4p specifically interacted with purified SWI/SNF, strongly suggest that Gcn4p independently recruits SWI/SNF and holoenzyme to its target promoters in the course of activating transcription.

Mol Cell, 1999 Oct, 4(4), 649 - 55
Activation domain-mediated targeting of the SWI/SNF complex to promoters stimulates transcription from nucleosome arrays; Neely KE et al.; The yeast SWI/SNF complex is required for the transcription of several yeast genes and has been shown to alter nucleosome structure in an ATP-dependent reaction . In this study, we show that the complex stimulated in vitro transcription from nucleosome templates in an activation domain-dependent manner . Transcription stimulation by SWI/SNF required an activation domain with which it directly interacts . The acidic activation domains of VP16, Gcn4, Swi5, and Hap4 interacted directly with the purified SWI/SNF complex and with the SWI/SNF complex in whole-cell extracts . The similarity of activation domain interactions and transcriptional stimulation between SWI/SNF and the SAGA histone acetyltransferase complex may account for their apparent overlapping functions in vivo.

Mol Cell, 1999 Oct, 4(4), 479 - 86
A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila; Sevrioukov EA et al.; Deep orange and carnation are two of the classic eye color genes in Drosophila . Here, we demonstrate that Deep orange is part of a protein complex that localizes to endosomal compartments . A second component of this complex is Carnation, a homolog of Sec1p-like regulators of membrane fusion . Because complete loss of deep orange function is lethal, the role of this complex in intracellular trafficking was analyzed in deep orange mutant clones . Retinal cells devoid of deep orange function completely lacked pigmentation and exhibited exaggerated multivesicular structures . Furthermore, a defect in endocytic trafficking was visualized in developing photoreceptor cells . These results provide direct evidence that eye color mutations of the granule group also disrupt vesicular trafficking to lysosomes.

Mol Cell, 1999 Oct, 4(4), 469 - 77
Ero1p oxidizes protein disulfide isomerase in a pathway for disulfide bond formation in the endoplasmic reticulum; Frand AR et al.; Native protein disulfide bond formation in the endoplasmic reticulum (ER) requires protein disulfide isomerase (PDI) and Ero1p . Here we show that oxidizing equivalents flow from Ero1p to substrate proteins via PDI . PDI is predominantly oxidized in wild-type cells but is reduced in an ero1-1 mutant . Direct dithiol-disulfide exchange between PDI and Ero1p is indicated by the capture of PDI-Ero1p mixed disulfides . Mixed disulfides can also be detected between PDI and the ER precursor of carboxypeptidase Y (CPY) . Further, PDI1 is required for the net formation of disulfide bonds in newly synthesized CPY, indicating that PDI functions as an oxidase in vivo . Together, these results define a pathway for protein disulfide bond formation in the ER . The PDI homolog Mpd2p is also oxidized by Ero1p.

Gene, 1999 Nov 1, 239(2), 333 - 40
Evidence for kinship between diverse G-protein coupled receptors; Josefsson LG; In an earlier publication we described similarities at the primary sequence level between the first probable plant G-protein coupled receptor (GPCR) and three GPCR families (families A, B and F according to Kolakowski's classification) that were previously considered evolutionarily unrelated . Here we analyze further the relatedness among different GPCR families.By using PSI-BLAST, which is a search algorithm that is more potent in detecting weak similarities, one finds additional similarities between GPCR families that have not previously been described . Based on these comparisons, it is possible to divide all the GPCR families into one large clade and two smaller ones . The large clade includes the rhodopsin family (family A), the glucagon receptor family (family B), cyclic AMP receptors (family F), an Arabidopsis thaliana receptor, the Frizzled family and probably also the STE3 pheromone receptors (family E) and vomeronasal receptors type 1 . The smaller clades consist of, in one case, BOSS and the GABA-B family of receptors (family C), and in the other the STE2 pheromone receptors (family D) alone . Although our findings are likely to reflect a common ancestry within each of these clades, whether or not two or all three of the clades also share an even more ancient ancestor between them remains an open question that cannot be answered from our present data.

Biochim Biophys Acta, 1999 Nov 16, 1422(3), 235 - 54
Mechanisms of protein translocation into mitochondria; Voos W et al.; Mitochondrial biogenesis utilizes a complex proteinaceous machinery for the import of cytosolically synthesized preproteins . At least three large multisubunit protein complexes, one in the outer membrane and two in the inner membrane, have been identified . These translocase complexes cooperate with soluble proteins from the cytosol, the intermembrane space and the matrix . The translocation of presequence-containing preproteins through the outer membrane channel includes successive electrostatic interactions of the charged mitochondrial targeting sequence with a chain of import components . Translocation across the inner mitochondrial membrane utilizes the energy of the proton motive force of the inner membrane and the hydrolysis of ATP . The matrix chaperone system of the mitochondrial heat shock protein 70 forms an ATP-dependent import motor by interaction with the polypeptide chain in transit and components of the inner membrane translocase . The precursors of integral inner membrane proteins of the metabolite carrier family interact with newly identified import components of the intermembrane space and are inserted into the inner membrane by a second translocase complex . A comparison of the full set of import components between the yeast Sacccharomyces cerevisiae and the nematode Caenorhabditis elegans demonstrates an evolutionary conservation of most components of the mitochondrial import machinery with a possible greater divergence for the import pathway of the inner membrane carrier proteins.

Biochim Biophys Acta, 1999 Nov 16, 1422(3), 207 - 34
Modelling G-protein-coupled receptors for drug design; Flower DR; The G-protein coupled receptors form a large and diverse multi-gene superfamily with many important physiological functions . As such, they have become important targets in pharmaceutical research . Molecular modelling and site-directed mutagenesis have played an important role in our increasing understanding of the structural basis of drug action at these receptors . Aspects of this understanding, how these techniques can be used within a drug-design programme, and remaining challenges for the future are reviewed.

Biochem Biophys Res Commun, 1999 Nov, 265(1), 170 - 6
The E-box of the human glycophorin B promoter is involved in the erythroid-specific expression of the GPB gene; Camara-Clayette V et al.; Previous studies performed on the glycophorin B (GPB) expression demonstrated that this gene is expressed in erythroid cells only and that the ubiquitous factor Ku70 is involved in the process . Here, we investigated the contribution of the -70 E-box sequence toward the GPB promoter expression . We found that the E-box bound two factors, the USF1/USF2 protein and an unidentified ubiquitous protein which was named factor U . Site-directed mutagenesis performed on the -70 E-box showed that the USF factor had an activating effect in CAT assays . Conversely, mutation of the -70 E-box that impaired the binding of factor U led to a positive CAT activity in nonerythroid cells and thus to the loss of the erythroid-specific expression of the GPB gene . This indicates that, in addition to the Ku70 factor, the extinction of the GPB promoter expression in nonerythroid cells depends also on the repressing effect of the factor U .

Biochem Biophys Res Commun, 1999 Nov, 265(1), 79 - 86
Biochemical characterization and expression analysis of neural thrombospondin-1-like proteins NELL1 and NELL2; Kuroda S et al.; Two closely related genes coding for NELL proteins (NELL1 and NELL2) have been cloned by the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C betaI (PKCbetaI) as bait . The rat NELL proteins show about 55% identity with each other and contain several protein motifs assigned to a secretion signal peptide, an NH(2)-terminal thrombospondin-1 (TSP-1)-like module, five von Willebrand factor C domains, and six epidermal growth factor-like domains; the NELL proteins share many protein motifs with TSP-1 . The NELL proteins expressed in COS-7 cells are homotrimeric glycoproteins and possess heparin-binding activity . Furthermore, while NELL1 and NELL2 show distinct subcellular localization in cytoplasm, they both are partially secreted into the culture medium of COS-7 cells . Although the NELL1 mRNA is faintly expressed in adult neural cells, the NELL2 mRNA is expressed abundantly, particularly in the pyramidal cells of rat hippocampus, showing neuronal high plasticity . During mouse embryogenesis, expression of the NELL2 mRNA is initiated 7-11 days postcoitum, simultaneously with neural plate formation . These results strongly suggest that the NELL2 protein, similar to but not identical with TSP-1, is involved in the growth and differentiation of neural cells . Additionally, the NELL1 and NELL2 mRNAs were found to be expressed abundantly in Burkitt's lymphoma Raji cells and colorectal adenocarcinoma SW480 cells, respectively . Thus, it is likely that the NELL proteins also participate in the growth, differentiation, and oncogenesis of cancer cell lines .

J Cell Sci, 1999 Nov, 112 ( Pt 22), 4123 - 34
Genetic interactions of Hrd3p and Der3p/Hrd1p with Sec61p suggest a retro-translocation complex mediating protein transport for ER degradation; Plemper RK et al.; The endoplasmic reticulum contains a quality control system that subjects misfolded or unassembled secretory proteins to rapid degradation via the cytosolic ubiquitin proteasome system . This requires retrograde protein transport from the endoplasmic reticulum back to the cytosol . The Sec61 pore, the central component of the protein import channel into the endoplasmic reticulum, was identified as the core subunit of the retro-translocon as well . As import of mutated proteins into the endoplasmic reticulum lumen is successfully terminated, a new targeting mechanism must exist that mediates re-entering of misfolded proteins into the Sec61 pore from the lumenal side de novo . The previously identified proteins Der3p/Hrd1p and, as we show here, Hrd3p of the yeast Saccharomyces cerevisiae, are localised in the endoplasmic reticulum membrane and are essential for the degradation of several substrates of the endoplasmic reticulum degradation machinery . Based on genetic studies we demonstrate that they functionally interact with each other and with Sec61p, probably establishing the central part of the retro-translocon . In the absence of Hrd3p, the otherwise stable protein Der3p/Hrd1p becomes rapidly degraded . This depends on a functional ubiquitin proteasome system and the presence of substrate molecules of the endoplasmic reticulum degradation system . When overexpressed, Der3p/Hrd1p accelerates CPY* degradation in Delta(hrd3) cells . Our data suggest a recycling process of Der3p/Hrd1p through Hrd3p . The retro-translocon seems to be build up at least by the Sec61 pore, Der3p/Hrd1p and Hrd3p and mediates both retrograde transport and ubiquitination of substrate molecules.

J Cell Sci, 1999 Nov, 112 ( Pt 22), 4031 - 9
Differential subcellular localization of DNA-dependent protein kinase components Ku and DNA-PKcs during mitosis; Koike M et al.; The Ku protein is a complex of two subunits, Ku70 and Ku80 . Ku plays an important role in DNA-PKcs-dependent double-strand break repair and V(D)J recombination, and in growth regulation, which is DNA-PKcs-independent . We studied the expression and the subcellular localization of Ku and DNA-PKcs throughout the cell cycle in several established human cell lines . Using immunofluorescence analysis and confocal laser scanning microscopy, we detected Ku70 and Ku80 in the nuclei in interphase cells . In mitotic cells (1) most of Ku protein was found diffused in the cytoplasm, (2) a fraction was detected at the periphery of condensed chromosomes, (3) no Ku protein was present in the chromosome interior . Association of Ku with isolated chromosomes was also observed . On the other hand, DNA-PKcs was detected in the nucleus in interphase cells and not at the periphery of condensed chromosomes during mitosis . Using indirect immunoprecipitation, we found that throughout the cell cycle, Ku70 and Ku80 were present as heterodimers, some in complex with DNA-PKcs . Our findings suggest that the localization of Ku at the periphery of metaphase chromosomes might be imperative for a novel function of Ku in the G(2)/M phase, which does not require DNA-PKcs.

Acta Biochim Pol, 1999, 46(2), 377 - 89
Structure and biogenesis of small nucleolar RNAs acting as guides for ribosomal RNA modification; Filipowicz W et al.; Maturation of pre-ribosomal RNA (pre-rRNA) in eukaryotic cells takes place in the nucleolus and involves a large number of cleavage events, which frequently follow alternative pathways . In addition, rRNAs are extensively modified, with the methylation of the 2'-hydroxyl group of sugar residues and conversion of uridines to pseudouridines being the most frequent modifications . Both cleavage and modification reactions of pre-rRNAs are assisted by a variety of small nucleolar RNAs (snoRNAs), which function in the form of ribonucleoprotein particles (snoRNPs) . The majority of snoRNAs acts as guides directing site-specific 2'-O-ribose methylation or pseudouridine formation . Over one hundred RNAs of this type have been identified to date in vertebrates and the yeast Saccharomyces cerevisiae . This number is readily explained by the findings that one snoRNA acts as a guide usually for one or at most two modifications, and human rRNAs contain 91 pseudouridines and 106 2'-O-methyl residues . In this article we review information about the biogenesis, structure and function of guide snoRNAs.

Acta Biochim Pol, 1999, 46(2), 315 - 24
Glycosylation defects corrected by the changes in GDPmannose level; Kruszewska J et al.; GDPMan is a key substrate in glycoprotein formation . This is especially true for lower eukaryotes where, in addition to the involvement in N-glycan biosynthesis and GPI-anchor formation, GDPMan takes part in the process which is unique for yeast and fungi i.e . O-mannosylation . Several lines of evidence have been presented that the level of GDPMan affects the process occurring in the Golgi compartment i.e . the elongation of outer mannose chain of glycoproteins in Saccharomyces cerevisiae . Results from our laboratory indicate that the availability of GDPMan affects also the early steps of glycoprotein formation ascribed to the endoplasmic reticulum, i.e . assembly of the dolichol-linked oligosaccharide as well as mannosyl-phosphodolichol (MPD) formation . The biochemical basis of carbohydrate deficient glycoprotein syndrome, a severe neurological disorder related to the GDPMan deficiency, is also discussed.

J Cell Biol, 1999 Nov 1, 147(3), 559 - 76
PAKa, a putative PAK family member, is required for cytokinesis and the regulation of the cytoskeleton in Dictyostelium discoideum cells during chemotaxis; Chung CY et al.; We have identified a Dictyostelium discoideum gene encoding a serine/threonine kinase, PAKa, a putative member of the Ste20/PAK family of p21-activated kinases, with a kinase domain and a long NH(2)-terminal regulatory domain containing an acidic segment, a polyproline domain, and a CRIB domain . PAKa colocalizes with myosin II to the cleavage furrow of dividing cells and the posterior of polarized, chemotaxing cells via its NH(2)-terminal domain . paka null cells are defective in completing cytokinesis in suspension . PAKa is also required for maintaining the direction of cell movement, suppressing lateral pseudopod extension, and proper retraction of the posterior of chemotaxing cells . paka null cells are defective in myosin II assembly, as the myosin II cap in the posterior of chemotaxing cells and myosin II assembly into cytoskeleton upon cAMP stimulation are absent in these cells, while constitutively active PAKa leads to an upregulation of myosin II assembly . PAKa kinase activity against histone 2B is transiently stimulated and PAKa incorporates into the cytoskeleton with kinetics similar to those of myosin II assembly in response to chemoattractant signaling . However, PAKa does not phosphorylate myosin II . We suggest that PAKa is a major regulator of myosin II assembly, but does so by negatively regulating myosin II heavy chain kinase.

Int Immunol, 1999 Nov, 11(11), 1753 - 61
Expression of the Bcl-2 family member A1 is developmentally regulated in T cells; Tomayko MM et al.; During T cell development, cells that fail to meet stringent selection criteria undergo programmed cell death . Thymocyte and peripheral T cell susceptibility to apoptosis is influenced by expression of Bcl-2 family members, some of which are expressed in a developmentally patterned manner . We previously showed developmentally regulated expression of A1, an anti-apoptotic Bcl-2 family member, among B cell developmental subsets . Here we show that cells of the T lineage also express A1 in a developmentally regulated manner . Both A1 mRNA and A1 protein are readily detectable in the thymus, and while present among DN cells, A1 mRNA is up-regulated to very high levels among double-positive (DP) thymocytes . It is then down-regulated to moderate levels among single-positive (SP) thymocytes, and finally expressed at approximately 25-fold lower levels among mature SP CD4(+) and CD8(+) lymph node T cells than among DP thymocytes . Furthermore, we find that in vitro TCR ligation up-regulates A1 expression among both DP and SP thymocytes . Together, these data show that A1 expression is developmentally regulated in T lymphocytes and is responsive to TCR signaling, suggesting that A1 may play a role in maintaining the viability of DP thymocytes.

Genetics, 1999 Nov, 153(3), 1117 - 30
A novel allele of RAD52 that causes severe DNA repair and recombination deficiencies only in the absence of RAD51 or RAD59; Bai Y et al.; With the use of an intrachromosomal inverted repeat as a recombination reporter, we have shown that mitotic recombination is dependent on the RAD52 gene, but reduced only fivefold by mutation of RAD51 . RAD59, a component of the RAD51-independent pathway, was identified previously by screening for mutations that reduced inverted-repeat recombination in a rad51 strain . Here we describe a rad52 mutation, rad52R70K, that also reduced recombination synergistically in a rad51 background . The phenotype of the rad52R70K strain, which includes weak gamma-ray sensitivity, a fourfold reduction in the rate of inverted-repeat recombination, elevated allelic recombination, sporulation proficiency, and a reduction in the efficiency of mating-type switching and single-strand annealing, was similar to that observed for deletion of the RAD59 gene . However, rad52R70K rad59 double mutants showed synergistic defects in ionizing radiation resistance, sporulation, and mating-type switching . These results suggest that Rad52 and Rad59 have partially overlapping functions and that Rad59 can substitute for this function of Rad52 in a RAD51 rad52R70K strain.

Dev Biol, 1999 Nov 15, 215(2), 358 - 74
Engineered truncations in the Drosophila mastermind protein disrupt Notch pathway function; Helms W et al.; The phenotypes and genetic interactions associated with mutations in the Drosophila mastermind (mam) gene have implicated it as a component of the Notch signaling pathway . However, its function and site of action within many tissues requiring Notch signaling have not been thoroughly investigated . To address these questions, we have constructed truncated versions of the Mam protein that elicit dominant phenotypes when expressed in imaginal tissues under GAL4-UAS regulation . By several criteria, these effects appear to phenocopy loss of function for the Notch pathway . When expressed in the notum, truncated Mam results in failure of lateral inhibition within proneural clusters and perturbations in cell fate specification within the sensory organ precursor cell lineage . Expression in the wing is associated with vein thickening and margin defects, including nicking and bristle loss . The truncation-associated wing margin phenotypes are modified by mutations in Notch and Wg pathway genes and are correlated with depressed expression of wg, cut, and vg . These data support the idea that Mam truncations have lost key effector domains and therefore behave as dominant-negative proteins . Coexpression of Delta or an activated form of Notch suppresses the effects of the Mam truncation, suggesting that Mam can function upstream of ligand-receptor interaction in the Notch pathway . This system should prove useful for the investigation of the role of Mam within the Notch pathway .

Appl Environ Microbiol, 1999 Nov, 65(11), 5059 - 65
Reactor-scale cultivation of the hyperthermophilic methanarchaeon Methanococcus jannaschii to high cell densities; Mukhopadhyay B et al.; For the hyperthermophilic and barophilic methanarchaeon Methanococcus jannaschii, we have developed a medium and protocols for reactor-scale cultivation that improved the final cell yield per liter from approximately 0.5 to approximately 7.5 g of packed wet cells ( approximately 1.8 g dry cell mass) under autotrophic growth conditions and to approximately 8.5 g of packed wet cells ( approximately 2 g dry cell mass) with yeast extract (2 g liter(-1)) and tryptone (2 g liter(-1)) as medium supplements . For growth in a sealed bottle it was necessary to add Se to the medium, and a level of 2 microM for added Se gave the highest final cell yield . In a reactor M . jannaschii grew without added Se in the medium; it is plausible that the cells received Se as a contaminant from the reactor vessel and the H(2)S supply . But, for the optimal performance of a reactor culture, an addition of Se to a final concentration of 50 to 100 microM was needed . Also, cell growth in a reactor culture was inhibited at much higher Se concentrations . These observations and the data from previous work with methanogen cell extracts (B . C . McBride and R . S . Wolfe, Biochemistry 10:4312-4317, 1971) suggested that from a continuously sparged reactor culture Se was lost in the exhaust gas as volatile selenides, and this loss raised the apparent required level of and tolerance for Se . In spite of having a proteinaceous cell wall, M . jannaschii withstood an impeller tip speed of 235.5 cms(-1), which was optimal for achieving high cell density and also was the higher limit for the tolerated shear rate . The organism secreted one or more acidic compounds, which lowered pH in cultures without pH control; this secretion continued even after cessation of growth.

J Biol Chem, 1999 Nov 5, 274(45), 32376 - 81
Analysis of the molecular mechanism for the antagonistic action of a novel 1alpha,25-dihydroxyvitamin D(3) analogue toward vitamin D receptor function; Ozono K et al.; We have recently reported that 23(S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) efficiently blocks the differentiation of HL-60 cells induced by 1alpha,25-dihydroxyvitamin D(3) (1alpha,25(OH)(2)D(3)) (Miura, D., Manabe, K., Ozono, K., Saito, M., Gao, Q., Norman, A . W., and Ishizuka, S . (1999) J . Biol . Chem . 274, 16392-16399) . To clarify the molecular mechanisms of this antagonism, we examined whether TEI-9647 antagonizes the genomic effects of 1alpha,25(OH)(2)D(3) . 10(-7) to 10(-9) M TEI-9647 inhibited the transactivation effect of 10(-8) M 1alpha,25(OH)(2)D(3) in a dose-dependent manner, while TEI-9647 alone did not activate the reporter activity driven by SV40 promoter containing two vitamin D response elements in Saos-2 cells . The antagonistic effect of TEI-9647 was also observed using the rat 24-hydroxylase gene promoter, but the effect was weaker in HeLa and COS-7 cells than in Saos-2 cells . TEI-9647 also exhibited antagonism in an assay system where the VDR fused to the GAL4 DNA-binding domain and the reporter plasmid containing the GAL4 binding site were used in Saos-2 cells, but did not in HeLa cells . TEI-9647 reduced the interaction between VDR and RXRalpha according to the results obtained from the mammalian two-hybrid system in Saos-2 cells, but did not in HeLa cells . The two-hybrid system also revealed that the interaction between VDR and SRC-1 was reduced by TEI-9647 in Saos-2 cells . These results demonstrate that the novel 1alpha,25(OH)(2)D(3) analogue, TEI-9647, is the first synthetic ligand for the VDR that efficiently antagonizes the action of 1alpha, 25(OH)(2)D(3), although the extent of its antagonism depends on cell type.

Science, 1999 Oct 29, 286(5441), 955 - 8
Epigenetic inheritance of active chromatin after removal of the main transactivator; Cavalli G et al.; The Drosophila Polycomb and trithorax group proteins act through chromosomal elements such as Fab-7 to maintain repressed or active gene expression, respectively . A Fab-7 element is switched from a silenced to a mitotically heritable active state by an embryonic pulse of transcription . Here, histone H4 hyperacetylation was found to be associated with Fab-7 after activation, suggesting that H4 hyperacetylation may be a heritable epigenetic tag of the activated element . Activated Fab-7 enables transcription of a gene even after withdrawal of the primary transcription factor . This feature may allow epigenetic maintenance of active states of developmental genes after decay of their early embryonic regulators.

EMBO J, 1999 Nov 1, 18(21), 6095 - 105
Cellular transcription factors recruit viral replication proteins to activate the Epstein-Barr virus origin of lytic DNA replication, oriLyt; Baumann M et al.; DNA replication of Epstein-Barr virus (EBV) during the productive phase of the life cycle of this herpesvirus depends on the cis-acting element oriLyt . It consists of two essential domains, the upstream and the downstream component . Whereas the upstream component contains several DNA-binding motifs for the viral activator protein BZLF1, the downstream component is known to be the binding site of several cellular proteins . We identified cellular transcription factors that bind synergistically to a functionally relevant subsequence of the downstream component, the TD element . Two of these transcription factors, ZBP-89 and Sp1, stimulate replication as shown by protein fusions with the GAL4 DNA-binding domain and a single GAL4 DNA-binding motif inserted into the TD element . In protein binding assays, we observed an interaction of Sp1 and ZBP-89 with the viral DNA polymerase and its processivity factor . Our data indicate that cellular transcriptional activators tether viral replication proteins to the lytic origin via direct protein-protein interactions to assemble the viral replication complex at oriLyt.

EMBO J, 1999 Nov 1, 18(21), 6106 - 18
HIV-1 tat transcriptional activity is regulated by acetylation; Kiernan RE et al.; The human immunodeficiency virus (HIV) trans- activator protein, Tat, stimulates transcription from the viral long-terminal repeats (LTR) through an RNA hairpin element, trans-activation responsive region (TAR) . We and others have shown that trans-activator protein (Tat)-associated histone acetyltransferases (TAHs), p300 and p300/CBP-associating factor (PCAF), assist functionally in the activation of chromosomally integrated HIV-1 LTR . Here, we show that p300 and PCAF also directly acetylate Tat . We defined two sites of acetylation located in different functional domains of Tat . p300 acetylated Lys50 in the TAR RNA binding domain, while PCAF acetylated Lys28 in the activation domain of Tat . In support of a functional role for acetylation in vivo, histone deacetylase inhibitor (trichostatin A) synergized with Tat in transcriptional activation of the HIV-1 LTR . Synergism was TAR-dependent and required the intact presence of both Lys28 and Lys50 . Mechanistically, acetylation at Lys28 by PCAF enhanced Tat binding to the Tat-associated kinase, CDK9/P-TEFb, while acetylation by p300 at Lys50 of Tat promoted the dissociation of Tat from TAR RNA that occurs during early transcription elongation . These data suggest that acetylation of Tat regulates two discrete and functionally critical steps in transcription, binding to an RNAP II CTD-kinase and release of Tat from TAR RNA.

EMBO J, 1999 Nov 1, 18(21), 6017 - 26
Degradation signals in the lysine-asparagine sequence space; Suzuki T et al.; The N-degrons, a set of degradation signals recognized by the N-end rule pathway, comprise a protein's destabilizing N-terminal residue and an internal lysine residue . We show that the strength of an N-degron can be markedly increased, without loss of specificity, through the addition of lysine residues . A nearly exhaustive screen was carried out for N-degrons in the lysine (K)-asparagine (N) sequence space of the 14-residue peptides containing either K or N (16 384 different sequences) . Of these sequences, 68 were found to function as N-degrons, and three of them were at least as active and specific as any of the previously known N-degrons . All 68 K/N-based N-degrons lacked the lysine at position 2, and all three of the strongest N-degrons contained lysines at positions 3 and 15 . The results support a model of the targeting mechanism in which the binding of the E3-E2 complex to the substrate's destabilizing N-terminal residue is followed by a stochastic search for a sterically suitable lysine residue . Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding.

EMBO J, 1999 Nov 1, 18(21), 6005 - 16
Cytoplasm to vacuole trafficking of aminopeptidase I requires a t-SNARE-Sec1p complex composed of Tlg2p and Vps45p; Abeliovich H et al.; Aminopeptidase I (API) is imported into the yeast vacuole/lysosome by a constitutive non-classical vesicular transport mechanism, the cytoplasm to vacuole targeting (Cvt) pathway . Newly synthesized precursor API is sequestered in double-membrane cytoplasmic Cvt vesicles . The Cvt vesicles fuse with the vacuole, releasing single-membrane Cvt bodies containing proAPI into the vacuolar lumen, and maturation of API occurs when the Cvt body is degraded, releasing mature API . Under starvation conditions, API is transported to the vacuole by macroautophagy, an inducible, non-selective mechanism that shares many similarities with the Cvt pathway . Here we show that Tlg2p, a member of the syntaxin family of t-SNARE proteins, and Vps45p, a Sec1p homologue, are required in the constitutive Cvt pathway, but not in inducible macroautophagy . Fractionation and protease protection experiments indicate that Tlg2p is required prior to or at the step of API segregation into the Cvt vesicle . Thus, the early Vps45-Tlg2p-dependent step of the Cvt pathway appears to be mechanistically distinct from the comparable stage in macroautophagy . Vps45p associates with both the Tlg2p and Pep12p t-SNAREs, but API maturation is not blocked in a pep12(ts) mutant, indicating that Vps45p independently regulates the function of multiple t-SNARES at distinct trafficking steps.

EMBO J, 1999 Nov 1, 18(21), 5953 - 62
The Sch9 protein kinase regulates Hsp90 chaperone complex signal transduction activity in vivo; Morano KA et al.; Basal and stress-induced synthesis of the components of the highly conserved heat shock protein Hsp90 chaperone complex require the heat shock transcription factor (HSF); Saccharomyces cerevisiae cells expressing the HSF allele HSF(1-583) reversibly arrest growth at 37 degrees C in the G(2)/M phase of the cell cycle due to diminished expression of these components . A suppressor mutant capable of restoring high-temperature growth to HSF(1-583) cells was identified, harboring a disruption of the SCH9 protein kinase gene, homologous to the protein kinase A and protein kinase B/Akt families of mammalian growth control kinases . Loss of Sch9 in HSF(1-583) cells derepresses Hsp90 signal transduction functions as demonstrated by restoration of transcriptional activity by the mammalian glucocorticoid receptor and the heme-dependent transcription factor Hap1, and by enhanced pheromone-dependent signaling through the Ste11 mitogen-activated protein kinase (MAPK) . Moreover, Sch9-deficient cells with normal levels of Hsp90 chaperone complex components display hyperactivation of the pheromone response MAPK pathway in the absence of pheromone . These results demonstrate that the evolutionarily conserved function of the Hsp90 chaperone complex as a signal transduction facilitator is modulated by a growth regulatory kinase.

EMBO J, 1999 Nov 1, 18(21), 5827 - 33
Retinoids activate proton transport by the uncoupling proteins UCP1 and UCP2; Rial E et al.; In mammalian brown adipose tissue, thermogenesis is explained by uncoupling mitochondrial respiration from ATP synthesis . Uncoupling protein-1 (UCP1) is responsible for this uncoupled state, because it allows proton re-entry into the matrix and thus dissipates the proton gradient generated by the respiratory chain . Proton transport by UCP1 is regulated negatively by nucleotides and positively by fatty acids . Adrenergic stimulation of brown adipocytes stimulates lipolysis and therefore enhances uncoupling and thermogenesis . Adrenergic stimulation also boosts ucp1 gene transcription . Since retinoic acid also promotes ucp1 gene transcription and its structure makes it a possible activator of UCP1, we hypothesized that retinoic acid, like noradrenaline, could have a dual action and trigger the activity of the protein UCP1 itself . Here we show that retinoic acid strongly increases proton transport by UCP1 in brown adipose tissue mitochondria and that it is much more potent than fatty acids . These data are corroborated with yeast mitochondria where UCP1 was introduced by genetic manipulation . The yeast expression system allows the comparison of the UCP1 with the newly described homologues UCP2 and UCP3 . The search for regulators of UCP2 has demonstrated that it is positively regulated by retinoids in a pH-dependent manner.

FEBS Lett, 1999 Oct 29, 460(2), 321 - 7
Identification and characterisation of novel polymorphisms in the CYP2A locus: implications for nicotine metabolism; Oscarson M et al.; The polymorphic human cytochrome P450 2A6 (CYP2A6) metabolises a number of drugs, activates a variety of precarcinogens and constitutes the major nicotine C-oxidase . A relationship between CYP2A6 genotype and smoking habits, as well as incidence of lung cancer, has been proposed . Two defective alleles have hitherto been identified, one of which is very common in Asian populations . Among Caucasians, an additional defective and frequently distributed allele (CYP2A6*3) has been suggested to play a protective role against nicotine addiction and cigarette consumption . Here, we have re-evaluated the genotyping method used for the CYP2A6*3 allele and found that a gene conversion in the 3' flanking region of 30-40% of CYP2A6*1 alleles results in genotype misclassification . In fact, no true CYP2A6*3 alleles were found among 100 Spaniards and 96 Chinese subjects . In one Spanish poor metaboliser of the CYP2A6 probe drug coumarin, we found two novel defective alleles . One, CYP2A6*5, encoded an unstable enzyme having a G479L substitution and the other was found to carry a novel type of CYP2A6 gene deletion (CYP2A6*4D) . The results imply the presence of numerous defective as well as active CYP2A6 alleles as a consequence of CYP2A6/CYP2A7 gene conversion events . We conclude that molecular epidemiological studies concerning CYP2A6 require validated genotyping methods for accurate detection of all known defective CYP2A6 alleles.

J Mol Biol, 1999 Oct 29, 293(3), 579 - 93
Interaction between NTF2 and xFxFG-containing nucleoporins is required to mediate nuclear import of RanGDP; Bayliss R et al.; Nuclear transport factor 2 (NTF2) is a small, homodimeric protein that binds to both RanGDP and xFxFG repeat-containing nucleoporins, such as yeast Nsp1p and vertebrate p62 . NTF2 is required for efficient nuclear protein import and has been shown to mediate the nuclear import of RanGDP . We have used the crystal structures of rat NTF2 and its complex with RanGDP to design a mutant, W7A-NTF2, in which the affinity for xFxFG-repeat nucleoporins is reduced while wild-type binding to RanGDP is retained . The 2.5 A resolution crystal structure of W7A-NTF2 is virtually superimposable upon the wild-type protein structure, indicating that the mutation had not introduced a more general conformational change . Therefore, our data suggest that the exposed side-chain of residue 7 is crucial to the interaction between NTF2 and xFxFG repeat-containing nucleoporins . Consistent with its reduced affinity for xFxFG nucleoporins, fluorescently labelled W7A-NTF2 binds less strongly to the nuclear envelope of permeabilized cultured cells than wild-type NTF2 and, when microinjected into Xenopus oocytes, colloidal gold coated with W7A-NTF2 binds less strongly to the central channel of nuclear pore complexes than wild-type NTF2-coated gold . Significantly, W7A-NTF2 only weakly stimulated the nuclear import of fluorescein-labelled RanGDP, providing direct evidence that an interaction between NTF2 and xFxFG repeat-containing nucleoporins is required to mediate the nuclear import of RanGDP .

Gene Expr, 1999, 8(1), 43 - 57
Functional analysis of the Ume3p/ Srb11p-RNA polymerase II holoenzyme interaction; Cooper KF et al.; The yeast C-type cyclin Ume3p/Srb11p and its cyclin-dependent kinase (Cdk) Ume5p are required for the full repression of genes involved in the stress response or meiosis . This cyclin-Cdk kinase copurifies with the RNA polymerase II holoenzyme complex, suggesting it functions through modification of the transcriptional machinery . This report describes two domains required for Ume3p-RNA Pol II holoenzyme association . One domain contains the highly conserved cyclin box that directs cyclin-Cdk interaction and requires Ume5p for holoenzyme binding . The second domain, termed HAD for holoenzyme associating domain, is located within the amino-terminal region of the cyclin and is sufficient for holoenzyme binding independent of Ume5p or the cyclin box . In addition to its role in RNA Pol II holoenzyme association, the HAD is also required for Ume3p-dependent repression in vivo . Finally, HAD mutations do not affect the ability of the Ume3p-Ume5p kinase to phosphorylate in vitro the carboxy-terminal domain (CTD) of RNA polymerase II, a reported target of cyclin C-Cdk activity . In conclusion, this study demonstrates that the association of the Ume3p to the holoenzyme is complex, involving two independent domains, both of which are required for full Ume3p-dependent repression in vivo . Furthermore, HAD-dependent repression does not appear to involve CTD phosphorylation, suggesting a different role for this domain in directing Ume3p-Ume5p activity.

Biol Chem, 1999 Sep, 380(9), 1133 - 8
Molecular characterization of a novel mammalian DnaJ-like Sec63p homolog; Skowronek MH et al.; We identified a human cDNA sequence encoding a polypeptide of 760 amino acids that shares 53% homology and 25.6% identity with the yeast DnaJ-like endoplasmic reticulum (ER) translocon component Sec63p . Three epitope-specific antisera revealed a protein of an apparent molecular mass of 83 kDa, both in human cell extracts and in dog pancreatic microsomes . Biochemical analyses show that it is an integral membrane protein of the rough ER, which has the DnaJ domain located in the ER lumen . The novel Sec63 protein could thus represent a key component of the mammalian ER protein translocation machinery.

Eur J Immunol, 1999 Oct, 29(10), 3077 - 88
A1 expression is stimulated by CD40 in B cells and rescues WEHI 231 cells from anti-IgM-induced cell death; Kuss AW et al.; Engagement of the antigen receptor on murine immature B cells leads to growth arrest followed by apoptosis . Concomitant signaling through CD40 sustains proliferation and rescues the cells from apoptosis . We show here that cross-linking CD40 stimulates the expression of A1, a member of the anti-apoptotic Bcl-2 family, in primary murine B lymphocytes . CD40-dependent stimulation of A1 was confirmed in WEHI 231 cells, an immature murine B cell lymphoma line . We transduced WEHI 231 cells with a bicistronic recombinant retroviral vector coding for A1 and a chimeric selection marker comprising the enhanced yellow fluorescent protein and the zeocin resistance protein . A1-transduced WEHI 231 cells showed a significant higher survival rate after engagement of the antigen receptor . In contrast, constitutive expression of A1 did not abrogate anti-IgM-induced c-myc down-regulation . Consistent with this, A1 did not release anti-IgM-induced cell cycle arrest . Our data indicate that CD40-stimulated A1 expression permits WEHI 231 cells to survive in the presence of anti-IgM antibodies and suggests a protective role for A1 in antigen receptor-mediated apoptosis in B cells.

Eur J Biochem, 1999 Nov, 266(1), 302 - 7
Two branches of the lupeol synthase gene in the molecular evolution of plant oxidosqualene cyclases; Shibuya M et al.; Two new triterpene synthase cDNAs, named as OEW and TRW, were cloned from olive leaves (Olea europaea) and from dandelion roots (Taraxacum officinale), respectively, by the PCR method with primers designed from the conserved sequences found in the known oxidosqualene cyclases . Their ORFs consisted of 2274 bp nucleotides and coded for 758 amino acid long polypeptides . They shared high sequence identity (78%) to each other, while they showed only about 60% identities to the known triterpene synthases LUPI (lupeol synthase clone from Arabidopsis thaliana) and PNY (beta-amyrin synthase clone from Panax ginseng) at amino acid level . To determine the enzyme functions of the translates, they were expressed in an ERG7 deficient yeast mutant . Accumulation of lupeol in the cells of yeast transformants proved both of these clones code for lupeol synthase proteins . An EST (expression sequence tag) clone isolated from Medicago truncatula roots as a homologue of cycloartenol synthase gene, exhibits high sequence identity (75-77%) to these two lupeol synthase cDNAs, suggesting it to be another lupeol synthase clone . Comparatively low identity (approximately 57%) of LUP1 from Arabidopsis thaliana to either one of these clones leaves LUP1 as a distinct clone among lupeol synthases . From these sequence comparisons, now we propose that two branches of lupeol synthase gene have been generated in higher plants during the course of evolution.

Eur J Biochem, 1999 Nov, 266(1), 293 - 301
The presence of the alternatively spliced A2 cassette in the vacuolar H+-ATPase subunit A prevents assembly of the V1 catalytic domain; Hernando N et al.; Vacuolar ATPases (V-ATPases) are multisubunit enzymes that couple the hydrolysis of ATP to the transport of H+ across membranes, and thus acidify several intracellular compartments and some extracellular spaces . Despite the high degree of genetic and pharmacological homogeneity of V-ATPases, cells differentially modulate the lumenal pH of organelles and, in some cells, V-ATPases are selectively targetted to the plasma membrane . Although the mechanisms underlying such differences are not known, the subunit isoform composition of V-ATPases could contribute to altered assembly, targeting or activity . We previously identified an alternatively spliced variant of the chicken A subunit in which a 30 amino acid cassette (A1) containing the Walker consensus sequence for ATP binding is replaced by a 24 amino acid cassette (A2) that lacks this feature . We have examined the ability of chimeric yeast/chicken A subunits containing either the A1 or the A2 cassette to restore the V-ATPase activity of yeast that lack the A subunit . The A1-containing chimeric subunit, but not the chimera that contains the A2 cassette, partially restores the ability of the mutated yeast to grow at neutral pH . Both chimeric proteins are expressed, although at lower levels than the similarly transfected yeast A subunit . The A2-containing subunit fails to associate with the vacuolar membrane or support the assembly of V-ATPase complexes . Thus, the substitution of the A1 sequence by A2 not only removes the Walker nucleotide binding sequence but also compromises the ability of the A subunit to assemble with other V-ATPase subunits.

Genes Dev, 1999 Oct 15, 13(20), 2704 - 12
Recruitment of Nanos to hunchback mRNA by Pumilio; Sonoda J et al.; Translational regulation of hunchback (hb) mRNA is essential for posterior patterning of the Drosophila embryo . This regulation is mediated by sequences in the 3'-untranslated region of hb mRNA (the Nanos response elements or NREs), as well as two trans-acting factors-Nanos and Pumilio . Pumilio recognizes the NREs via a conserved binding motif . The mechanism of Nanos action has not been clear . In this report we use protein-protein and protein-RNA interaction assays in yeast and in vitro to show that Nanos forms a ternary complex with the RNA-binding domain of Pumilio and the NRE . Mutant forms of the NRE, Nos, and Pum that do not regulate hb mRNA normally in embryos do not assemble normally into a ternary complex . In particular, recruitment of Nos is dependent on bases in the center of the NRE, on the carboxy-terminal Cys/His domain of Nos, and on residues in the eighth repeat of the Pum RNA-binding domain . These residues differ in a closely related human protein that also binds to the NRE but cannot recruit Drosophila Nos . Taken together, these findings suggest models for how Nos and Pum collaboratively target hb mRNA . More generally, they suggest that Pum-like proteins from other species may also act by recruiting cofactors to regulate translation.

Cancer Res, 1999 Oct 15, 59(20), 5068 - 74
Germ-line msh6 mutations in colorectal cancer families; Kolodner RD et al.; Hereditary nonpolyposis colorectal carcinoma (HNPCC) is due primarily to inherited mutations in two mismatch repair genes, MSH2 and MLH1, whereas germ-line mutations in other mismatch repair genes are rare . We examined the frequency of germ-line msh6 mutations in a population-based series of 140 colorectal cancer patients, including 45 sporadic cases, 91 familial non-HNPCC cases, and 4 HNPCC cases . Among the 91 population-based familial non-HNPCC cases, germ-line msh6 mutations were found in 6 patients (7.1% of probands analyzed; median age at diagnosis, 61 years) . These mutations included a splice site mutation, a frameshift mutation, two missense mutations that were demonstrated to be loss of function mutations, and two missense mutations for which functional studies were not possible . In contrast, germ-line msh6 mutations were not found in any of the 45 sporadic cases and the 4 HNPCC cases in the population-based series or in the second series of 58 clinic-based, primarily HNPCC families . Our data suggest that germ-line msh6 mutations predispose individuals to primarily late-onset, familial colorectal carcinomas that do not fulfill classic criteria for HNPCC.

Nucleic Acids Res, 1999 Nov 15, 27(22), 4468 - 75
The human REV1 gene codes for a DNA template-dependent dCMP transferase; Lin W et al.; DNA is frequently damaged by various physical and chemical agents . DNA damage can lead to mutations during replication . In the yeast Saccharomyces cerevisiae, the damage-induced mutagenesis pathway requires the Rev1 protein . We have isolated a human cDNA homologous to the yeast REV1 gene . The human REV1 cDNA consists of 4255 bp and codes for a protein of 1251 amino acid residues with a calculated molecular weight of 138 248 Da . The human REV1 gene is localized between 2q11.1 and 2q11.2 . We show that the human REV1 protein is a dCMP transferase that specifically inserts a dCMP residue opposite a DNA template G . In addition, the human REV1 transferase is able to efficiently and specifically insert a dCMP opposite a DNA template apurinic/apyrimidinic (AP) site or a uracil residue . These results suggest that the REV1 transferase may play a critical role during mutagenic translesion DNA synthesis bypassing a template AP site in human cells . Consistent with its role as a fundamental mutagenic protein, the REV1 gene is ubiquitously expressed in various human tissues.

Nucleic Acids Res, 1999 Nov 15, 27(22), 4427 - 35
Analysis of the histone acetyltransferase B complex of maize embryos; Lusser A et al.; Purified histone acetyltransferase B (HAT-B) from maize consists of two subunits, p50 and p45 . Cloning of the cDNA and genomic DNA encoding the catalytic subunit p50 revealed a consensus motif reminiscent of other acetyltransferases . Internal peptide sequences and immunological studies identified p45 as a protein related to the Retinoblastoma associated protein Rbap . Antibodies against recombinant p50 were able to immunoprecipitate the enzymatic activity of p50 as well as p45 . Consistent with the idea that HAT-B is involved in acetylation of newly synthesized histone H4 during DNA replication, mRNA and protein levels are correlated with S-phases during embryo germination . Inhibition of histone deacetylases by HC toxin or Trichostatin A caused a decrease of the in vivo expression of HAT-B mRNA . Regardless of its predominant cytoplasmic localization, a significant proportion of HAT-B-p50 is present in nuclei, irrespective of the cell cycle stage, suggesting an additional nuclear function.

Nucleic Acids Res, 1999 Nov 15, 27(22), 4344 - 52
Cloning and analysis of a Toxoplasma gondii histone acetyltransferase: a novel chromatin remodelling factor in Apicomplexan parasites; Hettmann C et al.; The yeast transcriptional adaptor GCN5 functions as a histone acetyltransferase, directly linking chromatin modification to transcriptional regulation . Homologues of yeast GCN5 have been found in Tetrahymena, Drosophila, Arabidopsis and human, suggesting that this pathway of chromatin remodelling is evolutionarily conserved . Consistent with this view, we have identified the Toxoplasma gondii homologue, referred to here as TgGCN5 . The gene codes for a protein of 474 amino acids with an estimated molecular mass of 53 kDa . The protein reveals two regions of close similarity with the GCN5 family members, the HAT domain and the bromodomain . Tg GCN5 occurs in a single copy in the T.gondii genome . The introduction of a second copy of TgGCN5 in T.gondii tachyzoites is toxic unless the HAT activity is disrupted by a single point mutation . Full TgGCN5 does not complement the growth defect in a yeast gcn5 (-)mutant strain, but a chimera comprising the T.gondii HAT domain fused to the remainder of yGCN5 does . These data show that T.gondii GNC5 is a histone acetyltransferase attesting to the significance of chromatin remodelling in gene regulation of Apicomplexa.

Proc Natl Acad Sci U S A, 1999 Oct 26, 96(22), 12935 - 40
Biosynthetic origin of conjugated double bonds: production of fatty acid components of high-value drying oils in transgenic soybean embryos; Cahoon EB et al.; Vegetable oils that contain fatty acids with conjugated double bonds, such as tung oil, are valuable drying agents in paints, varnishes, and inks . Although several reaction mechanisms have been proposed, little is known of the biosynthetic origin of conjugated double bonds in plant fatty acids . An expressed sequence tag (EST) approach was undertaken to characterize the enzymatic basis for the formation of the conjugated double bonds of alpha-eleostearic (18:3Delta(9cis, 11trans,13trans)) and alpha-parinaric (18:4Delta(9cis,11trans, 13trans,15cis)) acids . Approximately 3,000 ESTs were generated from cDNA libraries prepared from developing seeds of Momordica charantia and Impatiens balsamina, tissues that accumulate large amounts of alpha-eleostearic and alpha-parinaric acids, respectively . From ESTs of both species, a class of cDNAs encoding a diverged form of the Delta(12)-oleic acid desaturase was identified . Expression of full-length cDNAs for the Momordica (MomoFadX) and Impatiens (ImpFadX) enzymes in somatic soybean embryos resulted in the accumulation of alpha-eleostearic and alpha-parinaric acids, neither of which is present in untransformed soybean embryos . alpha-Eleostearic and alpha-parinaric acids together accounted for as much as 17% (wt/wt) of the total fatty acids of embryos expressing MomoFadX . These results demonstrate the ability to produce fatty acid components of high-value drying oils in transgenic plants . These findings also demonstrate a previously uncharacterized activity for Delta(12)-oleic acid desaturase-type enzymes that we have termed "conjugase."

Proc Natl Acad Sci U S A, 1999 Oct 26, 96(22), 12679 - 84
The MAPKKK Ste11 regulates vegetative growth through a kinase cascade of shared signaling components; Lee BN et al.; In haploid Saccharomyces cerevisiae, the mating and invasive growth (IG) pathways use the same mitogen-activated protein kinase kinase kinase kinase (MAPKKKK, Ste20), MAPKKK (Ste11), MAPKK (Ste7), and transcription factor (Ste12) to promote either G(1) arrest and fusion or foraging in response to distinct stimuli . This exquisite specificity is the result of pathway-specific receptors, G proteins, scaffold protein, and MAPKs . It is currently not thought that the shared signaling components function under the basal conditions of vegetative growth . We tested this hypothesis by searching for mutations that cause lethality when the STE11 gene is deleted . Strikingly, we found that Ste11, together with Ste20, Ste7, Ste12, and the IG MAPK Kss1, functions in a third pathway that promotes vegetative growth and is essential in an och1 mutant that does not synthesize mannoproteins . We term this pathway the STE vegetative growth (SVG) pathway . The SVG pathway functions, in part, to promote cell wall integrity in parallel with the protein kinase C pathway . During vegetative growth, the SVG pathway is inhibited by the mating MAPK Fus3 . By contrast, the SVG pathway is constitutively activated in an och1 mutant, suggesting that it senses intracellular changes arising from the loss of mannoproteins . We predict that general proliferative functions may also exist for other MAPK cascades thought only to perform specialized functions.

Proc Natl Acad Sci U S A, 1999 Oct 26, 96(22), 12542 - 7
The importin/karyopherin Kap114 mediates the nuclear import of TATA-binding protein; Morehouse H et al.; Two high copy suppressors of temperature-sensitive TATA-binding protein (TBP) mutants were isolated . One suppressor was TIF51A, which encodes eukaryotic translation initiation factor 5A . The other high copy suppressor, YGL241W, also known as KAP114, is one of 14 importin/karyopherin proteins in yeast . These proteins mediate the transport of specific macromolecules into and out of the nucleus . Cells lacking Kap114 partially mislocalize TBP to the cytoplasm . Kap114 binds TBP in vitro, and binding is disrupted in the presence of GTPgammaS . Therefore, Kap114 is an importer of TBP into the nucleus, but alternative import pathways must also exist.

Proc Natl Acad Sci U S A, 1999 Oct 26, 96(22), 12454 - 8
Ku is associated with the telomere in mammals; Hsu HL et al.; Telomeres are specialized DNA/protein complexes that comprise the ends of eukaryotic chromosomes . The highly expressed Ku heterodimer, composed of 70 and 80 K(d) subunits (Ku70 and Ku80), is the high-affinity DNA binding component of the DNA-dependent protein kinase . Ku is critical for nonhomologous DNA double-stranded break repair and site-specific recombination of V(D)J gene segments . Ku also plays an important role in telomere maintenance in yeast . Herein, we report, using an in vivo crosslinking method, that human and hamster telomeric DNAs specifically coimmunoprecipitate with human Ku80 after crosslinking . Localization of Ku to the telomere does not depend on the DNA-dependent protein kinase catalytic component . These findings suggest a direct link between Ku and the telomere in mammalian cells.

Genomics, 1999 Oct 15, 61(2), 156 - 69
Cloning, characterization, and localization of mouse and human SPO11; Romanienko PJ et al.; Spo11 is a meiosis-specific protein in yeast that has been found covalently bound to DNA double-strand breaks (DSBs) during the early stages of meiosis . These DSBs initiate homologous recombination, which is required for proper segregation of chromosomes and the generation of genetic diversity during meiosis . Here we report the cloning, characterization, tissue expression, and chromosomal localization of both mouse and human homologues of Spo11 . The putative mouse and human proteins are 82% identical and share approximately 25% identity with other family members . Northern blot analysis revealed testis-specific expression for both genes, but RT-PCR results showed ubiquitous expression of at least a portion of Spo11 in mouse . Human SPO11 was also detected in several somatic tissues . Mouse Spo11 was localized to chromosome 2H4, and human SPO11 was localized to chromosome 20q13.2-q13.3, a region amplified in some breast and ovarian tumors.

Acta Crystallogr D Biol Crystallogr, 1999 Nov, 55(11), 1822 - 6
Structure of a phosphoglycerate mutase:3-phosphoglyceric acid complex at 1.7 A; Crowhurst GS et al.; The crystal structure of the tetrameric glycolytic enzyme phosphoglycerate mutase from the yeast Saccharomyces cerevisiae has been determined to 1.7 A resolution in complex with the sugar substrate . The difference map indicates that 3-phosphoglycerate is bound at the base of a 12 A cleft, positioning C2 of the substrate within 3.5 A of the primary catalytic residue, histidine 8.

Biochem Biophys Res Commun, 1999 Oct 22, 264(2), 388 - 94
Single-strand breaks in agarose-embedded chromatin of nonapoptotic cells; Varga T et al.; Loop-size chromatin fragmentation frequently observed upon apoptotic cell death is thought to be initiated by ss nicks . Here we show that the agarose-embedded, deproteinized chromatin of normal, non-apoptotic murine and human cells, as well as yeast protoplasts, falls apart to 50-300 kb ss fragments upon heat denaturation, as revealed by urea-TAE field-inversion agarose gel electrophoresis resolving ss and ds fragments alike . These data were in line with S1digestion experiments . The nicks (gaps) observed are best explained either by enzymatic cleavages occurring upon cell lysis instantaneously or by preexisting discontinuities becoming manifest upon heat denaturation . These discontinuities go unnoticed in the usual nondenaturaing circumstances but seem to be inevitably present in any DNA preparation . The loop-size ds DNA fragmentation in apoptosis may be based on these pre-existing or "ready-to-go" (upon cell lysis) ss discontinuities of the normal cellular chromatin .

Results Probl Cell Differ, 2000, 27, 119 - 30
The MAP kinase cascade that includes MAPKKK-related protein kinase NPK1 controls a mitotic proces in plant cells; Nishihama R et al.; The tobacco NPK1 cDNA was the first-isolated plant cDNA for a homolog of mitogen-activated protein kinase kinase kinases (MAPKKKs) . The kinase domain of the NPK1 protein can replace the functions of MAPKKKs in yeasts, while the amino acid sequence of the kinase-unrelated region does not have any homology to those of MAPKKKs from other organisms . Transcription of the NPK1 gene takes place in meristematic tissues or immature organs in a tobacco plant . During a tobacco cell cycle, transcriptional and translational products of NPK1 are present from S to M phase and decrease after the M phase . Expression of the NACK1 gene, which is predicted to encode a novel kinesin-like microtubule-based motor protein capable of activating NPK1, is specific to M phase, suggesting that activation of NPK1 occurs in M phase . Characterization of cDNAs for a MAPKK and a MAPK which can act downstream of NPK1 makes a proposition that the MAP kinase pathway involving NPK1 regulates a mitotic process associated with microtubules.

Appl Microbiol Biotechnol, 1999 Sep, 52(3), 401 - 9
Genetic transformation of a Rhizomucor pusillus mutant defective in asparagine-linked glycosylation: production of a milk-clotting enzyme in a less-glycosylated form; Yamazaki H et al.; Rhizomucor pusillus 1116R3 has a defect in alg2 encoding a mannosyltransferase in the asparagine (N)-linked oligosaccharide biosynthetic pathway and produces proteins in less-glycosylated forms . For development of a genetic transformation system for this zygomycete, an uracil auxotroph (mutant 1116U17) as the host strain was derived by ultraviolet (UV) mutagenesis as 5-fluoroorotic acid-resistant colonies and the orotidine-5'-monophosphate (OMP) decarboxylase (pyr4) gene as a selection marker was cloned from the wild-type strain R . pusillus F27 by the polymerase chain reaction with primers designed on the basis of the pyr4 sequences from other fungi . The amino acid sequence of R . pusillus Pyr4 deduced from the nucleotide sequence showed high homology with the OMP decarboxylases from various fungi . The pyr4 gene on pUC19 (plasmid pRPPyr4) was introduced into protoplasts of R . pusillus 1116U17 by polyethylene glycol-assisted transformation . Transformation under optimized conditions yielded 5 Ura+ transformants with 1 microgram pRPPyr4 DNA and 1 x 10(7) viable protoplasts . Southern blot analysis of the genomic DNA from the transformants showed that multiple copies of the pRPPyr4 sequence were integrated into the genome by homologous recombination at the pyr4 locus . For the purpose of production of a milk-clotting aspartic proteinase (MPP) in a less-glycosylated form, mpp from the wild-type strain was cloned in pRPPyr4 and introduced into protoplasts of R . pusillus 1116U17 . Transformants obtained in this way contained multiple copies of mpp at the chromosomal mpp locus and produced MPP as a mixture of molecules having no sugar chains and Man0-1GlcNAc2 at the two N-linked glycosylation sites in an amount about 12 times larger than the parent strain . The transformation system for R . pusillus 1116U17 would be useful for production of proteins with truncated N-linked oligosaccharide chains.

Mol Pharmacol, 1999 Nov, 56(5), 895 - 901
Activating mutation of adenylyl cyclase reverses its inhibition by G proteins; Zimmermann G et al.; We have implemented a yeast genetic selection developed previously by our laboratory to identify mutant mammalian type V adenylyl cyclases insensitive to inhibition by G(ialpha.) One mutation isolated was localized to the first cytoplasmic domain at a Phe residue (position 400), which is conserved in all nine isoforms of membrane-bound mammalian adenylyl cyclase . Biochemical characterization of the F400Y mutant revealed a dramatic conversion of the G(ialpha) response from inhibitory to stimulatory . This mutation results in additional activating effects . The mutant exhibits an enhanced sensitivity toward activation by either G(salpha) or forskolin . Synergism between G(salpha) and forskolin is not observed for the F400Y mutant, presumably because the mutant already is in the sensitized state . Additionally, an enhancement of the basal unstimulated activity was observed . This mutation, which is the first demonstration of an activating point in a mammalian adenylyl cyclase, mimics a sensitized conformation of the wild-type enzyme that underlies the synergism between stimulatory inputs, and additionally, removes the inhibitory regulatory input provided by G(ialpha) . Because sensitizing adenylyl cyclase toward its stimulators can have profound biological implications, this raises the possibility that naturally occurring mutations resembling those at the Phe400 residue may be associated with human disease states.

J Biol Chem, 1999 Oct 29, 274(44), 31671 - 8
A highly conserved signal controls degradation of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase in eukaryotes; Gardner RG et al.; Sterol synthesis by the mevalonate pathway is modulated, in part, through feedback-regulated degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) . In both mammals and yeast, a non-sterol isoprenoid signal positively regulates the rate of HMGR degradation . To define more precisely the molecule that serves as the source of this signal, we have conducted both pharmacological and genetic manipulations of the mevalonate pathway in yeast . We now demonstrate that farnesyl diphosphate (FPP) is the source of the positive signal for Hmg2p degradation in yeast . This FPP-derived signal does not act by altering the endoplasmic reticulum degradation machinery in general . Rather, the FPP-derived signal specifically modulates Hmg2p stability . In mammalian cells, an FPP-derived molecule also serves as a positive signal for HMGR degradation . Thus, both yeast and mammalian cells employ the same strategy for regulation of HMGR degradation, perhaps by conserved molecular processes.

J Biol Chem, 1999 Oct 29, 274(44), 31641 - 7
Reciprocal signaling between heterotrimeric G proteins and the p21-stimulated protein kinase; Wang J et al.; p21-activated protein kinase (PAK)-1 phosphorylated Galpha(z), a member of the Galpha(i) family that is found in the brain, platelets, and adrenal medulla . Phosphorylation approached 1 mol of phosphate/mol of Galpha(z) in vitro . In transfected cells, Galpha(z) was phosphorylated both by wild-type PAK1 when stimulated by the GTP-binding protein Rac1 and by constitutively active PAK1 mutants . In vitro, phosphorylation occurred only at Ser(16), one of two Ser residues that are the major substrate sites for protein kinase C (PKC) . PAK1 did not phosphorylate other Galpha subunits (i1, i2, i3, o, s, or q) . PAK1-phosphorylated Galpha(z) was resistant both to RGSZ1, a G(z)-selective GTPase-activating protein (GAP), and to RGS4, a relatively nonselective GAP for the G(i) and G(q) families of G proteins . Phosphorylation of Ser(27) by PKC did not alter sensitivity to either GAP . The previously described inhibition of G(z) GAPs by PKC is therefore mediated by phosphorylation of Ser(16) . Phosphorylation of either Ser(16) by PAK1 or Ser(27) by PKC decreased the affinity of Galpha(z) for Gbetagamma; phosphorylation of both residues by PKC caused no further effect . PAK1 thus regulates Galpha(z) function by attenuating the inhibitory effects of both GAPs and Gbetagamma . In this context, the kinase activity of PAK1 toward several protein substrates was directly inhibited by Gbetagamma, suggesting that PAK1 acts as a Gbetagamma-regulated effector protein . This inhibition of mammalian PAK1 by Gbetagamma contrasts with the stimulation of the PAK homolog Ste20p in Saccharomyces cerevisiae by the Gbetagamma homolog Ste4p/Ste18p.

J Biol Chem, 1999 Oct 29, 274(44), 31583 - 7
Phospho-carboxyl-terminal domain binding and the role of a prolyl isomerase in pre-mRNA 3'-End formation; Morris DP et al.; A phospho-carboxyl-terminal domain (CTD) affinity column created with yeast CTD kinase I and the CTD of RNA polymerase II was used to identify Ess1/Pin1 as a phospho-CTD-binding protein . Ess1/Pin1 is a peptidyl prolyl isomerase involved in both mitotic regulation and pre-mRNA 3'-end formation . Like native Ess1, a GSTEss1 fusion protein associates specifically with the phosphorylated but not with the unphosphorylated CTD . Further, hyperphosphorylated RNA polymerase II appears to be the dominant Ess1 binding protein in total yeast extracts . We demonstrate that phospho-CTD binding is mediated by the small WW domain of Ess1 rather than the isomerase domain . These findings suggest a mechanism in which the WW domain binds the phosphorylated CTD of elongating RNA polymerase II and the isomerase domain reconfigures the CTD though isomerization of proline residues perhaps by a processive mechanism . This process may be linked to a variety of pre-mRNA maturation events that use the phosphorylated CTD, including the coupled processes of pre-mRNA 3'-end formation and transcription termination.

J Biol Chem, 1999 Oct 29, 274(44), 31565 - 70
Generation of a dominant-negative mutant of endothelial PAS domain protein 1 by deletion of a potent C-terminal transactivation domain; Maemura K et al.; Endothelial PAS domain protein 1 (EPAS1) is a basic helix-loop-helix/PAS domain transcription factor that is preferentially expressed in vascular endothelial cells . EPAS1 shares high homology with hypoxia-inducible factor-1alpha (HIF-1alpha) and, like HIF-1alpha, has been shown to bind to the HIF-1-binding site and to activate its downstream genes such as vascular endothelial growth factor (VEGF) and erythropoietin . In this report, we show that EPAS1 increased VEGF gene expression through the HIF-1-binding site . This transactivation was enhanced further by cotransfection of an aryl hydrocarbon receptor nuclear translocator expression plasmid . Deletion analysis of EPAS1 revealed a potent activation domain (amino acids 486-639) essential for EPAS1 to transactivate the VEGF promoter . We confirmed the ability of this domain to activate transcription using a Gal4 fusion protein system . Because a truncated EPAS1 protein lacking the transactivation domain at amino acids 486-639 eliminated induction of the VEGF promoter by wild-type EPAS1, the truncated protein functions as a dominant-negative mutant . Most important, infection of the cells with an adenoviral construct expressing this mutant inhibited the induction of VEGF mRNA under conditions that mimic hypoxia . Our results suggest that EPAS1 is an important regulator of VEGF gene expression . Since VEGF plays a crucial role in angiogenesis, the ability of dominant-negative EPAS1 to inhibit VEGF promoter activity raises the possibility of a novel approach to inhibiting pathological angiogenesis.

J Biol Chem, 1999 Oct 29, 274(44), 31485 - 92
Functional characterization of ABC10alpha, an essential polypeptide shared by all three forms of eukaryotic DNA-dependent RNA polymerases; Rubbi L et al.; ABC10alpha is a small polypeptide shared by the three yeast RNA polymerases . Homologous polypeptides in higher eukaryotes have a zinc-binding CX(2)C . CX(2)C motif and a conserved basic C-terminal end . These features are also found in archaeal gene products that may encode an RNA polymerase subunit . The CX(2)C . CX(2)C motif is partly dispensable, since only its first cysteine is essential for growth, whereas the basic C-terminal end is critical in vivo . A mutant in the latter domain has an RNA polymerase III-specific defect and, in vitro, impairs RNA polymerase III assembly . Polymerase activity was, however, not affected in various faithful transcription assays . The mutant is suppressed by a high gene dosage of the second largest subunit of RNA polymerase III, whereas the homologous subunits of RNA polymerase I and II have aggravating effects . In a two-hybrid assay, ABC10alpha binds to the C-terminal half of the second largest subunit of RNA polymerase I, in a way that requires the integrity of the CX(2)C . CX(2)C motif . Thus, ABC10alpha appears to interact directly with the second largest subunit during polymerase assembly . This interaction is presumably a major rate-limiting step in assembly, since diploid cells containing only one functional gene copy for ABC10alpha have a partial growth defect.

J Biol Chem, 1999 Oct 29, 274(44), 31209 - 16
Ubiquinone at center N is responsible for triphasic reduction of cytochrome b in the cytochrome bc(1) complex; Snyder CH et al.; We have examined the pre-steady state reduction kinetics of the Saccharomyces cerevisiae cytochrome bc(1) complex by menaquinol in the presence and absence of endogenous ubiquinone to elucidate the mechanism of triphasic cytochrome b reduction . With cytochrome bc(1) complex from wild type yeast, cytochrome b reduction was triphasic, consisting of a rapid partial reduction phase, an apparent partial reoxidation phase, and a slow rereduction phase . Absorbance spectra taken by rapid scanning spectroscopy at 1-ms intervals before, during, and after the apparent reoxidation phase showed that this was caused by a bona fide reoxidation of cytochrome b and not by any negative spectral contribution from cytochrome c(1) . With cytochrome bc(1) complex from a yeast mutant that cannot synthesize ubiquinone, cytochrome b reduction by either menaquinol or ubiquinol was rapid and monophasic . Addition of ubiquinone restored triphasic cytochrome b reduction, and the duration of the reoxidation phase increased as the ubiquinone concentration increased . When reduction of the cytochrome bc(1) complex through center P was blocked, cytochrome b reduction through center N was biphasic and was slowed by the addition of exogenous ubiquinone . These results show that ubiquinone residing at center N in the oxidized cytochrome bc(1) complex is responsible for the triphasic reduction of cytochrome b.

Curr Biol, 1999 Oct 21, 9(20), 1187 - 90
The COP9 signalosome is essential for development of Drosophila melanogaster; Freilich S et al.; The COP9 signalosome (originally described as the COP9 complex) is an essential multi-subunit repressor of light-regulated development in plants {1} {2} . It has also been identified in mammals, though its role remains obscure {3} {4} {5} . This complex is similar to the regulatory lid of the proteasome and eIF3 {5} {9} {10} {11} {12} and several of its subunits are known to be involved in kinase signaling pathways {4} {6} {7} {8} . No proteins homologous to COP9 signalosome components were identified in the Saccharomyces cerevisiae genome, suggesting that the COP9 signalosome is specific for multi-cellular differentiation {13} . In order to reveal the developmental function of the COP9 signalosome in animals, we have isolated Drosophila melanogaster genes encoding eight subunits of the COP9 signalosome, and have shown by co-immunoprecipitation and gel-filtration analysis that these proteins are components of the Drosophila COP9 signalosome . Yeast two-hybrid assays indicated that several of these proteins interact, some through the PCI domain . Disruption of one of the subunits by either a P-element insertion or deletion of the gene caused lethality at the late larval or pupal stages . This lethality is probably a result of numerous pleiotropic effects . Our results indicate that the COP9 signalosome is conserved in invertebrates and that it has an essential role in animal development.

Curr Biol, 1999 Oct 21, 9(20), 1177 - 9
Identification of a family of human F-box proteins; Cenciarelli C et al.; F-box proteins are an expanding family of eukaryotic proteins characterized by an approximately 40 aminoacid motif, the F box (so named because cyclin F was one of the first proteins in which this motif was identified) {1} . Some F-box proteins have been shown to be critical for the controlled degradation of cellular regulatory proteins {2} {3} . In fact, F-box proteins are one of the four subunits of ubiquitin protein ligases called SCFs . The other three subunits are the Skp1 protein; one of the cullin proteins (Cul1 in metazoans and Cdc53 or Cul A in the yeast Saccharomyces cerevisiae); and the recently identified Roc1 protein (also called Rbx1 or Hrt1) . SCF ligases bring ubiquitin conjugating enzymes (either Ubc3 or Ubc4) to substrates that are specifically recruited by the different F-box proteins . The need for high substrate specificity and the large number of known F-box proteins in yeast and worms {2} {4} suggest the existence of a large family of mammalian F-box proteins . Using Skp1 as a bait in a yeast two-hybrid screen and by searching DNA databases, we identified a family of 26 human F-box proteins, 25 of which were novel . Some of these proteins contained WD-40 domains or leucine-rich repeats; others contained either different protein-protein interaction modules or no recognizable motifs . We have named the F-box proteins that contain WD-40 domains Fbws, those containing leucine-rich repeats, Fbls, and the remaining ones Fbxs . We have further characterized representative members of these three classes of F-box proteins.

Curr Biol, 1999 Oct 21, 9(20), 1183 - 6
The guanine-nucleotide-exchange factor Cdc24p is targeted to the nucleus and polarized growth sites; Toenjes KA et al.; Generation of cellular asymmetry or cell polarity plays a critical role in cell-cycle-regulated morphogenetic processes involving the actin cytoskeleton . The GTPase Cdc42 regulates actin rearrangements and signal transduction pathways in all eukaryotic cells {1}, and the temporal and spatial regulation of Cdc42p depends on the activity and targeting of its guanine-nucleotide exchange factor (GEF) . Cdc24p, the Saccharomyces cerevisiae GEF for Cdc42p, is found in a particulate fraction and localizes to the plasma membrane {2} {3} at sites of polarized growth {4} . We show that Cdc24p labeled with green fluorescent protein (GFP-Cdc24p) was targeted to pre-bud sites, the tips and sides of enlarging buds, and mating projections in pheromone-treated cells . Unexpectedly, GFP-Cdc24p also localized to the nucleus and GFP-Cdc24p levels diminished before nuclear division followed by its reappearance in divided nuclei and mother-bud necks during cytokinesis . The Cdc24p amino-terminal 283 amino acids were necessary and sufficient for nuclear localization, which depended on the cyclin-dependent-kinase inhibitor Far1p . The Cdc24p carboxy-terminal 289 amino acids were necessary and sufficient for targeting to the pre-bud site, bud, mother-bud neck, and mating projection . Targeting was independent of the Cdc24p-binding proteins Far1p, the GTPase Rsr1p/Bud1p, the scaffold protein Bem1p, and the G(beta) subunit Ste4p . These data are consistent with a temporal and spatial regulation of Cdc24p-dependent activation of Cdc42p during the cell cycle.

Curr Biol, 1999 Oct 21, 9(20), R779 - 82
Protein translocation: is Hsp70 pulling my chain?
Jensen RE, Johnson AE.
Hsp70 proteins in the lumen of the endoplasmic reticulum and in the mitochondrial matrix are thought to drive the translocation of proteins into each organelle . Recent experiments aimed at distinguishing between two models for Hsp70 function appear to reach opposite conclusions.

Exp Gerontol, 1999 Aug, 34(5), 645 - 58
Effect of age on DNA binding of the ku protein in irradiated human peripheral blood mononuclear cells (PBMC); Frasca D et al.; DNA binding of the ku protein was investigated in peripheral blood mononuclear cells (PBMC) from 24 subjects of different ages (20-89 years old) displaying age-related changes in DNA repair, mitotic responsiveness, and cytokine production . Ku is an heterodimeric protein composed of two subunits of 70 and 80 kDa, which is involved in the earliest steps of DNA damage recognition . DNA binding of ku 70/80 was found unchanged in normal PBMC from aging subjects but progressively declined in x-ray-irradiated PBMC from young to adult, and elderly subjects . This finding was concomitant with the age-related fall of DNA repair in the whole population.

Biochemistry, 1999 Oct 5, 38(40), 13414 - 22
Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers; Altamirano CV et al.; Human butyrylcholinesterase (BChE) in serum is composed predominantly of tetramers . The tetramerization domain of each subunit is contained within 40 C-terminal residues . To identify key residues within this domain participating in tetramer stabilization, the interaction between C-terminal 46 residue peptides was quantitated in the yeast two-hybrid system . The wild-type peptide interacted strongly with another wild-type peptide in the yeast two-hybrid system . The C571A mutant peptides interacted to a similar degree as the wild-type . However, the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) and C571 were altered to alanines showed only 12% of the interaction seen with the wild-type peptide . The seven mutations (aromatics-off) were incorporated into the complete BChE molecule, with or without the C571A mutation, and expressed in 293T and CHO-K1 cells . Expression of wild-type BChE in these cell lines yielded 10% tetramers . The aromatics-off mutant formed dimers and monomers but no tetramers . The aromatics-off/C571A mutant yielded only monomers . Addition of poly-L-proline to culture medium, or coexpression with the N-terminus of COLQ including the proline-rich attachment domain (Q(N)PRAD), increased the amount of tetrameric wild-type BChE from 10 to 70%, but had no effect on the G534stop (lacking 41 C-terminal residues) and the aromatics-off mutants . Recombinant BChE produced by coexpression with Q(N)PRAD was purified by column chromatography . The purified tetramers contained the FLAG-tagged Q(N)PRAD peptide . These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues and that poly-L-proline and PRAD act through these aromatic residues to induce tetramerization.

Insect Biochem Mol Biol, 1999 Oct, 29(10), 915 - 30
Transcriptional activation of the cloned Heliothis virescens (Lepidoptera) ecdysone receptor (HvEcR) by muristeroneA; Martinez A et al.; Ecdysteroids play an important role during insect development . We report here the isolation and characterisation of an Ecdysone receptor (EcR) homologue from Heliothis virescens (HvEcR) and present evidence supporting the HvEcR active role as an active component of the native insect receptor . Alignment of the deduced amino acid sequence of HvEcR with those of EcRs from other species confirmed its membership of this family and showed that it is closely related to the B1 isoform of Drosophila melanogaster . Northern blot analysis showed that two transcripts (6.0 and 6.5 kb) were recognised by a probe spanning the DNA and ligand binding domains of the HvEcR . Genomic Southern blots showed that the HvEcR is encoded by a single copy gene . Two lines of evidence towards the functional activity of the HvEcR are presented . In vitro transcribed and translated HvEcR showed specific binding to hsp27 and pall response elements in the presence of CfUSP . Stable expression of HvEcR in 293 cells induced reporter gene activity in the presence of muristeroneA in a dose dependant manner while dexamethasone failed to activate.

Biochem J, 1999 Nov 1, 343 Pt 3, 673 - 80
Evidence for an interaction of the metalloprotease-disintegrin tumour necrosis factor alpha convertase (TACE) with mitotic arrest deficient 2 (MAD2), and of the metalloprotease-disintegrin MDC9 with a novel MAD2-related protein, MAD2beta; Nelson KK et al.; Metalloprotease-disintegrins are a family of transmembrane glycoproteins that have a role in fertilization, sperm migration, myoblast fusion, neural development and ectodomain shedding . In the present study we used the yeast two-hybrid system to search for proteins that interact with the cytoplasmic domain of two metalloprotease-disintegrins, tumour necrosis factor alpha convertase (TACE; ADAM17) and MDC9 (ADAM9; meltrin gamma) . We have identified mitotic arrest deficient 2 (MAD2) as a binding partner of the TACE cytoplasmic domain, and a novel MAD2-related protein, MAD2beta, as a binding partner of the MDC9 cytoplasmic domain . MAD2beta has 23% sequence identity with MAD2, which is a component of the spindle assembly (or mitotic) checkpoint mechanism . Northern blot analysis of human tissues indicates that MAD2beta mRNA is expressed ubiquitously . The interaction of the TACE and MDC9 cytoplasmic domains with their binding partners has been confirmed biochemically . The independent identification of MAD2 and MAD2beta as potential interacting partners of distinct metalloprotease-disintegrins raises the possibility of a link between metalloprotease-disintegrins and the cell cycle, or of functions for MAD2 and MAD2beta that are not related to cell cycle control.

Biochem J, 1999 Nov 1, 343 Pt 3, 653 - 62
Conserved charged residues in the leucine-rich repeat domain of the Ran GTPase activating protein are required for Ran binding and GTPase activation; Haberland J et al.; GTPase activating proteins (GAPs) for Ran, a Ras-related GTPase participating in nucleocytoplasmic transport, have been identified in different species ranging from yeast to man . All RanGAPs are characterized by a conserved domain consisting of eight leucine-rich repeats (LRRs) interrupted at two positions by so-called separating regions, the latter being unique for RanGAPs within the family of LRR proteins . The cytosolic RanGAP activity is essential for the Ran GTPase cycle which in turn provides directionality in nucleocytoplasmic transport, but the structural basis for the interaction between Ran and its GAP has not been elucidated . In order to gain a better understanding of this interaction we generated a number of mutant RanGAPs carrying amino acid substitutions in the LRR domain and analysed their complex formation with Ran as well as their ability to stimulate the intrinsic GTPase activity of the G protein . We show that conserved charged residues present in the separating regions of the LRR domain are indispensable for efficient Ran binding and GAP activity . These separating regions contain three conserved arginines which could possibly serve as catalytic residues similar to the arginine fingers identified in GAPs for other small GTPases . However, mutations in two of these arginines do not affect the GAP activity and replacement of the third conserved arginine (Arg91 in human RanGAP) severely interferes not only with GAP activity but also with Ran binding . This indicates that RanGAP-stimulated GTP hydrolysis on Ran does not involve a catalytic arginine residue but requires certain charged residues of the LRR domain of the GAP for mediating the protein-protein interaction.

Methods, 1999 Oct, 19(2), 338 - 49
Genetic approaches to the study of protein-protein interactions; Appling DR; This article describes genetic approaches to the study of heterologous protein-protein interactions, focusing on the yeast Saccharomyces cerevisiae as a useful eukaryotic model system . Several methods are described that can be used to search for new interactions, including extragenic suppression, multicopy suppression, synthetic lethality, and transdominant inhibition . Strategies for screening, genetic characterization, and clone identification are described, along with recent examples from the literature . In addition, genetic methods are discussed that can be used to further characterize a newly discovered protein-protein interaction . These include the creation of mutant libraries of a given protein by chemical mutagenesis or polymerase chain reaction, the production of dominant-negative mutants, and strategies for introducing these mutant alleles back into yeast for analysis . Although these genetic methods are quite powerful, they are often just a starting point for further biochemical or cell biological experiments .

Mutat Res, 1999 Sep 13, 435(1), 23 - 33
Protein complexes in nucleotide excision repair; Araujo SJ et al.; The main pathway by which mammalian cells remove DNA damage caused by UV light and some other mutagens is nucleotide excision repair (NER) . The best characterised components of the human NER process are those proteins defective in the inherited disorder xeroderma pigmentosum (XP) . The proteins known to be involved in the first steps of the NER reaction (damage recognition and incision-excision) are heterotrimeric RPA, XPA, the 6 to 9 subunit TFIIH, XPC-hHR23B, XPG, and ERCC1-XPF . Many interactions between these proteins have been found in recent years using different methods both in mammalian cells and for the homologous proteins in yeast . There are virtually no quantitative measurements of the relative strengths of these interactions . Higher order associations between these proteins in solution and even the existence of a complete "repairosome" complex have been reported, which would have implications both for the mechanism of repair and for the interplay between NER and other cellular processes . Nevertheless, evidence for a completely pre-assembled functional repairosome in solution is inconclusive and the order of action of repair factors on damaged DNA is uncertain.

Plant Mol Biol, 1999 Aug, 40(6), 959 - 67
The mat-r open reading frame is transcribed from a non-canonical promoter and contains an internal promoter to co-transcribe exons nad1e and nad5III in wheat mitochondria; Farre JC et al.; The expression of the mat-r locus (mat-r-nad1e-nad5III) was studied in wheat mitochondria . Transcription initiation sites were mapped by S1 protection, primer extension and capping experiments . Two different transcription initiation sites were found . One, non-canonical promoter of low expression level generates a transcript containing the complete mat-r open reading frame (orf), suggesting that this form is the maturase-reverse transcriptase mRNA . A second transcription initiation site, found within the coding region of the mat-r orf, directs the transcription of an abundant co-transcript containing the carboxy-terminal region of the mat-r orf, exon e of the nad1 gene, exon III of the nad5 gene and their respective trains-introns . The co-transcript promoter carries the consensus motif of plant mitochondrial promoters . Analysis of transcript sequences reveals the presence of editing sites in analogous positions in both nad1e and nad5III trans-introns, suggesting that RNA editing is necessary for the trans-splicing process.

Genes Cells, 1999 Aug, 4(8), 425 - 44
High copy number suppression of the meiotic arrest caused by a dmc1 mutation: REC114 imposes an early recombination block and RAD54 promotes a DMC1-independent DSB repair pathway; Bishop DK et al.; BACKGROUND: DMC1, the meiosis-specific eukaryotic homologue of bacterial recA, is required for completion of meiotic recombination and cell cycle progression past prophase . In a dmc1 mutant, double strand break recombination intermediates accumulate and cells arrest in prophase . We isolated genes which, when present at high copy numbers, suppress the meiotic arrest phenotype conferred by dmc1 mutations . RESULTS: Among the genes isolated were two which suppress arrest by altering the recombination process . REC114 suppresses formation of double strand break (DSB) recombination intermediates . The low viability of spores produced by dmc1 mutants carrying high copy numbers of REC114 is rescued when reductional segregation is bypassed by mutation of spo13 . High copy numbers of RAD54 suppress dmc1 arrest, promote DSB repair, and allow formation of viable spores following reductional segregation . Analysis of the combined effects of a null mutation in RED1, a gene required for meiotic chromosome structure, with null mutations in RAD54 and DMC1 shows that RAD54, while not normally important for repair of DSBs during meiosis, is required for efficient repair of breaks by the intersister recombination pathway that operates in red1 dmc1 double mutants . CONCLUSIONS: Over-expression of REC114 suppresses meiotic arrest by preventing formation of DSBs . High copy numbers of RAD54 activate a DMC1-independent mechanism that promotes repair of DSBs by homology-mediated recombination . The ability of RAD54 to promote DMC1-independent recombination is proposed to involve suppression of a constraint that normally promotes recombination between homologous chromatids rather than sisters.

J Cell Biol, 1999 Oct 18, 147(2), 351 - 66
The kinesin-related protein, HSET, opposes the activity of Eg5 and cross-links microtubules in the mammalian mitotic spindle; Mountain V et al.; We have prepared antibodies specific for HSET, the human homologue of the KAR3 family of minus end-directed motors . Immuno-EM with these antibodies indicates that HSET frequently localizes between microtubules within the mammalian metaphase spindle consistent with a microtubule cross-linking function . Microinjection experiments show that HSET activity is essential for meiotic spindle organization in murine oocytes and taxol-induced aster assembly in cultured cells . However, inhibition of HSET did not affect mitotic spindle architecture or function in cultured cells, indicating that centrosomes mask the role of HSET during mitosis . We also show that (acentrosomal) microtubule asters fail to assemble in vitro without HSET activity, but simultaneous inhibition of HSET and Eg5, a plus end-directed motor, redresses the balance of forces acting on microtubules and restores aster organization . In vivo, centrosomes fail to separate and monopolar spindles assemble without Eg5 activity . Simultaneous inhibition of HSET and Eg5 restores centrosome separation and, in some cases, bipolar spindle formation . Thus, through microtubule cross-linking and oppositely oriented motor activity, HSET and Eg5 participate in spindle assembly and promote spindle bipolarity, although the activity of HSET is not essential for spindle assembly and function in cultured cells because of centrosomes.

J Cell Biol, 1999 Oct 18, 147(2), 235 - 46
The karyopherin Kap122p/Pdr6p imports both subunits of the transcription factor IIA into the nucleus; Titov AA et al.; We discovered a nuclear import pathway mediated by the product of the previously identified Saccharomyces cerevisiae gene PDR6 (pleiotropic drug resistance) . This gene product functions as a karyopherin (Kap) for nuclear import . Consistent with previously proposed nomenclature, we have renamed this gene KAP122 . Kap122p was localized both to the cytoplasm and the nucleus . As a prominent import substrate of Kap122p, we identified the complex of the large and small subunit (Toa1p and Toa2p, respectively) of the general transcription factor IIA (TFIIA) . Recombinant GST-Kap122p formed a complex with recombinant His(6)-Toa1p/Toa2p . In wild-type cells, Toa1p and Toa2p were localized to the nucleus . Consistent with Kap122p being the principal Kap for import of the Toa1p-Toa2p complex, we found that deletion of KAP122 results in increased cytoplasmic localization of both Toa1p and Toa2p . Deletion of KAP122 is not lethal, although deletion of TOA1 and TOA2 is . Together these data suggest that Kap122p is the major Kap for the import of Toa1p-Toa2p into the nucleus . Like other substrate-Kap complexes, the Toa1p/Toa2p/Kap122p complex isolated from yeast cytosol or reconstituted from recombinant proteins, was dissociated by RanGTP but not RanGDP . Kap122p bound to nucleoporins, specifically, to the peptide repeat-containing fragments of Nup1p and Nup2p.

J Clin Invest, 1999 Oct, 104(8), 1097 - 105
Overproduction of Th2-specific chemokines in NC/Nga mice exhibiting atopic dermatitis-like lesions; Vestergaard C et al.; We have examined the expression of chemokines and their receptors in the atopic dermatitis-like (AD-like) lesions of NC/Nga mice . Such lesions develop when the mice are kept in conventional conditions, but not when they are kept isolated from specific pathogens . The thymus- and activation-regulated chemokine TARC is unexpectedly highly expressed in the basal epidermis of 14-week-old mice with lesions, whereas it is not expressed in the skin without lesions . Production of TARC by keratinocytes was confirmed by culturing murine keratinocytic cell line cells (PAM212) with TNF-alpha, IFN-gamma, or IL-1beta . Expression of another Th2 chemokine, macrophage-derived chemokine (MDC), was observed in the skin from mice kept in both conventional and pathogen-free conditions, but expression of MDC was increased severalfold in the skin with lesions . The cellular origin of MDC was identified to be dermal dendritic cells . Infiltration of the skin by IL-4-producing T cells and mast cells, and the increase of CCR4 mRNA in the skin, coincided with the development of AD lesions . These observations indicate that TARC and MDC actively participate in the pathogenesis of AD-like lesions in NC/Nga mice and that these Th2 chemokines could be novel targets for intervention therapy of AD in humans.

DNA Seq, 1998, 9(5-6), 323 - 8
Cloning and characterisation of a prohibitin gene from infective larvae of the parasitic nematode Toxocara canis; Loukas A et al.; Infective larvae of the parasitic nematode Toxocara canis express an mRNA (Tc-pro-1) encoding a predicted protein that shares significant homology with prohibitin, a protein involved in inhibition of cell proliferation - The closest homologues of Tc-pro-1 include an expressed sequence tag (EST) from Caenorhabditis elegans and Drosophila L2Cc, a protein thought to be essential for larval development and moulting . Other homologues include prohibitin from rat and human and an EST from Saccharomyces cerevisiae . Parasite life cycles generally include periods of developmental arrest, which in the larvae of T . canis may persist for many years without loss of metabolic activity . This report of the first full-length gene encoding prohibitin from a parasitic nematode raises interesting suggestions about the potential role of prohibitin in diapause and in the regulation of moulting in development.

Mol Cell Biol, 1999 Nov, 19(11), 7782 - 91
Evidence for distinct substrate specificities of importin alpha family members in nuclear protein import; Kohler M et al.; Importin alpha plays a pivotal role in the classical nuclear protein import pathway . Importin alpha shuttles between nucleus and cytoplasm, binds nuclear localization signal-bearing proteins, and functions as an adapter to access the importin beta-dependent import pathway . In contrast to what is found for importin beta, several isoforms of importin alpha, which can be grouped into three subfamilies, exist in higher eucaryotes . We describe here a novel member of the human family, importin alpha7 . To analyze specific functions of the distinct importin alpha proteins, we recombinantly expressed and purified five human importin alpha's along with importin alpha from Xenopus and Saccharomyces cerevisiae . Binding affinity studies showed that all importin alpha proteins from humans or Xenopus bind their import receptor (importin beta) and their export receptor (CAS) with only marginal differences . Using an in vitro import assay based on permeabilized HeLa cells, we compared the import substrate specificities of the various importin alpha proteins . When the substrates were tested singly, only the import of RCC1 showed a strong preference for one family member, importin alpha3, whereas most of the other substrates were imported by all importin alpha proteins with similar efficiencies . However, strikingly different substrate preferences of the various importin alpha proteins were revealed when two substrates were offered simultaneously.

Mol Cell Biol, 1999 Nov, 19(11), 7697 - 704
The TFIIIC90 subunit of TFIIIC interacts with multiple components of the RNA polymerase III machinery and contains a histone-specific acetyltransferase activity; Hsieh YJ et al.; Human transcription factor IIIC (hTFIIIC) is a multisubunit complex that directly recognizes promoter elements and recruits TFIIIB and RNA polymerase III . Here we describe the cDNA cloning and characterization of the 90-kDa subunit (hTFIIIC90) that is present within a DNA-binding subcomplex (TFIIIC2) of TFIIIC . hTFIIIC90 has no specific homology to any of the known yeast TFIIIC subunits . Immunodepletion and immunoprecipitation studies indicate that hTFIIIC90 is a bona fide subunit of TFIIIC2 and absolutely required for RNA polymerase III transcription . hTFIIIC90 shows interactions with the hTFIIIC220, hTFIIIC110, and hTFIIIC63 subunits of TFIIIC, the hTFIIIB90 subunit of TFIIIB, and the human RPC39 (hRPC39) and hRPC62 subunits of an initiation-specific subcomplex of RNA polymerase III . These interactions may facilitate both TFIIIB and RNA polymerase III recruitment to the preinitiation complex by TFIIIC . We show that hTFIIIC90 has an intrinsic histone acetyltransferase activity with a substrate specificity for histone H3.

Mol Cell Biol, 1999 Nov, 19(11), 7630 - 8
RPH1 and GIS1 are damage-responsive repressors of PHR1; Jang YK et al.; The Saccharomyces cerevisiae DNA repair gene PHR1 encodes a photolyase that catalyzes the light-dependent repair of pyrimidine dimers . PHR1 expression is induced at the level of transcription by a variety of DNA-damaging agents . The primary regulator of the PHR1 damage response is a 39-bp sequence called URS(PHR1) which is the binding site for a protein(s) that constitutes the damage-responsive repressor PRP . In this communication, we report the identification of two proteins, Rph1p and Gis1p, that regulate PHR1 expression through URS(PHR1) . Both proteins contain two putative zinc fingers that are identical throughout the DNA binding region, and deletion of both RPH1 and GIS1 is required to fully derepress PHR1 in the absence of damage . Derepression of PHR1 increases the rate and extent of photoreactivation in vivo, demonstrating that the damage response of PHR1 enhances cellular repair capacity . In vitro footprinting and binding competition studies indicate that the sequence AG(4) (C(4)T) within URS(PHR1) is the binding site for Rph1p and Gis1p and suggests that at least one additional DNA binding component is present in the PRP complex.

Mol Cell Biol, 1999 Nov, 19(11), 7621 - 9
p57(Kip2) stabilizes the MyoD protein by inhibiting cyclin E-Cdk2 kinase activity in growing myoblasts; Reynaud EG et al.; We show that expression of p57(Kip2), a potent tight-binding inhibitor of several G(1) cyclin-cyclin-dependent kinase (Cdk) complexes, increases markedly during C2C12 myoblast differentiation . We examined the effect of p57(Kip2) on the activity of the transcription factor MyoD . In transient transfection assays, transcriptional transactivation of the mouse muscle creatine kinase promoter by MyoD was enhanced by the Cdk inhibitors . In addition, p57(Kip2), p21(Cip1), and p27(Kip1) but not p16(Ink4a) induced an increased level of MyoD protein, and we show that MyoD, an unstable nuclear protein, was stabilized by p57(Kip2) . Forced expression of p57(Kip2) correlated with hypophosphorylation of MyoD in C2C12 myoblasts . A dominant-negative Cdk2 mutant arrested cells at the G(1) phase transition and induced hypophosphorylation of MyoD . Furthermore, phosphorylation of MyoD by purified cyclin E-Cdk2 complexes was inhibited by p57(Kip2) . In addition, the NH2 domain of p57(Kip2) necessary for inhibition of cyclin E-Cdk2 activity was sufficient to inhibit MyoD phosphorylation and to stabilize it, leading to its accumulation in proliferative myoblasts . Taken together, our data suggest that repression of cyclin E-Cdk2-mediated phosphorylation of MyoD by p57(Kip2) could play an important role in the accumulation of MyoD at the onset of myoblast differentiation.

Mol Cell Biol, 1999 Nov, 19(11), 7600 - 9
Structural and functional cross-talk between a distant enhancer and the epsilon-globin gene promoter shows interdependence of the two elements in chromatin; McDowell JC et al.; We investigated the requirements for enhancer-promoter communication by using the human beta-globin locus control region (LCR) DNase I-hypersensitive site 2 (HS2) enhancer and the epsilon-globin gene in chromatinized minichromosomes in erythroid cells . Activation of globin genes during development is accompanied by localized alterations of chromatin structure, and CACCC binding factors and GATA-1, which interact with both globin promoters and the LCR, are believed to be critical for globin gene transcription activation . We found that an HS2 element mutated in its GATA motif failed to remodel the epsilon-globin promoter or activate transcription yet HS2 nuclease accessibility did not change . Accessibility and transcription were reduced at promoters with mutated GATA-1 or CACCC sites . Strikingly, these mutations also resulted in reduced accessibility at HS2 . In the absence of a globin gene, HS2 is similarly resistant to nuclease digestion . In contrast to observations in Saccharomyces cerevisiae, HS2-dependent promoter remodeling was diminished when we mutated the TATA box, crippling transcription . This mutation also reduced HS2 accessibility . The results indicate that the epsilon-globin promoter and HS2 interact both structurally and functionally and that both upstream activators and the basal transcription apparatus contribute to the interaction . Further, at least in this instance, transcription activation and promoter remodeling by a distant enhancer are not separable.

Mol Cell Biol, 1999 Nov, 19(11), 7568 - 76
Mutations in VPS16 and MRT1 stabilize mRNAs by activating an inhibitor of the decapping enzyme; Zhang S et al.; Decapping is a rate-limiting step in the decay of many yeast mRNAs; the activity of the decapping enzyme therefore plays a significant role in determining RNA stability . Using an in vitro decapping assay, we have identified a factor, Vps16p, that regulates the activity of the yeast decapping enzyme, Dcp1p . Mutations in the VPS16 gene result in a reduction of decapping activity in vitro and in the stabilization of both wild-type and nonsense-codon-containing mRNAs in vivo . The mrt1-3 allele, previously shown to affect the turnover of wild-type mRNAs, results in a similar in vitro phenotype . Extracts from both vps16 and mrt1 mutant strains inhibit the activity of purified Flag-Dcp1p . We have identified a 70-kDa protein which copurifies with Flag-Dcp1p as the abundant Hsp70 family member Ssa1p/2p . Intriguingly, the interaction with Ssa1p/2p is enhanced in strains with mutations in vps16 or mrt1 . We propose that Hsp70s may be involved in the regulation of mRNA decapping.

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