J Exp Bot, 2001 Apr, 52(357), 663 - 8 Down-regulation of a ripening-related beta-galactosidase gene (TBG1) in transgenic tomato fruits; Carey AT et al.; Exo-galactanase/beta-galactosidase (EC 3.2.1.23) activity is thought to be responsible for the loss of galactosyl residues from the cell walls of ripening tomatoes . Transgenic tomato plants (Lycopersicon esculentum Mill cv . Ailsa Craig) with reduced exo-galactanase/beta-galactosidase mRNA were generated to test this hypothesis and to investigate the role of the enzyme in fruit softening . A previously identified tomato beta-galactosidase cDNA clone, TBG1, was used in the experiments . Heterologous expression of the clone in yeast demonstrated that TBG1 could release galactosyl residues from tomato cell wall galactans . Transgenic plants showed a reduction in TBG1 mRNA to 10% of normal levels in the ripening fruits . However, despite the reduction in message, total beta-galactosidase and exo-galactanase activities were unaffected . Furthermore, there was no apparent effect on levels of cell wall galactosyl residues when compared with the control . It was concluded that during the ripening of tomato fruits a family of beta-galactosidases capable of degrading cell wall galactans are active and down-regulation of TBG1 message to 10% was insufficient to alter the degree of galactan degradation. J Biol Chem, 2001 Aug 31, 276(35), 32642 - 7 Epub 2001 Jun 18. Thrombin receptor signaling to cytoskeleton requires Hsp90; Pai KS et al.; Thrombin is a serine protease that evokes various cellular responses involved in injury and repair of the nervous system through the activation of protease-activated receptor-1 (PAR-1) . Signals that modulate cell morphology precede most PAR-1 effects, but the initial signal transduction molecules are not known . Using the yeast two-hybrid system, we identified Hsp90, a chaperone with known signaling properties, as a binding partner of PAR-1 . The interaction was confirmed by glutathione S-transferase pull-down, overlay, and co-immunoprecipitation assays . Morphological assays in mouse astrocytes were carried out to evaluate the importance of Hsp90 during cytoskeletal signaling . Reducing Hsp90 levels by antisense treatment or disruption of the Hsp90.PAR-1 complex by the Hsp90-specific drug geldanamycin attenuated thrombin-mediated astrocyte shape changes . Furthermore, overexpression of the PAR-1 cytoplasmic tail abrogated thrombin-induced cytoskeletal changes in neuronal cells . Treatment with geldanamycin specifically inhibited activation of RhoA without affecting thrombin-mediated intracellular calcium release, revealing the regulation of a distinct signaling pathway by Hsp90 . Taken together, these studies demonstrate that Hsp90 may be essential for PAR-1-mediated signaling to the cytoskeleton. Curr Biol, 2001 Apr 3, 11(7), R253 - 6 Meiotic recombination: breaking the genome to save it; Lichten M; Recombination ensures the correct segregation of chromosomes to gametes during meiosis . Recent studies point to a universal mechanism for initiating meiotic recombination: the formation of double-strand DNA breaks by Spo11p. Curr Biol, 2001 Apr 3, 11(7), 508 - 13 Activation of the anaphase-promoting complex and degradation of cyclin B is not required for progression from Meiosis I to II in Xenopus oocytes; Taieb FE et al.; Sister chromatid separation and cyclin degradation in mitosis depend on the association of the anaphase-promoting complex (APC) with the Fizzy protein (Cdc20), leading to the metaphase/anaphase transition and exit from mitosis {1--3} . In Xenopus, after metaphase of the first meiotic division, only partial cyclin degradation occurs, and chromosome segregation during anaphase I proceeds without sister chromatid separation {4--7} . We investigated the role of xFizzy during meiosis using an antisense depletion approach . xFizzy accumulates to high levels in Meiosis I, and injection of antisense oligonucleotides to xFizzy blocks nearly all APC-mediated cyclin B degradation and Cdc2/cyclin B (MPF) inactivation between Meiosis I and II . However, even without APC activation, xFizzy-ablated oocytes progress to Meiosis II as shown by cyclin E synthesis, further accumulation of cyclin B, and evolution of the metaphase I spindle to a metaphase II spindle via a disc-shaped aggregate of microtubules known to follow anaphase I {8} . Inhibition of the MAPK pathway by U0126 in antisense-injected oocytes prevents cyclin B accumulation beyond the level that is present at metaphase I . Full synthesis and accumulation can be restored in the presence of U0126 by the expression of a constitutively active form of the MAPK target, p90(Rsk) . Thus, p90(Rsk) is sufficient not only to partially inhibit APC activity {7}, but also to stimulate cyclin B synthesis in Meiosis II. Curr Biol, 2001 Apr 3, 11(7), 482 - 8 CDC-42 controls early cell polarity and spindle orientation in C . elegans; Gotta M et al.; BACKGROUND: Generation of asymmetry in the one-cell embryo of C . elegans establishes the anterior--posterior axis (A-P), and is necessary for the proper identity of early blastomeres . Conserved PAR proteins are asymmetrically distributed and are required for the generation of this early asymmetry . The small G protein Cdc42 is a key regulator of polarity in other systems, and recently it has been shown to interact with the mammalian homolog of PAR-6 . The function of Cdc42 in C . elegans had not yet been investigated, however . RESULTS: Here, we show that C . elegans cdc-42 plays an essential role in the polarity of the one-cell embryo and the proper localization of PAR proteins . Inhibition of cdc-42 using RNA interference results in embryos with a phenotype that is nearly identical to par-3, par-6, and pkc-3 mutants, and asymmetric localization of these and other PAR proteins is lost . We further show that C . elegans CDC-42 physically interacts with PAR-6 in a yeast two-hybrid system, consistent with data on the interaction of human homologs . CONCLUSIONS: Our results show that CDC-42 acts in concert with the PAR proteins to control the polarity of the C . elegans embryo, and provide evidence that the interaction of CDC-42 and the PAR-3/PAR-6/PKC-3 complex has been evolutionarily conserved as a functional unit. FEBS Lett, 2001 Jun 8, 498(2-3), 150 - 6 Nuclear export of mRNA; Zenklusen D et al.; Export of mRNA through nuclear pore complexes (NPC) is preceded by multiple and well coordinated processing steps, resulting in the formation of an export competent ribonucleoprotein complex (mRNP) . Numerous factors involved in the translocation of the mRNP through the NPC and its release into the cytoplasm have been isolated mainly through genetic approaches in yeast, and putative functional homologues have been identified in metazoan systems . Understanding the mechanism of mRNA export relies, in part, on the functional characterization of these factors and the establishment of a complete network of molecular interactions . Here we summarize recent progress in the characterization of yeast and mammalian components implicated in the export of an mRNA from the nucleus to the cytoplasm. FEBS Lett, 2001 Jun 8, 498(2-3), 140 - 4 Transcriptomes, transcription activators and microarrays; Devaux F et al.; Gene-specific transcription activators are among the main factors which specifically shape the transcriptome profiles . It is tempting to take advantage of their properties to decipher the genome expression circuitry . The advent of microarray technology has offered fantastic opportunities to quickly analyze the expression profiles dictated by specific transcription factors . This review will first focus on the strategies which have been devised to control the activity of transcription factors and in the second part on the microarray experiments which addressed the role of these transcription factors in the genome-wide expression profile . This last part will mainly consider the case of the yeast Saccharomyces cerevisiae genome . All the collected data are available through the on-line database yTAFNET . yTAFNET is designed to help the characterization of connections between the different yeast regulatory networks. Exp Cell Res, 2001 Jul 1, 267(1), 126 - 34 hVPS41 is expressed in multiple isoforms and can associate with vesicles through a RING-H2 finger motif; McVey Ward D et al.; Vps41p, the protein encoded by the yeast gene VPS41, has been shown to mediate formation of AP-3 transport vesicles from the Golgi apparatus and to facilitate the docking and fusion of lysosomal vesicles . Although both of these activities involve transient association with membrane structures, the mechanisms that mediate those interactions have not been determined . Orthologues of VPS41 have been identified in humans, Drosophila, tomato, and Arabidopsis; the degree of sequence similarity among these genes suggests a highly conserved function . Here we provide evidence that hVps41, the human homologue of Vps41p, is expressed in two isoforms that differ in that one contains a C-terminal RING-H2 sequence motif . Transient expression analysis suggests that this RING-H2 domain is responsible for membrane association . This observation was further supported by the cytosolic localization of site-specific mutants . A truncated construct containing only the hVps41 RING-H2 domain was found to associate with a class of intracellular vesicles that originated from the Golgi and showed partial coincidence with the delta subunit of the adaptor protein complex-3 . Together with information from the homologous yeast system, these results suggest that hVps41 may also be involved in the formation and fusion of transport vesicles from the Golgi . J Biol Chem, 2001 Aug 17, 276(33), 30773 - 8 Epub 2001 Jun 15. Atp11p and Atp12p are assembly factors for the F(1)-ATPase in human mitochondria; Wang ZG et al.; Atp11p and Atp12p were first described as proteins required for assembly of the F(1) component of the mitochondrial ATP synthase in Saccharomyces cerevisiae (Ackerman, S . H., and Tzagoloff, A . (1990) Proc . Natl . Acad . Sci . U . S . A . 87, 4986-4990) . Here we report the isolation of the cDNAs and the characterization of the human genes for Atp11p and Atp12p and show that the human proteins function like their yeast counterparts . Human ATP11 spans 24 kilobase pairs in 9 exons and maps to 1p32.3-p33, while ATP12 contains > or =8 exons and localizes to 17p11.2 . Both genes are broadly conserved in eukaryotes and are expressed in a wide range of tissues, which suggests that Atp11p and Atp12p are essential housekeeping proteins of human cells . The information reported herein will be useful in the evaluation of patients with ascertained deficiencies in the ATP synthase, in which the underlying biochemical defect is unknown and may reside in a protein that influences the assembly of the enzyme. Gene, 2001 Jun 13, 271(1), 99 - 108 Cloning and characterization of two Arabidopsis genes that belong to the RAD21/REC8 family of chromosome cohesin proteins; Dong F et al.; Sister chromatid cohesion is required for proper chromosome segregation during cell division . One group of proteins that is essential for sister chromatid cohesion during mitosis and meiosis is the RAD21/REC8 family of cohesin proteins . Two cohesin proteins are found in yeast; one that functions mainly in mitosis while the other participates in meiosis . In contrast, only one cohesin gene appears to be present in Drosophila . In previous studies we identified an Arabidopsis cohesin protein that is required for meiosis . In this report we describe the isolation and characterization of two additional Arabidopsis cohesin genes . The structure of the genes suggests that they arose via a gene duplication event followed by extensive sequence evolution . Transcripts for the two genes are present throughout the plant and are highest in regions of active cell division, suggesting that the proteins may participate in chromosome cohesion during mitosis. Biochim Biophys Acta, 2001 Jun 15, 1526(3), 301 - 9 Study of the mode of action of endopolygalacturonase from Fusarium moniliforme; Bonnin E et al.; One endopolygalacturonase from Fusarium moniliforme was purified from the culture broth of a transformed strain of Saccharomyces cerevisiae . Its kinetic parameters and mode of action were studied on galacturonic acid oligomers and homogalacturonan . The dimer was not a substrate for the enzyme . The enzyme was shown to follow Michaelis-Menten behaviour towards the other substrates tested . Affinity and maximum rate of hydrolysis increased with increasing chain length, up to the hexamer or heptamer, for which V(max) was in the same range as with homogalacturonan . The enzyme was demonstrated to have a multi-chain attack mode of action and its active site included five subsites ranging from -3 to +2 . The final products of hydrolysis of homogalacturonan were the monomer and the dimer of galacturonic acid. Virus Res, 2001 Aug, 76(2), 183 - 9 The H1 double-stranded RNA genome of Ustilago maydis virus-H1 encodes a polyprotein that contains structural motifs for capsid polypeptide, papain-like protease, and RNA-dependent RNA polymerase; Kang J et al.; The Ustilago maydis viral (UmV) genome consists of three distinct size groups of double-stranded RNA (dsRNA) segments: H (heavy), M (medium), and L (light) . The H segments have been suggested to encode all essential viral proteins, but without any molecular evidences . As a preliminary step to understand viral genomic organization and the molecular mechanism governing gene expression in UmV, we determined the complete nucleotide sequence of the H1 dsRNA genome in P1 viral killer subtype . The H1 dsRNA genome (designated UmV-H1) contained a single open reading frame that encodes a polyprotein of 1820 residues, which is predicted to be autocatalytically processed by a viral papain-like protease to generate viral proteins . The amino-terminal region is the capsid polypeptide with a predicted molecular mass of 79.9 kDa . The carboxy-terminal region is the RNA-dependent RNA polymerase (RDRP) that has a high sequence homology to those of the totiviruses . The H2 dsRNA also encodes a distinct RDRP, suggesting that UmV is a complex virus system like the Saccharomyces cerevisiae viruses ScV-L1 and -La. EMBO J, 2001 Jun 15, 20(12), 3272 - 81 Role of the non-homologous DNA end joining pathway in the early steps of retroviral infection; Li L et al.; Early after infection, the retroviral RNA genome is reverse transcribed to generate a linear cDNA copy, then that copy is integrated into a chromosome of the host cell . We report that unintegrated viral cDNA is a substrate for the host cell non-homologous DNA end joining (NHEJ) pathway, which normally repairs cellular double-strand breaks by end ligation . NHEJ activity was found to be required for an end-ligation reaction that circularizes a portion of the unintegrated viral cDNA in infected cells . Consistent with this, the NHEJ proteins Ku70 and Ku80 were found to be bound to purified retroviral replication intermediates . Cells defective in NHEJ are known to undergo apoptosis in response to retroviral infection, a response that we show requires reverse transcription to form the cDNA genome but not subsequent integration . We propose that the double-strand ends present in unintegrated cDNA promote apoptosis, as is known to be the case for chromosomal double-strand breaks, and cDNA circularization removes the pro-apoptotic signal. EMBO J, 2001 Jun 15, 20(12), 3167 - 76 Cell signaling can direct either binary or graded transcriptional responses; Biggar SR et al.; Transcriptional control is generally thought to operate as a binary switch, a behavior that might explain observations such as monoallelic gene expression, stochastic phenotypic changes and bimodal gene activation kinetics . By measuring the activity of the single-copy GAL1 promoter in single cells, we found that changes in the activities of either the transcriptional activator, Gal4 (by simple recruitment with synthetic ligands), or the transcriptional repressor, Mig1, generated graded (non-binary) changes in gene expression that were proportional to signal intensity . However, in the context of the endogenous glucose-responsive signaling pathway, these transcription factors formed part of a binary transcriptional response . Genetic studies demonstrated that this binary response resulted from regulation of a second repressor, Gal80, whereas regulation of Mig1 by a distinct signaling pathway generated graded changes in GAL1 promoter activity . Surprisingly, isogenetic cells can respond to glucose with either binary or graded changes in gene expression, depending on growth conditions . Our studies demonstrate that a given promoter can adapt either binary or graded behavior, and identify the Mig1 and Gal80 genes as necessary for binary versus graded behavior of the Gal1 promoter. EMBO J, 2001 Jun 15, 20(12), 3145 - 55 Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion; Veit M et al.; Activated fatty acids stimulate budding and fusion in several cell-free assays for vesicular transport . This stimulation is thought to be due to protein palmitoylation, but relevant substrates have not yet been identified . We now report that Vac8p, a protein known to be required for vacuole inheritance, becomes palmitoylated when isolated yeast vacuoles are incubated under conditions that allow membrane fusion . Similar requirements for Vac8p palmitoylation and vacuole fusion, the inhibition of vacuole fusion by antibodies to Vac8p and the strongly reduced fusion of vacuoles lacking Vac8p suggest that palmitoylated Vac8p is essential for homotypic vacuole fusion . Strikingly, palmitoylation of Vac8p is blocked by the addition of antibodies to Sec18p (yeast NSF) only . Consistent with this, a portion of Vac8p is associated with the SNARE complex on vacuoles, which is lost during Sec18p- and ATP-dependent priming . During or after SNARE complex disassembly, palmitoylation occurs and anchors Vac8p to the vacuolar membrane . We propose that palmitoylation of Vac8p is regulated by the same machinery that controls membrane fusion. EMBO J, 2001 Jun 15, 20(12), 3124 - 31 Sec61p-independent degradation of the tail-anchored ER membrane protein Ubc6p; Walter J et al.; Tail-anchored proteins are distinct from other membrane proteins as they are thought to insert into the endoplasmic reticulum (ER) membrane independently of Sec61p translocation pores . These pores not only mediate import but are also assumed to catalyze export of proteins in a process called ER-associated protein degradation (ERAD) . In order to examine the Sec61p dependence of the export of tail-anchored proteins, we analyzed the degradation pathway of a tail-anchored ER membrane protein, the ubiquitin-conjugating enzyme 6 (Ubc6p) . In contrast to other ubiquitin conjugating enzymes (Ubcs), Ubc6p is naturally short-lived . Its proteolysis is mediated specifically by the unique Ubc6p tail region . Degradation further requires the activity of Cue1p-assembled Ubc7p, and its own catalytic site cysteine . However, it occurs independently of the other ERAD components Ubc1p, Hrd1p/Der3p, Hrd3p and Der1p . In contrast to other natural ERAD substrates, proteasomal mutants accumulate a membrane-bound degradation intermediate of Ubc6p . Most interestingly, mutations in SEC61 do not reduce the turnover of full-length Ubc6p nor cause a detectable accumulation of degradation intermediates . These data are in accordance with a model in which tail-anchored proteins can be extracted from membranes independently of Sec61p. Biochim Biophys Acta, 2001 May 31, 1536(2-3), 85 - 96 The unfolded protein response and Alzheimer's disease; Imaizumi K et al.; Disruption of calcium homeostasis, inhibition of protein glycosylation, and reduction of disulfide bonds provoke accumulation of unfolded protein in the endoplasmic reticulum (ER), and are therefore a type of 'ER stress' . Normal cells respond to ER stress by increasing transcription of genes encoding ER-resident chaperones such as GRP78/BiP, GRP94 and protein disulfide isomerase to facilitate protein folding . This induction system is termed the unfolded protein response . Familial Alzheimer's disease-linked presenilin-1 (PS1) mutation downregulates the unfolded protein response and leads to vulnerability to ER stress . The mechanisms by which mutant PS1 affects the ER stress response are attributed to the inhibited activation of ER stress transducers such as IRE1, PERK and ATF6. Biochim Biophys Acta, 2001 May 28, 1519(1-2), 70 - 7 Molecular cloning and functional characterization of two murine cDNAs which encode Ubc variants involved in DNA repair and mutagenesis; Franko J et al.; Ubiquitin-conjugating enzyme (Ubc) variants share structural similarity with Ubcs but lack the essential cysteine residue required to form a thioester bond with ubiquitin . Yeast Mms2 is a Ubc variant and plays an important role in error-free DNA postreplication repair to protect cells from killing by DNA damaging agents and mutagenesis . Ironically, one of two known Mms2 homologs, CROC1, has been linked to cell immortalization and tumorigenesis . To further investigate cellular roles played by mammalian Mms2 homologs, we report here the molecular cloning, tissue distribution and functional characterization of two mouse cDNAs encoding mMMS2 and mCROC1 . Unlike human CROC1, the mCROC1 gene does not encode two alternative transcripts in most tissues . Instead, nonoverlapping sequences were found in two distinct cDNA clones that together would constitute a full-length open reading frame homologous to CROC1B . Both mMMS2 and the C-terminal mCROC1 core domain are able to complement the yeast mms2 mutant functionally and are able to interact with Ubc13 in a yeast two-hybrid assay, indicating that they are true yeast Mms2 homologs and may play a similar role in DNA postreplication repair . We propose several hypotheses to reconcile the seemingly contradictory observations regarding roles of the two mammalian Mms2 homologs in tumorigenesis and carcinogenesis. Mutat Res, 2001 Jul 1, 478(1-2), 1 - 21 Recognition of cisplatin adducts by cellular proteins; Kartalou M et al.; Cisplatin is a widely used chemotherapeutic agent . It reacts with nucleophilic bases in DNA and forms 1,2-d(ApG), 1,2-d(GpG) and 1,3-d(GpTpG) intrastrand crosslinks, interstrand crosslinks and monofunctional adducts . The presence of these adducts in DNA is through to be responsible for the therapeutic efficacy of cisplatin . The exact signal transduction pathway that leads to cell cycle arrest and cell death following treatment with the drug is not known but cell death is believed to be mediated by the recognition of the adducts by cellular proteins . Here we describe the structural information available for cisplatin and related platinum adducts, the interactions of the adducts with cellular proteins and the implications of these interactions for cell survival. Mol Genet Genomics, 2001 May, 265(3), 497 - 507 Stability and inheritance of methylation states at PstI sites in Pisum; Knox MR et al.; The nuclear genome of pea is heavily methylated and the stability of this methylation pattern is unknown . In this study we investigated the stability of DNA methylation and its contribution to restriction fragment polymorphism as judged by AFLP fragment differences . To do this the results of a conventional AFLP analysis were compared with those obtained by a related procedure performed on pre-amplified DNA which provided a 5-methylcytosine-free DNA template (SDAFLP) . Genetic mapping in a recombinant inbred (RI) population showed that polymorphisms attributable to different methylation states of PstI sites were abundant, and generally appeared to be stably inherited, although occasional failures of the inheritance of methylation states have been found . Assessments of genetic diversity by AFLP and SDAFLP were in general agreement with each other and with the currently accepted phylogeny of Pisum, but within cultivated groups the number of differences appeared to be exaggerated by AFLP . The data suggest that epigenetic differences may have played a role in the domestication of pea. Curr Genet, 2001 Apr, 39(2), 77 - 82 Over-expression of the INO2 regulatory gene alters regulation of an INO1-lacZ reporter gene but does not affect regulation of INO1 expression; Miller LL et al.; The yeast INO2 gene encodes a transcriptional activator . Inositol and choline repress transcription of the INO2 gene, and its target genes . That is, INO2 transcription is auto-regulated . This observation prompted two separate investigations to determine if regulation of INO2 is required for regulation of its target genes . One study, using northern blot hybridization, showed that constitutive INO2 transcription did not affect regulation of the INO1 gene, while another study revealed that it severely dampened regulation of an INO1-lacZ gene . By repeating both assays from a single yeast strain we demonstrate that this disparity is due to the different reporter systems. Curr Genet, 2001 Apr, 39(2), 62 - 7 Ssb1 chaperone is a {PSI+} prion-curing factor; Chacinska A et al.; Yeast SUP7' or SUP11 nonsense suppressors have no phenotypic expression in strains deficient in the isopentenylation of A37 in tRNA . Here we show that such strains spontaneously produce cells with a nonsense suppressor phenotype which is related to the cytoplasmically inherited determinant and manifests all the key features of the {PSI+} prion . A screen of a multicopy yeast genomic library for genes that inactivate the {PSI+}-related suppressor phenotype resulted in the isolation of the SSB1 gene . Moreover, we demonstrate that multicopy plasmid encoding the Ssb1 chaperone cures cells of the {PSI+} prion. Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7325 - 30 Epub 2001 Jun 12. Protein kinase Cdc15 activates the Dbf2-Mob1 kinase complex; Mah AS et al.; Exit from mitosis in budding yeast requires inactivation of cyclin-dependent kinases through mechanisms triggered by the protein phosphatase Cdc14 . Cdc14 activity, in turn, is regulated by a group of proteins, the mitotic exit network (MEN), which includes Lte1, Tem1, Cdc5, Cdc15, Dbf2/Dbf20, and Mob1 . The direct biochemical interactions between the components of the MEN remain largely unresolved . Here, we investigate the mechanisms that underlie activation of the protein kinase Dbf2 . Dbf2 kinase activity depended on Tem1, Cdc15, and Mob1 in vivo . In vitro, recombinant protein kinase Cdc15 activated recombinant Dbf2, but only when Dbf2 was bound to Mob1 . Conserved phosphorylation sites Ser-374 and Thr-544 (present in the human, Caenorhabditis elegans, and Drosophila melanogaster relatives of Dbf2) were required for DBF2 function in vivo, and activation of Dbf2-Mob1 by Cdc15 in vitro . Although Cdc15 phosphorylated Dbf2, Dbf2-Mob1, and Dbf2(S374A/T544A)-Mob1, the pattern of phosphate incorporation into Dbf2 was substantially altered by either the S374A T544A mutations or omission of Mob1 . Thus, Cdc15 promotes the exit from mitosis by directly switching on the kinase activity of Dbf2 . We propose that Mob1 promotes this activation process by enabling Cdc15 to phosphorylate the critical Ser-374 and Thr-544 phosphoacceptor sites of Dbf2. Gene, 2001 May 30, 270(1-2), 31 - 40 Mouse fatty acid transport protein 4 (FATP4): characterization of the gene and functional assessment as a very long chain acyl-CoA synthetase; Herrmann T et al.; FATP4 (SLC27A4) is a member of the fatty acid transport protein (FATP) family, a group of evolutionarily conserved proteins that are involved in cellular uptake and metabolism of long and very long chain fatty acids . We cloned and characterized the murine FATP4 gene and its cDNA . From database analysis we identified the human FATP4 genomic sequence . The FATP4 gene was assigned to mouse chromosome 2 band B, syntenic to the region 9q34 encompassing the human gene . The open reading frame was determined to be 1929 bp in length, encoding a polypeptide of 643 amino acids . Within the coding region, the exon-intron structures of the murine FATP4 gene and its human counterpart are identical, revealing a high similarity to the FATP1 gene . The overall amino acid identity between the deduced murine and human FATP4 polypeptides is 92.2%, and between the murine FATP1 and FATP4 polypeptides is 60.3% . Northern analysis showed that FATP4 mRNA was expressed most abundantly in small intestine, brain, kidney, liver, skin and heart . Transfection of FATP4 cDNA into COS1 cells resulted in a 2-fold increase in palmitoyl-CoA synthetase (C16:0) and a 5-fold increase in lignoceroyl-CoA synthetase (C24:0) activity from membrane extracts, indicating that the FATP4 gene encodes an acyl-CoA synthetase with substrate specificity biased towards very long chain fatty acids. Mol Biol Cell, 2001 Jun, 12(6), 1671 - 85 Drosophila heterochromatin protein 1 (HP1)/origin recognition complex (ORC) protein is associated with HP1 and ORC and functions in heterochromatin-induced silencing; Shareef MM et al.; Heterochromatin protein 1 (HP1) is a conserved component of the highly compact chromatin of higher eukaryotic centromeres and telomeres . Cytogenetic experiments in Drosophila have shown that HP1 localization into this chromatin is perturbed in mutants for the origin recognition complex (ORC) 2 subunit . ORC has a multisubunit DNA-binding activity that binds origins of DNA replication where it is required for origin firing . The DNA-binding activity of ORC is also used in the recruitment of the Sir1 protein to silence nucleation sites flanking silent copies of the mating-type genes in Saccharomyces cerevisiae . A fraction of HP1 in the maternally loaded cytoplasm of the early Drosophila embryo is associated with a multiprotein complex containing Drosophila melanogaster ORC subunits . This complex appears to be poised to function in heterochromatin assembly later in embryonic development . Here we report the identification of a novel component of this complex, the HP1/ORC-associated protein . This protein contains similarity to DNA sequence-specific HMG proteins and is shown to bind specific satellite sequences and the telomere-associated sequence in vitro . The protein is shown to have heterochromatic localization in both diploid interphase and mitotic chromosomes and polytene chromosomes . Moreover, the gene encoding HP1/ORC-associated protein was found to display reciprocal dose-dependent variegation modifier phenotypes, similar to those for mutants in HP1 and the ORC 2 subunit. Biotechnol Bioeng, 2001, 76(1), 86 - 90 Glutamate synthesis via photoreduction of NADP+ by photostable chlorophyllide coupled with polyethylene-glycol; Asada H et al.; Chlorophyllide a was coupled with alpha-(3-aminopropyl)-omega-methoxypoly(oxyethylene) (PEG-NH2) to form a PEG-chlorophyllide conjugate through an acid-amide bond . The conjugate catalyzed the reduction of methylviologen in the presence of 2-mercaptoethanol . It also catalyzed the photoreduction of NADP+ or NAD+ in the presence of ascorbate as an electron donor and ferredoxin-NADP+ reductase as the coupling enzyme . Utilizing the reducing power of NADPH generated by PEG-chlorophyllide conjugate under illumination, glutamate was synthesized from 2-oxoglutarate and NH4+ in the presence of glutamate dehydrogenase . PEG-chlorophyllide conjugate was quite stable toward light illumination compared with chlorophyll a . The increase in the molecular weight of PEG in the PEG-chlorophyllide conjugates was accompanied by the enhancement of photostability of the conjugate and also by the increased solubility in the aqueous solution . J Biol Chem, 2001 Aug 10, 276(32), 30483 - 9 Epub 2001 Jun 08. Distinct functional surface regions on ubiquitin; Sloper-Mould KE et al.; The characterized functions of the highly conserved polypeptide ubiquitin are to target proteins for proteasome degradation or endocytosis . The formation of a polyubiquitin chain of at least four units is required for efficient proteasome binding . By contrast, monoubiquitin serves as a signal for the endocytosis of plasma membrane proteins . We have defined surface residues that are important for ubiquitin's vital functions in Saccharomyces cerevisiae . Surprisingly, alanine scanning mutagenesis showed that only 16 of ubiquitin's 63 surface residues are essential for vegetative growth in yeast . Most of the essential residues localize to two hydrophobic clusters that participate in proteasome recognition and/or endocytosis . The others reside in or near the tail region, which is important for conjugation and deubiquitination . We also demonstrate that the essential residues comprise two distinct functional surfaces: residues surrounding Phe(4) are required for endocytosis, whereas residues surrounding Ile(44) are required for both endocytosis and proteasome degradation. J Biol Chem, 2001 Aug 3, 276(31), 29051 - 8 Epub 2001 Jun 08. Biochemical studies of Zmpste24-deficient mice; Leung GK et al.; Genetic studies in Saccharomyces cerevisiae identified two genes, STE24 and RCE1, involved in cleaving the three carboxyl-terminal amino acids from isoprenylated proteins that terminate with a CAAX sequence motif . Ste24p cleaves the carboxyl-terminal "-AAX" from the yeast mating pheromone a-factor, whereas Rce1p cleaves the -AAX from both a-factor and Ras2p . Ste24p also cleaves the amino terminus of a-factor . The mouse genome contains orthologues for both yeast RCE1 and STE24 . We previously demonstrated, with a gene-knockout experiment, that mouse Rce1 is essential for development and that Rce1 is entirely responsible for the carboxyl-terminal proteolytic processing of the mouse Ras proteins . In this study, we cloned mouse Zmpste24, the orthologue for yeast STE24 and showed that it could promote a-factor production when expressed in yeast . Then, to assess the importance of Zmpste24 in development, we generated Zmpste24-deficient mice . Unlike the Rce1 knockout mice, Zmpste24-deficient mice survived development and were fertile . Since no natural substrates for mammalian Zmpste24 have been identified, yeast a-factor was used as a surrogate substrate to investigate the biochemical activities in membranes from the cells and tissues of Zmpste24-deficient mice . We demonstrate that Zmpste24-deficient mouse membranes, like Ste24p-deficient yeast membranes, have diminished CAAX proteolytic activity and lack the ability to cleave the amino terminus of the a-factor precursor . Thus, both enzymatic activities of yeast Ste24p are conserved in mouse Zmpste24, but these enzymatic activities are not essential for mouse development or for fertility. J Biol Chem, 2001 Aug 10, 276(32), 29792 - 7 Epub 2001 Jun 13. The brain exocyst complex interacts with RalA in a GTP-dependent manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene; Brymora A et al.; Ral is a small GTPase involved in critical cellular signaling pathways . The two isoforms, RalA and RalB, are widely distributed in different tissues, with RalA being enriched in brain . The best characterized RalA signaling pathways involve RalBP1 and phospholipase D . To investigate RalA signaling in neuronal cells we searched for RalA-binding proteins in brain . We found at least eight proteins that bound RalA in a GTP-dependent manner . Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) identified these as the components of the exocyst complex . The yeast exocyst is a regulator of polarized secretion, docking vesicles to regions of the plasma membrane involved in active exocytosis . We identified the human FLJ10893 protein as the mammalian homologue of the yeast exocyst protein Sec3p . The exocyst complex did not contain the previously identified exocyst component rSec15, but a new homologue of both yeast Sec15p and rSec15, called KIAA0919 . Western blots confirmed that two rat exocyst proteins, rSec6 and rSec8, bound active RalA in nerve terminals, as did RalBP1 . Phospholipase D bound RalA in a nucleotide-independent manner . This places the RalA signaling system in mammalian nerve terminals, where the exocyst may act as an effector for activated RalA in directing sites of exocytosis. Methods, 2001 Jul, 24(3), 218 - 29 The tandem affinity purification (TAP) method: a general procedure of protein complex purification; Puig O et al.; Identification of components present in biological complexes requires their purification to near homogeneity . Methods of purification vary from protein to protein, making it impossible to design a general purification strategy valid for all cases . We have developed the tandem affinity purification (TAP) method as a tool that allows rapid purification under native conditions of complexes, even when expressed at their natural level . Prior knowledge of complex composition or function is not required . The TAP method requires fusion of the TAP tag, either N- or C-terminally, to the target protein of interest . Starting from a relatively small number of cells, active macromolecular complexes can be isolated and used for multiple applications . Variations of the method to specifically purify complexes containing two given components or to subtract undesired complexes can easily be implemented . The TAP method was initially developed in yeast but can be successfully adapted to various organisms . Its simplicity, high yield, and wide applicability make the TAP method a very useful procedure for protein purification and proteome exploration . J Chromatogr A, 2001 May 11, 917(1-2), 55 - 61 Bioskin as an affinity matrix for the separation of glycoproteins; Vicente C et al.; Bioskin is a natural product produced by a mixed culture of Acetobacter xylinum, Saccharomyces cerevisiae and S . pombe cultured on media containing sucrose . It is of fibrillar nature able to retain some proteins, such as cytochrome c, by adsorption, and mainly composed of glucosamine and N-acetyl-D-glucosamine . This makes it possible that, at an adequate pH value, proteins charged as polyanionic molecules, such as catalase, can be retained by ionic adsorption using the positively charged amino groups of the matrix . In addition, bioskin can also be used as an affinity matrix to retain glycoproteins able to perform specific affinity reactions with the amino sugars of the matrix, such as invertase, fetuin or ovalbumin . Its possible use as a chromatographic support is discussed. Anal Chem, 2001 Jun 1, 73(11), 2578 - 86 Phosphoprotein isotope-coded affinity tag approach for isolating and quantitating phosphopeptides in proteome-wide analyses; Goshe MB et al.; A method has been developed that utilizes phosphoprotein isotope-coded affinity tags (PhIAT) that combines stable isotope and biotin labeling to enrich and quantitatively measure differences in the O-phosphorylation states of proteins . The PhIAT labeling approach involves hydroxide ion-mediated beta-elimination of the O-phosphate moiety and the addition of 1,2-ethanedithiol containing either four alkyl hydrogens (EDT-D0) or four alkyl deuteriums (EDT-D4) followed by biotinylation of the EDT-D0/D4 moiety using (+)-biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine . The PhIAT reagent, which contains the nucleophilic sulfhydryl and isotopic label covalently linked to a biotin moiety, was synthesized and has the potential utility to reduce the O-phosphorylation derivatization into a one-step process . The PhIAT labeling approach was initially demonstrated using the model phosphoprotein beta-casein . After proteolytic digestion, the PhIAT-labeled peptides were affinity isolated using immobilized avidin and analyzed using capillary reversed-phase liquid chromatography-mass spectrometry . PhIAT-labeled beta-casein peptides corresponding to peptides containing known sites of O-phosphorylation were isolated and identified . The PhIAT labeling method was also applied to a yeast protein extract . The PhIAT labeling technique provides a reliable method for making quantitative measurements of differences in the O-phosphorylation state of proteins. Inflammation, 2001 Jun, 25(3), 165 - 9 An intact cytoskeleton is required for prolonged respiratory burst activity during neutrophil phagocytosis; Granfeldt D et al.; The temporal relationship between phagocytosis and respiratory burst activity was investigated . Neutrophil uptake of yeast particles was synchronized and the kinetics of the oxidative burst was determined using an isoluminol/luminol amplified chemiluminescence system . The reactive oxygen species were mainly generated intracellularly (defined as the activity that remained in an luminol-enhanced system in the presence of superoxide dismutase and catalase) . Following phagocytosis, the intracellular response rapidly reached a level close to the maximum and the activity was almost constant for the first 10 to 15 min . The response then slowly declined . The presence of cytochalasin B, an inhibitor of actin polymerization, greatly reduced the respiratory burst activity, and this was true also when the inhibitor was added after completion of uptake of yeast particles . Our results thus show that there is a continuous production of oxygen metabolites long after phagocytosis is completed . There is also a requirement for an intact cytoskeleton for prolonged superoxide production inside the phagosome. Plant Physiol, 2001 Jun, 126(2), 910 - 20 Cloning and expression pattern of a gene encoding an alpha-xylosidase active against xyloglucan oligosaccharides from Arabidopsis; Sampedro J et al.; An alpha-xylosidase active against xyloglucan oligosaccharides was purified from cabbage (Brassica oleracea var . capitata) leaves . Two peptide sequences were obtained from this protein, the N-terminal and an internal one, and these were used to identify an Arabidopsis gene coding for an alpha-xylosidase that we propose to call AtXYL1 . It has been mapped to a region of chromosome I between markers at 100.44 and 107.48 cM . AtXYL1 comprised three exons and encoded a peptide that was 915 amino acids long, with a potential signal peptide of 22 amino acids and eight possible N-glycosylation sites . The protein encoded by AtXYL1 showed the signature regions of family 31 glycosyl hydrolases, which comprises not only alpha-xylosidases, but also alpha-glucosidases . The alpha-xylosidase activity is present in apoplastic extractions from Arabidopsis seedlings, as suggested by the deduced signal peptide . The first eight leaves from Arabidopsis plants were harvested to analyze alpha-xylosidase activity and AtXYL1 expression levels . Both increased from older to younger leaves, where xyloglucan turnover is expected to be higher . When this gene was introduced in a suitable expression vector and used to transform Saccharomyces cerevisiae, significantly higher alpha-xylosidase activity was detected in the yeast cells . alpha-Glucosidase activity was also increased in the transformed cells, although to a lesser extent . These results show that AtXYL1 encodes for an apoplastic alpha-xylosidase active against xyloglucan oligosaccharides that probably also has activity against p-nitrophenyl-alpha-D-glucoside. Plant Cell, 2001 Jun, 13(6), 1369 - 82 Nodule-specific regulation of phosphatidylinositol transfer protein expression in Lotus japonicus; Kapranov P et al.; Phosphatidylinositol transfer proteins (PITPs) modulate signal transduction pathways and membrane-trafficking functions in eukaryotes . Here, we describe the characterization of a gene family from Lotus japonicus that encodes a novel class of plant PITP-like proteins (LjPLPs) and that is regulated in an unusual nodule-specific manner . Members of this gene family were identified based on their nucleotide sequence homology with a previously described cDNA, LjNOD16, which encodes the L . japonicus late nodulin Nlj16 . Nlj16 or highly related amino acid sequences are shown to constitute C-terminal domains of LjPLPs and are suggested to function as specific plasma membrane targeting modules . The expression patterns of one member of this gene family (LjPLP-IV) revealed that LjNOD16 mRNA synthesis in nodules is the result of the transcriptional activity of a nodule-specific promoter located in an intron of the LjPLP-IV gene . This intron-borne bidirectional promoter also generates nodule-specific antisense transcripts derived from the N-terminal PITP domain coding region of the LjPLP-IV gene . We propose that Nlj16 protein synthesis and LjPLP-IV antisense transcript generation are components of an elaborate mechanism designed to control LjPLP synthesis and/or functioning in nodules. J Cell Biol, 2001 Jun 11, 153(6), 1151 - 60 Tom40, the pore-forming component of the protein-conducting TOM channel in the outer membrane of mitochondria; Ahting U et al.; Tom40 is the main component of the preprotein translocase of the outer membrane of mitochondria (TOM complex) . We have isolated Tom40 of Neurospora crassa by removing the receptor Tom22 and the small Tom components Tom6 and Tom7 from the purified TOM core complex . Tom40 is organized in a high molecular mass complex of approximately 350 kD . It forms a high conductance channel . Mitochondrial presequence peptides interact specifically with Tom40 reconstituted into planar lipid membranes and decrease the ion flow through the pores in a voltage-dependent manner . The secondary structure of Tom40 comprises approximately 31% beta-sheet, 22% alpha-helix, and 47% remaining structure as determined by circular dichroism measurements and Fourier transform infrared spectroscopy . Electron microscopy of purified Tom40 revealed particles primarily with one center of stain accumulation . They presumably represent an open pore with a diameter of approximately 2.5 nm, similar to the pores found in the TOM complex . Thus, Tom40 is the core element of the TOM translocase; it forms the protein-conducting channel in an oligomeric assembly. J Biol Chem, 2001 Aug 10, 276(32), 30002 - 10 Epub 2001 Jun 11. pEg2 aurora-A kinase, histone H3 phosphorylation, and chromosome assembly in Xenopus egg extract; Scrittori L et al.; In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10 . In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs . We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10 . The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation . Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts . These data identify pEg2 as a good candidate for mitotic histone H3 kinase . However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs . This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae. Mol Microbiol, 2001 Jun, 40(5), 1165 - 74 The Candida glabrata Amt1 copper-sensing transcription factor requires Swi/Snf and Gcn5 at a critical step in copper detoxification; Koch KA et al.; The yeast Candida glabrata rapidly autoactivates transcription of the AMT1 gene in response to potentially toxic copper levels through the copper-inducible binding of the Amt1 transcription factor to a metal response element (MRE) within a positioned nucleosome . Our previous studies have characterized the role of a 16 bp homopolymeric dA:dT DNA structural element in facilitating rapid Amt1 access to the AMT1 promoter nucleosomal MRE . In this study, we have used the genetically more facile yeast Saccharomyces cerevisiae to identify additional cellular factors that are important for promoting rapid autoactivation of the AMT1 gene in response to toxic copper levels . We demonstrate that the Swi/Snf nucleosome remodelling complex and the histone acetyltransferase Gcn5 are both essential for AMT1 gene autoregulation, and that the requirement for these chromatin remodelling factors is target gene specific . Chromatin accessibility measurements performed in vitro and in vivo indicate that part of the absolute requirement for these factors is derived from their involvement in facilitating nucleosomal access to the AMT1 promoter MRE . Additionally, these data implicate the involvement of Swi/Snf and Gcn5 at multiple levels of AMT1 gene autoregulation. Mol Microbiol, 2001 May, 40(4), 1009 - 19 RNA polymerase II and TBP occupy the repressed CYC1 promoter; Martens C et al.; Saccharomyces cerevisiae CYC1 gene expression has been studied in great detail with regard to the response to oxygen availability and carbon source . In the absence of oxygen and the presence of glucose, the CYC1 gene is completely repressed . Chromatin structure is thought to play an important role in CYC1 gene regulation, as nucleosome depletion results in 94-fold derepression . In addition, the CYC1 core promoter has been used extensively in hybrid constructs to study activation by heterologous transcription factors . Therefore, we set out to map the chromatin structure of the CYC1 promoter and determine its role in CYC1 gene regulation . We report here that the repressed CYC1 promoter contains no positioned nucleosomes over the core promoter . However, we did find TFIID and RNA polymerase II bound in a complex on the repressed promoter . These results indicate that recruitment of TFIID and RNA polymerase II are not rate-limiting steps in CYC1 activation. Biochemistry, 2001 Jun 19, 40(24), 7301 - 8 Role of phylogenetically conserved amino acids in folding of Na,K-ATPase; Jorgensen JR et al.; This paper focuses on the amino acid sequence 708-TGDGVNDSPALKK in pig kidney Na,K-ATPase as one of the best conserved among P-type ATPases . In Ca-ATPase this sequence forms a strand-loop-helix structure as part of a Rossman fold next to the phosphorylation site . Substitution of polar residues in the investigated sequence interfered with high-level accumulation of mutant protein . Mutant alpha1-subunit protein only accumulated in membranes from yeast cells grown at 15 degrees C whereas wild-type protein accumulated at both 15 and 35 degrees C . A systematic screen for the molecular mechanism behind lack of accumulation of mutant protein at 35 degrees C showed that transcription and translation were unaffected by the mutations . To demonstrate in vivo protein folding problems, an unfolded protein response reporter system was constructed in yeast . In this strain, only expression of mutant Na,K-ATPase alpha1-subunit caused induction of the unfolded protein response at 35 degrees C, indicating folding problems in the ER . Lowering the expression temperature to 15 degrees C prevented induction of the unfolded protein response after mutant protein expression, indicating correct folding at this temperature . At the permissive temperature mutant proteins were able to escape the endoplasmic reticulum quality control, reach the plasma membrane, and bind ouabain with high affinity . Since mutants in the 708-TGDGVNDSPALKK segment had a thermo inactivation profile identical to that of wild type, they were classified as temperature-sensitive synthesis mutants . The results indicate that this segment contributes side chains of importance for overall folding and maturation of Na,K-ATPase and all other P-type ATPases. J Mol Biol, 2001 Jun 15, 309(4), 845 - 53 The crystal structure of delta(3)-delta(2)-enoyl-CoA isomerase; Mursula AM et al.; The active-site geometry of the first crystal structure of a Delta(3)-Delta(2)-enoyl-coenzyme A (CoA) isomerase (the peroxisomal enzyme from the yeast Saccharomyces cerevisiae) shows that only one catalytic base, Glu158, is involved in shuttling the proton from the C2 carbon atom of the substrate, Delta(3)-enoyl-CoA, to the C4 atom of the product, Delta(2)-enoyl-CoA . Site-directed mutagenesis has been performed to confirm that this glutamate residue is essential for catalysis . This Delta(3)-Delta(2)-enoyl-CoA isomerase is a hexameric enzyme, consisting of six identical subunits . It belongs to the hydratase/isomerase superfamily of enzymes which catalyze a wide range of CoA-dependent reactions . The members of the hydratase/ isomerase superfamily have only a low level of sequence identity . Comparison of the crystal structure of the Delta(3)-Delta(2)-enoyl-CoA isomerase with the other structures of this superfamily shows only one region of large structural variability, which is in the second turn of the spiral fold and which is involved in defining the shape of the binding pocket . J Biol Chem, 2001 Aug 17, 276(33), 31321 - 31 Epub 2001 Jun 07. Mutational analysis of conserved residues in the GCN5 family of histone acetyltransferases; Langer MR et al.; GCN5 is a critical transcriptional co-activator and is the defining member of a large superfamily of N-acetyltransferases . GCN5 catalyzes the transfer of an acetyl group from acetyl-CoA to the epsilon-amino of lysine 14 within the core H3 histone protein . Previous biochemical analyses have indicated a fully ordered kinetic mechanism . Recent structural studies have implicated several conserved residues in catalysis and substrate binding . Here the roles of Glu-173, His-145, and Asp-214 in yeast GCN5 have been evaluated using site-directed mutagenesis, steady state and pre-steady state kinetics, pH analysis, isotope partitioning, and equilibrium binding studies . The results with wild type and E173Q, H145A, and D214A mutants are consistent with chemical catalysis being rate-determining in turnover . All mutants exhibited K(d) values (3.5-8.5 microm) for AcCoA that were similar to wild type enzyme, indicating no functional role for these residues in AcCoA binding . The E173Q mutant demonstrated a approximately 500-600-fold decreases in k(cat) and k(cat)/K(m),(H3), consistent with Glu-173 acting as the general base catalyst as proposed previously . No significant effect was observed on substrate binding steps . His-145 was identified as a residue in the peptide binding cleft that must be unprotonated (pK(a) = 5.8) for peptide binding and likely hydrogen-bonds to the Ser-10 hydroxyl of histone H3 . His-145 also contributes to lowering the pK(a) value (by 0.8 units) of general base Glu-173 through a water-mediated hydrogen bond to the carboxylate side chain . Analysis of D214A revealed an obligate protein isomerization step that occurs after AcCoA binding and permits efficient peptide binding . Asp-214 is part of a conformationally flexible loop that mediates the isomerization by stabilizing distinct conformers of the protein. Trends Endocrinol Metab, 2001 Jul, 12(5), 184 - 6 Molecular scaffold protein and cellular responses; Keenan SM et al.; Mitogen-activated kinases (MAPK) regulate many diverse cellular processes, including growth, differentiation and responses to stress . The organization of MAPKs through the use of scaffolding proteins is crucial for the selective activation of these kinases by different stimuli . Recent studies identify beta-arrestins as members of the family of MAPK scaffold proteins . beta-Arrestins not only shut off signaling by uncoupling G-protein-coupled receptors (GPCRs) from their heterotrimeric G proteins, but also contribute to the specificity of GPCRs signaling by recruiting and activating selective MAPKs. Phytochemistry, 2001 Jul, 57(5), 743 - 7 Antifungal polysulphides from Petiveria alliacea L; Benevides PJ et al.; Bioactivity-directed fractionation of the CH(2)Cl(2)/MeOH (2:1, v/v) extract of the roots of Petiveria alliacea, using mutant yeast strains of Saccharomyces cerevisiae and fungi Cladosporium cladosporioides and C . sphaerospermum led to the isolation of dipropyl disulphide (1), dibenzyl sulphide (2), dibenzyl disulphide (3), dibenzyl trisulphide (4), dibenzyl tetrasulphide (5), benzylhydroxymethyl sulphide (6) and di(benzyltrithio) methane (7) . Of these, 5-7 are new compounds and this is the first report of the natural occurrence of 2 and 3. Biochem Biophys Res Commun, 2001 Jun 15, 284(3), 757 - 62 A transgenic mouse expressing human CYP4B1 in the liver; Imaoka S et al.; The human CYP4B1 protein was expressed in the liver of a transgenic mouse line under the control of the promoter of the human apolipoprotein E (apo E) gene . Hepatic microsomes of transgenic mice catalyzed omega-hydroxylation of lauric acid and also activated 2-aminofluorene (2-AF), which is a typical substrate for CYP4B1, to mutagenic compounds detected by an umu gene expression assay . These activities observed in transgenic mouse were efficiently inhibited by CYP4B1 antibody . However, such inhibition was not observed in control mice . This is the first report to indicate catalytic activities of human CYP4B1 . For further characterization of human CYP4B1, a fusion protein of CYP4B1 and NADPH-P450 reductase was expressed in yeast cells . It was able to activate 2-AF and was also able to catalyze omega-hydroxylation of lauric acid . This transgenic mouse line and the recombinant fusion protein provide a useful tool to study human CYP4B1 and its relation to chemical toxicity and carcinogenesis . Biochemistry, 2001 Jun 5, 40(22), 6688 - 98 Mg2+-induced tRNA folding; Serebrov V et al.; Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments . By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased . The last two transitions were not accompanied by changes in the number of base pairs . The observed transitions were attributed to Mg(2+) binding to four distinct types of sites . The first two types are strong sites with K(diss) of 4 and 16 microM . The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM . Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each . Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change . The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases . Fast and slow kinetic phases of tRNA refolding were observed . Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+) . Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+) . A multistep scheme for Mg(2+)-induced tRNA folding is discussed. Nature, 2001 Jun 7, 411(6838), 713 - 6 Defects in mismatch repair promote telomerase-independent proliferation; Rizki A et al.; Mismatch repair has a central role in maintaining genomic stability by repairing DNA replication errors and inhibiting recombination between non-identical (homeologous) sequences . Defects in mismatch repair have been linked to certain human cancers, including hereditary non-polyposis colorectal cancer (HNPCC) and sporadic tumours . A crucial requirement for tumour cell proliferation is the maintenance of telomere length, and most tumours achieve this by reactivating telomerase . In both yeast and human cells, however, telomerase-independent telomere maintenance can occur as a result of recombination-dependent exchanges between often imperfectly matched telomeric sequences . Here we show that loss of mismatch-repair function promotes cellular proliferation in the absence of telomerase . Defects in mismatch repair, including mutations that correspond to the same amino-acid changes recovered from HNPCC tumours, enhance telomerase-independent survival in both Saccharomyces cerevisiae and a related budding yeast with a degree of telomere sequence homology that is similar to human telomeres . These results indicate that enhanced telomeric recombination in human cells with mismatch-repair defects may contribute to cell immortalization and hence tumorigenesis. J Biol Chem, 2001 Aug 10, 276(32), 30208 - 15 Epub 2001 Jun 06. p65-activated histone acetyltransferase activity is repressed by glucocorticoids: mifepristone fails to recruit HDAC2 to the p65-HAT complex; Ito K et al.; Glucocorticoids acting through their specific receptor can either enhance or repress gene transcription . Dexamethasone represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor expression through a combination of direct inhibition of p65-associated histone acetyltransferase (HAT) activity and by recruiting histone deacetylase 2 (HDAC2) to the p65-HAT complex . Here we show that mifepristone, a glucocorticoid receptor partial agonist, has no ability to induce gene expression but represses interleukin-1beta-stimulated histone acetylation and granulocyte-macrophage colony-stimulating factor release by 50% maximally . Mifepristone was able to inhibit p65-associated HAT activity to the same extent as dexamethasone but failed to inhibit the natural promoter to an equal extent due to an inability to recruit HDAC2 to the p65-associated HAT complex . These data suggest that the maximal repressive actions of glucocorticoids require recruitment of HDAC2 to a p65-HAT complex . These data also suggest that pharmacological manipulation of specific histone acetylation status is a potentially useful approach for the treatment of inflammatory diseases. J Biol Chem, 2001 Aug 3, 276(31), 29067 - 71 Epub 2001 Jun 06. Architecture of the human origin recognition complex; Dhar SK et al.; All the human homologs of the six subunits of Saccharomyces cerevisiae origin recognition complex have been reported so far . However, not much has been reported on the nature and the characteristics of the human origin recognition complex . In an attempt to purify recombinant human ORC from insect cells infected with baculoviruses expressing HsORC subunits, we found that human ORC2, -3, -4, and -5 form a core complex . HsORC1 and HsORC6 subunits did not enter into this core complex, suggesting that the interaction of these two subunits with the core ORC2-5 complex is extremely labile . We found that the C-terminal region of ORC2 interacts directly with the N-terminal region of ORC3 . The C-terminal region of ORC3 was, however, necessary to bring ORC4 and ORC5 into the core complex . A fragment containing the N-terminal 200 residues of ORC3 (ORC3N) competitively inhibited the ORC2-ORC3 interaction . Overexpression of this fragment in U2OS cells blocked the cells in G(1), providing the first evidence that a mammalian ORC subunit is important for the G(1)-S transition in mammalian cells. Bioinformatics, 2001 Jun, 17(6), 520 - 5 Missing value estimation methods for DNA microarrays; Troyanskaya O et al.; MOTIVATION: Gene expression microarray experiments can generate data sets with multiple missing expression values . Unfortunately, many algorithms for gene expression analysis require a complete matrix of gene array values as input . For example, methods such as hierarchical clustering and K-means clustering are not robust to missing data, and may lose effectiveness even with a few missing values . Methods for imputing missing data are needed, therefore, to minimize the effect of incomplete data sets on analyses, and to increase the range of data sets to which these algorithms can be applied . In this report, we investigate automated methods for estimating missing data . RESULTS: We present a comparative study of several methods for the estimation of missing values in gene microarray data . We implemented and evaluated three methods: a Singular Value Decomposition (SVD) based method (SVDimpute), weighted K-nearest neighbors (KNNimpute), and row average . We evaluated the methods using a variety of parameter settings and over different real data sets, and assessed the robustness of the imputation methods to the amount of missing data over the range of 1--20% missing values . We show that KNNimpute appears to provide a more robust and sensitive method for missing value estimation than SVDimpute, and both SVDimpute and KNNimpute surpass the commonly used row average method (as well as filling missing values with zeros) . We report results of the comparative experiments and provide recommendations and tools for accurate estimation of missing microarray data under a variety of conditions. Bioinformatics, 2001 Jun, 17(6), 495 - 508 Aligning gene expression time series with time warping algorithms; Aach J et al.; motivation: Increasingly, biological processes are being studied through time series of RNA expression data collected for large numbers of genes . Because common processes may unfold at varying rates in different experiments or individuals, methods are needed that will allow corresponding expression states in different time series to be mapped to one another . Results: We present implementations of time warping algorithms applicable to RNA and protein expression data and demonstrate their application to published yeast RNA expression time series . Programs executing two warping algorithms are described, a simple warping algorithm and an interpolative algorithm, along with programs that generate graphics that visually present alignment information . We show time warping to be superior to simple clustering at mapping corresponding time states . We document the impact of statistical measurement noise and sample size on the quality of time alignments, and present issues related to statistical assessment of alignment quality through alignment scores . We also discuss directions for algorithm improvement including development of multiple time series alignments and possible applications to causality searches and non-temporal processes ('concentration warping'). Annu Rev Biochem, 2001, 70, 677 - 701 Function, structure, and mechanism of intracellular copper trafficking proteins; Huffman DL et al.; Genetic, biochemical, and spectroscopic studies have established a new function for an intracellular protein, i.e., guiding and inserting a copper cofactor into the active site of a target enzyme . Studies of these new proteins have revealed a fundamental aspect of copper physiology, namely the vast overcapacity of the cytoplasm for copper sequestration . This finding framed the mechanistic, energetic, and structural aspects of intracellular copper trafficking proteins . One hallmark of the copper chaperones is the similarity of the protein fold between the chaperone and its target enzyme . The surface residues presented by each partner, however, are quite different, and some initial findings concerning the complementarity of these interfaces have led to mechanistic insights . The copper chaperones appear to lower the activation barrier for metal transfer into specific protein-binding sites . The manner in which they facilitate metal insertion appears to involve a docking of the metal donor and acceptor sites in close proximity to one another . Although the intimate mechanism is still open, it appears that a low activation barrier for metal transfer is achieved by a network of coordinate-covalent, electrostatic, and hydrogen bonding interactions in the vicinity of the metal-binding site itself. Annu Rev Biochem, 2001, 70, 475 - 501 Transcriptional coactivator complexes; Naar AM et al.; The last two decades have witnessed a tremendous expansion in our knowledge of the mechanisms employed by eukaryotic cells to control gene activity . A critical insight to transcriptional control mechanisms was provided by the discovery of coactivators, a diverse array of cellular factors that connect sequence-specific DNA binding activators to the general transcriptional machinery, or that help activators and the transcriptional apparatus to navigate through the constraints of chromatin . A number of coactivators have been isolated as large multifunctional complexes, and biochemical, genetic, molecular, and cellular strategies have all contributed to uncovering many of their components, activities, and modes of action . Coactivator functions can be broadly divide into two classes: (a) adaptors that direct activator recruitment of the transcriptional apparatus, (b) chromatin-remodeling or -modifying enzymes . Strikingly, several distinct coactivator complexes nonetheless share many subunits and appear to be assembled in a modular fashion . Such structural and functional modularity could provide the cell with building blocks from which to construct a versatile array of coactivator complexes according to its needs . The extent of functional interplay between these different activities in gene-specific transcriptional regulation is only now becoming apparent, and will remain an active area of research for years to come. Annu Rev Biochem, 2001, 70, 81 - 120 Histone acetyltransferases; Roth SY et al.; Transcriptional regulation in eukaryotes occurs within a chromatin setting and is strongly influenced by nucleosomal barriers imposed by histone proteins . Among the well-known covalent modifications of histones, the reversible acetylation of internal lysine residues in histone amino-terminal domains has long been positively linked to transcriptional activation . Recent biochemical and genetic studies have identified several large, multisubunit enzyme complexes responsible for bringing about the targeted acetylation of histones and other factors . This review discusses our current understanding of histone acetyltransferases (HATs) or acetyltransferases (ATs): their discovery, substrate specificity, catalytic mechanism, regulation, and functional links to transcription, as well as to other chromatin-modifying activities . Recent studies underscore unexpected connections to both cellular regulatory processes underlying normal development and differentiation, as well as abnormal processes that lead to oncogenesis . Although the functions of HATs and the mechanisms by which they are regulated are only beginning to be understood, these fundamental processes are likely to have far-reaching implications for human biology and disease. Bioseparation, 2000, 9(5), 269 - 76 Extraction of peptide tagged cutinase in detergent-based aqueous two-phase systems; Rodenbrock A et al.; Detergent-based aqueous two-phase systems have the advantage to require only one auxiliary chemical to induce phase separation above the cloud point . In a systematic study the efficiency of tryptophan-rich peptide tags was investigated to enhance the partitioning of an enzyme to the detergent-rich phase using cutinase as an example . Up to 90% enzyme activity could be extracted in a single step from whole broth of recombinant Saccharomyces cerevisiae expressing cutinase variants carrying a (WP)4 tag . In contrast, the extraction yield of wild type cutinase was 2-3% only . The detergent concentration and the temperature are the main parameters to optimize the extraction yield . Considering availability, extraction yields, and price the detergent Agrimul NRE 1205 served best for enzyme recovery. J Hum Genet, 2001, 46(6), 314 - 9 Identification of single-nucleotide polymorphisms (SNPs) of human N-acetyltransferase genes NAT1, NAT2, AANAT, ARD1 and L1CAM in the Japanese population; Sekine A et al.; By direct sequencing of regions of the human genome containing five genes belonging to the acetyltransferase family, arylamine N-acetyltransferase (NAT1), arylamine N-acetyltransferase (NAT2), arylalkylamine N-acetyltransferase (AANAT), L1 cell adhesion molecule (L1CAM), and the human homolog of Saccharomyces cerevisiae N-acetyltransferase ARD1, we identified 53 single-nucleotide polymorphisms (SNPs) and two insertion/ deletion polymorphisms in 48 healthy Japanese volunteers . NAT1 and NAT2 are so-called drug-metabolizing enzymes . In the NAT1 gene we found two SNPs and a 3-bp insertion/ deletion polymorphism that corresponded to the NAT1*3, *10, and *18A/*18B alleles reported in other populations . The frequencies of NAT1* alleles in our Japanese subjects were 52.6% for NAT1*4, 1.0% for NAT1*3, 40.6% for NAT1*10, 2.6% for NAT1*18A and 3.1% for NAT1*18B . In the NAT2 gene we found 32 SNPs and a 1-bp insertion/ deletion polymorphism; 6 SNPs within the coding region were reported previously and belonged to the slow acetylator group (NAT2*5, NAT2*6 and NAT2*7), and 2 of the 8 SNPs in the 5' flanking region were reported in the dbSNP of GenBank, but the remaining 24 SNPs and the insertion/deletion polymorphism were novel . The frequencies of NAT2* alleles in Japanese (51.3% for NAT2*4, 1.6% for *5B, 26.1% for *6A, 2.2% for *6B, 1.2% for *7A, 10.1% for *7B, 7.4% for *12A, and 1.1% for *13) were significantly different from those reported in Caucasian populations . In the AANAT gene we found 4 novel SNPs: 2 in the 5' flanking region, 1 in exon 4, and 1 in intron 3 . In the two genes belonging to the N-terminal N-acetyltransferase family, we identified 9 SNPs, 7 of them novel, for ARD1, and six novel SNPs for L1CAM . Variations at these loci may contribute to an understanding of the way in which different genotypes may affect the activities of human N-acetyltransferases, especially as regards the therapeutic efficacy of certain drugs and antibiotics. Proc Natl Acad Sci U S A, 2001 Jul 3, 98(14), 8133 - 8 Epub 2001 Jun 05. The role of haustoria in sugar supply during infection of broad bean by the rust fungus Uromyces fabae; Voegele RT et al.; Biotrophic plant pathogenic fungi differentiate specialized infection structures within the living cells of their host plants . These haustoria have been linked to nutrient uptake ever since their discovery . We have for the first time to our knowledge shown that the flow of sugars from the host Vicia faba to the rust fungus Uromyces fabae seems to occur largely through the haustorial complex . One of the most abundantly expressed genes in rust haustoria, the expression of which is negligible in other fungal structures, codes for a hexose transporter . Functional expression of the gene termed HXT1 in Saccharomyces cerevisiae and Xenopus laevis oocytes assigned a substrate specificity for D-glucose and D-fructose and indicated a proton symport mechanism . Abs against HXT1p exclusively labeled haustoria in immunofluorescence microscopy and the haustorial plasma membrane in electron microscopy . These results suggest that the fungus concentrates this transporter in haustoria to take advantage of a specialized compartment of the haustorial complex . The extrahaustorial matrix, delimited by the plasma membranes of both host and parasite, constitutes a newly formed apoplastic compartment with qualities distinct from those of the bulk apoplast . This organization might facilitate the competition of the parasite with natural sink organs of the host. Proc Natl Acad Sci U S A, 2001 Jun 19, 98(13), 7206 - 11 Epub 2001 Jun 05. Identification and characterization of a lysosomal transporter for small neutral amino acids; Sagne C et al.; In eukaryotic cells, lysosomes represent a major site for macromolecule degradation . Hydrolysis products are eventually exported from this acidic organelle into the cytosol through specific transporters . Impairment of this process at either the hydrolysis or the efflux step is responsible of several lysosomal storage diseases . However, most lysosomal transporters, although biochemically characterized, remain unknown at the molecular level . In this study, we report the molecular and functional characterization of a lysosomal amino acid transporter (LYAAT-1), remotely related to a family of H+-coupled plasma membrane and synaptic vesicle amino acid transporters . LYAAT-1 is expressed in most rat tissues, with highest levels in the brain where it is present in neurons . Upon overexpression in COS-7 cells, the recombinant protein mediates the accumulation of neutral amino acids, such as gamma-aminobutyric acid, l-alanine, and l-proline, through an H+/amino acid symport . Confocal microscopy on brain sections revealed that this transporter colocalizes with cathepsin D, an established lysosomal marker . LYAAT-1 thus appears as a lysosomal transporter that actively exports neutral amino acids from lysosomes by chemiosmotic coupling to the H+-ATPase of these organelles . Homology searching in eukaryotic genomes suggests that LYAAT-1 defines a subgroup of lysosomal transporters in the amino acid/auxin permease family. Mol Cell Biol, 2001 Jul, 21(13), 4311 - 20 Essential roles of Snf5p in Snf-Swi chromatin remodeling in vivo; Geng F et al.; Snf-Swi, the prototypical ATP-dependent nucleosome-remodeling complex, regulates transcription of a subset of yeast genes . With the exception of Snf2p, the ATPase subunit, the functions of the other components are unknown . We have investigated the role of the conserved Snf-Swi core subunit Snf5p through characterization of two conditional snf5 mutants . The mutants contain single amino acid alterations of invariant or conserved residues that abolish Snf-Swi-dependent transcription by distinct mechanisms . One mutation impairs Snf-Swi assembly and, consequently, its stable association with a target promoter . The other blocks a postrecruitment catalytic remodeling step . These findings suggest that Snf5p coordinates the assembly and nucleosome-remodeling activities of Snf-Swi. Mol Cell Biol, 2001 Jul, 21(13), 4276 - 91 In vivo action of the HRD ubiquitin ligase complex: mechanisms of endoplasmic reticulum quality control and sterol regulation; Gardner RG et al.; Ubiquitination is used to target both normal proteins for specific regulated degradation and misfolded proteins for purposes of quality control destruction . Ubiquitin ligases, or E3 proteins, promote ubiquitination by effecting the specific transfer of ubiquitin from the correct ubiquitin-conjugating enzyme, or E2 protein, to the target substrate . Substrate specificity is usually determined by specific sequence determinants, or degrons, in the target substrate that are recognized by the ubiquitin ligase . In quality control, however, a potentially vast collection of proteins with characteristic hallmarks of misfolding or misassembly are targeted with high specificity despite the lack of any sequence similarity between substrates . In order to understand the mechanisms of quality control ubiquitination, we have focused our attention on the first characterized quality control ubiquitin ligase, the HRD complex, which is responsible for the endoplasmic reticulum (ER)-associated degradation (ERAD) of numerous ER-resident proteins . Using an in vivo cross-linking assay, we directly examined the association of the separate HRD complex components with various ERAD substrates . We have discovered that the HRD ubiquitin ligase complex associates with both ERAD substrates and stable proteins, but only mediates ubiquitin-conjugating enzyme association with ERAD substrates . Our studies with the sterol pathway-regulated ERAD substrate Hmg2p, an isozyme of the yeast cholesterol biosynthetic enzyme HMG-coenzyme A reductase (HMGR), indicated that the HRD complex discerns between a degradation-competent "misfolded" state and a stable, tightly folded state . Thus, it appears that the physiologically regulated, HRD-dependent degradation of HMGR is effected by a programmed structural transition from a stable protein to a quality control substrate. Mol Cell Biol, 2001 Jul, 21(13), 4246 - 55 Evidence of p53-dependent cross-talk between ribosome biogenesis and the cell cycle: effects of nucleolar protein Bop1 on G(1)/S transition; Pestov DG et al.; Bop1 is a novel nucleolar protein involved in rRNA processing and ribosome assembly . We have previously shown that expression of Bop1Delta, an amino-terminally truncated Bop1 that acts as a dominant negative mutant in mouse cells, results in inhibition of 28S and 5.8S rRNA formation and deficiency of newly synthesized 60S ribosomal subunits (Z . Strezoska, D . G . Pestov, and L . F . Lau, Mol . Cell . Biol . 20:5516-5528, 2000) . Perturbation of Bop1 activities by Bop1Delta also induces a powerful yet reversible cell cycle arrest in 3T3 fibroblasts . In the present study, we show that asynchronously growing cells are arrested by Bop1Delta in a highly concerted fashion in the G(1) phase . Kinase activities of the G(1)-specific Cdk2 and Cdk4 complexes were downregulated in cells expressing Bop1Delta, whereas levels of the Cdk inhibitors p21 and p27 were concomitantly increased . The cells also displayed lack of hyperphosphorylation of retinoblastoma protein (pRb) and decreased expression of cyclin A, indicating their inability to progress through the restriction point . Inactivation of functional p53 abrogated this Bop1Delta-induced cell cycle arrest but did not restore normal rRNA processing . These findings show that deficiencies in ribosome synthesis can be uncoupled from cell cycle arrest and reveal a new role for the p53 pathway as a mediator of the signaling link between ribosome biogenesis and the cell cycle . We propose that aberrant rRNA processing and/or ribosome biogenesis may cause "nucleolar stress," leading to cell cycle arrest in a p53-dependent manner. Mol Cell Biol, 2001 Jul, 21(13), 4233 - 45 New function of CDC13 in positive telomere length regulation; Meier B et al.; Two roles for the Saccharomyces cerevisiae Cdc13 protein at the telomere have previously been characterized: it recruits telomerase to the telomere and protects chromosome ends from degradation . In a synthetic lethality screen with YKU70, the 70-kDa subunit of the telomere-associated Yku heterodimer, we identified a new mutation in CDC13, cdc13-4, that points toward an additional regulatory function of CDC13 . Although CDC13 is an essential telomerase component in vivo, no replicative senescence can be observed in cdc13-4 cells . Telomeres of cdc13-4 mutants shorten for about 150 generations until they reach a stable level . Thus, in cdc13-4 mutants, telomerase seems to be inhibited at normal telomere length but fully active at short telomeres . Furthermore, chromosome end structure remains protected in cdc13-4 mutants . Progressive telomere shortening to a steady-state level has also been described for mutants of the positive telomere length regulator TEL1 . Strikingly, cdc13-4/tel1Delta double mutants display shorter telomeres than either single mutant after 125 generations and a significant amplification of Y' elements after 225 generations . Therefore CDC13, TEL1, and the Yku heterodimer seem to represent distinct pathways in telomere length maintenance . Whereas several CDC13 mutants have been reported to display elongated telomeres indicating that Cdc13p functions in negative telomere length control, we report a new mutation leading to shortened and eventually stable telomeres . Therefore we discuss a key role of CDC13 not only in telomerase recruitment but also in regulating telomerase access, which might be modulated by protein-protein interactions acting as inhibitors or activators of telomerase activity. J Biol Chem, 2001 Aug 31, 276(35), 32597 - 605 Epub 2001 Jun 04. The binding interaction of HMG-1 with the TATA-binding protein/TATA complex; Das D et al.; High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II . In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex . By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex . HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant . In support of the binding target of HMG-1 being the N terminus of hTBP, the N-terminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation . Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds . These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55-95) in hTBP is primarily responsible for stable complex formation . In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site . Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophila but not yeast . This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP. J Biol Chem, 2001 Aug 10, 276(32), 29628 - 31 Epub 2001 Jun 04. A multiprotein complex that interacts with RNA polymerase II elongator; Li Y et al.; A three-subunit Hap complex that interacts with the RNA polymerase II Elongator was isolated from yeast . Deletions of genes for two Hap subunits, HAP1 and HAP3, confer pGKL killer-insensitive and weak Elongator phenotypes . Preferential interaction of the Hap complex with free rather than RNA polymerase II-associated Elongator suggests a role in the regulation of Elongator activity. J Biol Chem, 2001 Jul 27, 276(30), 28541 - 5 Epub 2001 Jun 04. Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains; Takatsu H et al.; GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment . By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor . A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction . A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes . These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors. Genes Dev, 2001 Jun 1, 15(11), 1406 - 18 Inhibition of early apoptotic events by Akt/PKB is dependent on the first committed step of glycolysis and mitochondrial hexokinase; Gottlob K et al.; The serine/threonine kinase Akt/PKB is a major downstream effector of growth factor-mediated cell survival . Activated Akt, like Bcl-2 and Bcl-xL, prevents closure of a PT pore component, the voltage-dependent anion channel (VDAC); intracellular acidification; mitochondrial hyperpolarization; and the decline in oxidative phosphorylation that precedes cytochrome c release . However, unlike Bcl-2 and Bcl-xL, the ability of activated Akt to preserve mitochondrial integrity, and thereby inhibit apoptosis, requires glucose availability and is coupled to its metabolism . Hexokinases are known to bind to VDAC and directly couple intramitochondrial ATP synthesis to glucose metabolism . We provide evidence that such coupling serves as a downstream effector function for Akt . First, Akt increases mitochondria-associated hexokinase activity . Second, the antiapoptotic activity of Akt requires only the first committed step of glucose metabolism catalyzed by hexokinase . Finally, ectopic hexokinase expression mimics the ability of Akt to inhibit cytochrome c release and apoptosis . We therefore propose that Akt increases coupling of glucose metabolism to oxidative phosphorylation and regulates PT pore opening via the promotion of hexokinase-VDAC interaction at the outer mitochondrial membrane. Genes Dev, 2001 Jun 1, 15(11), 1361 - 72 Pds1 phosphorylation in response to DNA damage is essential for its DNA damage checkpoint function; Wang H et al.; In Saccharomyces cerevisiae, Pds1 is an anaphase inhibitor and plays an essential role in DNA damage and spindle checkpoint pathways . Pds1 is phosphorylated in response to DNA damage but not spindle disruption, indicating distinct mechanisms delaying anaphase entry . Phosphorylation of Pds1 is Mec1 and Chk1 dependent in vivo . Here, we show that Pds1 is phosphorylated at multiple sites in vivo in response to DNA damage by Chk1 . Mutation of the Chk1 phosphorylation sites on Pds1 abolished most of its DNA damage-inducible phosphorylation and its checkpoint function, whereas its anaphase inhibitor functions and spindle checkpoint functions remain intact . Loss of Pds1 phosphorylation correlates with APC-dependent Pds1 destruction in response to DNA damage . We also show that APC(Cdc20) is active in preanaphase arrested cells after DNA damage . This suggests that Pds1 is stabilized by phosphorylation in response to DNA damage, but APC(Cdc20) activity is not altered . Our results indicate that phosphorylation of Pds1 by Chk1 is the key function of Chk1 required to prevent anaphase entry. FEBS Lett, 2001 Jun 1, 498(1), 110 - 5 The predicted beta12-beta13 loop is important for inhibition of PP2Acalpha by the antitumor drug fostriecin; Evans DR et al.; The potential anticancer agent fostriecin (FOS) is a potent inhibitor of the protein Ser/Thr phosphatases PP2A and PP4 and a weaker inhibitor of PP1 . Random mutagenesis and automated screening in yeast identified residues in human PP2Acalpha important for inhibitory FOS binding . A C269S substitution in the predicted beta12-beta13 loop decreased the FOS sensitivity of intact cells and increased the IC(50) of PP2Acalpha by 10-fold in vitro . Changing PP2Acalpha Cys-269 to phenylalanine, the equivalent residue in PP1, and the Y267G and G270D substitutions caused a similar effect . The results provide information relevant to the design of novel protein Ser/Thr phosphatase inhibitory drugs. Mol Cell, 2001 May, 7(5), 1013 - 23 Evolutionarily conserved interaction between CstF-64 and PC4 links transcription, polyadenylation, and termination; Calvo O et al.; Tight connections exist between transcription and subsequent processing of mRNA precursors, and interactions between the transcription and polyadenylation machineries seem especially extensive . Using a yeast two-hybrid screen to identify factors that interact with the polyadenylation factor CstF-64, we uncovered an interaction with the transcriptional coactivator PC4 . Both human proteins have yeast homologs, Rna15p and Sub1p, respectively, and we show that these two proteins also interact . Given evidence that certain polyadenylation factors, including Rna15p, are necessary for termination in yeast, we show that deletion or overexpression of SUB1 suppresses or enhances, respectively, both growth and termination defects detected in an rna15 mutant strain . Our findings provide an additional, unexpected connection between transcription and polyadenylation and suggest that PC4/Sub1p, via its interaction with CstF-64/Rna15p, possesses an evolutionarily conserved antitermination activity. Mol Cell, 2001 May, 7(5), 981 - 91 The 19S regulatory particle of the proteasome is required for efficient transcription elongation by RNA polymerase II; Ferdous A et al.; It is generally thought that the primary or even sole activity of the 19S regulatory particle of the 26S proteasome is to facilitate the degradation of polyubiquitinated proteins by the 20S-core subunit . However, we present evidence that the 19S complex is required for efficient elongation of RNA polymerase II (RNAP II) in vitro and in vivo . First, yeast strains carrying alleles of SUG1 and SUG2, encoding 19S components, exhibit phenotypes indicative of elongation defects . Second, in vitro transcription is inhibited by antibodies raised against Sug1, or by heat-inactivating temperature-sensitive Sug1 mutants with restoration of elongation by addition of immunopurified 19S complex . Finally, Cdc68, a known elongation factor, coimmunoprecipitates with the 19S complex, indicating a physical interaction . Inhibition of the 20S proteolytic core of the proteasome has no effect on elongation . This work defines a nonproteolytic role for the 19S complex in RNAP II transcription. Cell, 2001 Jun 1, 105(5), 637 - 44 Immunity to K1 killer toxin: internal TOK1 blockade; Sesti F et al.; K1 killer strains of Saccharomyces cerevisiae harbor RNA viruses that mediate secretion of K1, a protein toxin that kills virus-free cells . Recently, external K1 toxin was shown to directly activate TOK1 channels in the plasma membranes of sensitive yeast cells, leading to excess potassium flux and cell death . Here, a mechanism by which killer cells resist their own toxin is shown: internal toxin inhibits TOK1 channels and suppresses activation by external toxin. Plant J, 2001 Apr, 26(2), 181 - 9 Arabidopsis IRT2 gene encodes a root-periphery iron transporter; Vert G et al.; Iron uptake from the soil is a tightly controlled process in plant roots, involving specialized transporters . One such transporter, IRT1, was identified in Arabidopsis thaliana and shown to function as a broad-range metal ion transporter in yeast . Here we report the cloning and characterization of the IRT2 cDNA, a member of the ZIP family of metal transporters, highly similar to IRT1 at the amino-acid level . IRT2 expression in yeast suppresses the growth defect of iron and zinc transport yeast mutants and enhances iron uptake and accumulation . However, unlike IRT1, IRT2 does not transport manganese or cadmium in yeast . IRT2 expression is detected only in roots of A . thaliana plants, and is upregulated by iron deficiency . By fusing the IRT2 promoter to the uidA reporter gene, we show that the IRT2 promoter is mainly active in the external cell layers of the root subapical zone, and therefore provide the first tissue localization of a plant metal transporter . Altogether, these data support a role for the IRT2 transporter in iron and zinc uptake from the soil in response to iron-limited conditions. Mol Reprod Dev, 2001 Jun, 59(2), 144 - 58 Expression of genes involved in mammalian meiosis during the transition from egg to embryo; Hwang SY et al.; The ooplasm of higher eukaryotes provides substances necessary for completing the last stages of meiosis and initiating the first mitotic division . These processes are firmly attuned to other events in the egg and newly formed embryo, such as switching from the use of maternal transcripts to the onset of zygotic transcription . In mammals little is known about the molecular mechanisms guiding this transition, largely due to the lack of information about genes expressed in the egg and early embryos . Studies of yeast mitosis have contributed much of what is known about the vertebrate cell cycle, and recent reports indicate that homologs of yeast DNA repair genes also function during mammalian gametogenesis . To examine whether this conservation can be expanded to include genes operative in oocyte meiosis, we performed a computer-based search for homologs of yeast genes that are induced during sporulation in C . elegans, Drosophila, and mammals . Results from this study suggest that yeast and higher eukaryotes share genes that coordinate the overall process of meiosis . However intriguing differences exist, reflecting the distinctive mechanisms governing the progression of meiosis in each organism . ESTs representing more than half of the mammalian homologs are present in mouse cDNA libraries that contains genes controlling the meiosis/mitosis transition . About 50% of these genes contain potential cis-elements for cytoplasmic polyadenylation in their 3'-UTR, suggesting the importance of controlled translation in the egg and zygote. Nat Cell Biol, 2001 Jun, 3(6), 531 - 7 Dynamics of the COPII coat with GTP and stable analogues; Antonny B et al.; We have developed an assay to monitor the assembly of the COPII coat onto liposomes in real time . We show that with Sar1pGTP bound to liposomes, a single round of assembly and disassembly of the COPII coat lasts a few seconds . The two large COPII complexes Sec23/24p and Sec13/31p bind almost instantaneously (in less than 1 s) to Sar1pGTP-doped liposomes . This binding is followed by a fast (less than 10 s) disassembly due to a 10-fold acceleration of the GTPase-activating protein activity of Sec23/24p by the Sec13/31p complex . Experiments with the phosphate analogue BeFx suggest that Sec23/24p provides residues directly involved in GTP hydrolysis on Sar1p. Blood, 2001 Jun 15, 97(12), 3763 - 7 Expression of the neuronal cyclin-dependent kinase 5 activator p35Nck5a in human monocytic cells is associated with differentiation; Chen F et al.; Although cyclin-dependent kinase 5 (Cdk5) is widely expressed in human tissues, its activator p35Nck5a is generally considered to be neuron specific . In addition to neuronal cells, active Cdk5 complexes have been reported in developing tissues, such as the embryonic muscle and ocular lens, and in human leukemia HL60 cells induced to differentiate by an exposure to 1,25-dihydroxyvitamin D(3); however, its activator in these cells has not been demonstrated . The results of this study indicate that p35Nck5a is associated with Cdk5 in monocytic differentiation of hematopoietic cells . Specifically, p35Nck5a is expressed in normal human monocytes and in leukemic cells induced to differentiate toward the monocytic lineage, but not in lymphocytes or cells induced to granulocytic differentiation by retinoic acid . It is present in a complex with Cdk5 that has protein kinase activity, and when ectopically expressed together with Cdk5 in undifferentiated HL60 cells, it induces the expression of CD14 and "nonspecific" esterase, markers of monocytic phenotype . These observations not only indicate a functional relationship between Cdk5 and p35Nck5a, but also support a role for this complex in monocytic differentiation . (Blood . 2001;97:3763-3767) Protein Expr Purif, 2001 Jun, 22(1), 1 - 10 Immunoaffinity purification and reconstitution of human alpha(2)-adrenergic receptor subtype C2 into phospholipid vesicles; Liitti S et al.; Large quantities of correctly folded, pure alpha(2)-adrenergic receptor protein are needed for structural analysis . We report here the first efficient method to purify human alpha(2)-adrenergic receptor subtype C2 to homogeneity from recombinant yeast Saccharomyces cerevisiae by one-step purification using a monoclonal antibody column (specific for alpha(2)C2) . We show that the adrenoceptor antagonist phentolamine stabilized the receptor during purification . We used a very effective chaotropic agent, NaSCN, to elute the receptor from the immunoaffinity column with an overall yield of 34% before reconstitution . Ligand binding of detergent-solubilized, immunoaffinity-purified receptors could not be demonstrated, but partial recovery of ligand binding activity was achieved when purified receptors were reconstituted into phospholipid vesicles . The reconstituted receptors still bound radioligand after storage on ice for 4 weeks . This purification procedure can be easily scaled-up and thus demonstrates the utility of a monoclonal antibody column and NaSCN elution to purify large quantities of G-protein-coupled receptors . Stroke, 2001 Jun, 32(6), 1401 - 7 Early decrease in dna repair proteins, Ku70 and Ku86, and subsequent DNA fragmentation after transient focal cerebral ischemia in mice; Kim GW et al.; BACKGROUND AND PURPOSE: Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway . To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice . METHODS: Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery . Ku protein expression was studied by immunohistochemistry and Western blot analysis . DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) . The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion . RESULTS: Immunohistochemistry showed constitutive expression of Ku proteins in control brains . The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours . Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P<0.001) . DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours . Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining . CONCLUSIONS: These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI. J Biol Chem, 2001 Aug 10, 276(32), 29782 - 91 Epub 2001 May 31. Marked stepwise differences within a common kinetic mechanism characterize TATA-binding protein interactions with two consensus promoters; Powell RM et al.; Binding of the TATA-binding protein (TBP) to promoter DNA bearing the TATA sequence is an obligatory initial step in RNA polymerase II transcription initiation . The interactions of Saccharomyces cerevisiae TBP with the E4 (TATATATA) and adenovirus major late (TATAAAAG) promoters have been modeled via global analysis of kinetic and thermodynamic data obtained using fluorescence resonance energy transfer . A linear two-intermediate kinetic mechanism describes the reaction of both of these consensus strong promoters with TBP . Qualitative features common to both interactions include tightly bound TBP-DNA complexes with similar solution geometries, simultaneous DNA binding and bending, and the presence of intermediate TBP-DNA conformers at high mole fraction throughout most of the reaction and at equilibrium . Despite very similar energetic changes overall, the stepwise entropic and enthalpic compensations along the two pathways differ markedly following the initial binding/bending event . Furthermore, TBP-E4 dissociation ensues from both replacement and displacement processes, in contrast to replacement alone for TBP-adenovirus major late promoter . A model is proposed that explicitly correlates these similarities and differences with the sequence-specific structural properties inherent to each promoter . This detailed mechanistic comparison of two strong promoters interacting with TBP provides a foundation for subsequent comparison between consensus and variant promoter sequences reacting with TBP. J Biol Chem, 2001 Aug 3, 276(31), 29268 - 74 Epub 2001 May 31. Proteomic analysis of nucleoporin interacting proteins; Allen NP et al.; The Saccharomyces cerevisiae nuclear pore complex is a supramolecular assembly of 30 nucleoporins that cooperatively facilitate nucleocytoplasmic transport . Thirteen nucleoporins that contain FG peptide repeats (FG Nups) are proposed to function as stepping stones in karyopherin-mediated transport pathways . Here, protein interactions that occur at individual FG Nups were sampled using immobilized nucleoporins and yeast extracts . We find that many proteins bind to FG Nups in highly reproducible patterns . Among 135 proteins identified by mass spectrometry, most were karyopherins and nucleoporins . The PSFG nucleoporin Nup42p and the GLFG nucleoporins Nup49p, Nup57p, Nup100p, and Nup116p exhibited generic interactions with karyopherins; each bound 6--10 different karyopherin betas, including importins as well as exportins . Unexpectedly, the same Nups also captured the hexameric Nup84p complex and Nup2p . In contrast, the FXFG nucleoporins Nup1p, Nup2p, and Nup60p were more selective and captured mostly the Kap95p.Kap60p heterodimer . When the concentration of Gsp1p-GTP was elevated in the extracts to mimic the nucleoplasmic environment, the patterns of interacting proteins changed; exportins exhibited enhanced binding to FG Nups, and importins exhibited reduced binding . The results demonstrate a global role for Gsp1p-GTP on karyopherin-nucleoporin interactions and provide a rudimentary map of the routes that karyopherins take as they cross the nuclear pore complex. EMBO J, 2001 Jun 1, 20(11), 2954 - 65 A subcomplex of three eIF3 subunits binds eIF1 and eIF5 and stimulates ribosome binding of mRNA and tRNA(i)Met; Phan L et al.; Yeast translation initiation factor 3 contains five core subunits (known as TIF32, PRT1, NIP1, TIF34 and TIF35) and a less tightly associated component known as HCR1 . We found that a stable subcomplex of His8-PRT1, NIP1 and TIF32 (PN2 subcomplex) could be affinity purified from a strain overexpressing these eIF3 subunits . eIF5, eIF1 and HCR1 co-purified with this subcomplex, but not with distinct His8-PRT1- TIF34-TIF35 (P45) or His8-PRT1-TIF32 (P2) sub complexes . His8-PRT1 and NIP1 did not form a stable binary subcomplex . These results provide in vivo evidence that TIF32 bridges PRT1 and NIP1, and that eIFs 1 and 5 bind to NIP1, in native eIF3 . Heat-treated prt1-1 extracts are defective for Met-tRNA(i)Met binding to 40S subunits, and we also observed defective 40S binding of mRNA, eIFs 1 and 5 and eIF3 itself in these extracts . We could rescue 40S binding of Met- tRNA(i)Met and mRNA, and translation of luciferase mRNA, in a prt1-1 extract almost as well with purified PN2 subcomplex as with five-subunit eIF3, whereas the P45 subcomplex was nearly inactive . Thus, several key functions of eIF3 can be carried out by the PRT1-TIF32-NIP1 subcomplex. EMBO J, 2001 Jun 1, 20(11), 2907 - 13 Engineered interphase chromosome loops guide intrachromosomal recombination; Kostriken R et al.; How large-scale topologies regulate interphase chromosome function remains an important question in eukaryotic cell biology . Looped structures are thought to modulate transcription by pairing promoters with distant control elements and to orchestrate intrachromosomal recombination events by pairing appropriate recombination partners . To explore the effects of chromosomal topology on intrachromosomal recombination, distinct loop geometries were engineered into chromosome III of the budding yeast Saccharomyces cerevisiae . These topologies were created by employing pairs of lac operator clusters to serve as pairing sites and a modified lac repressor to perform the role of a protein cross-bridge . The influence of these engineered loops on the selection of donor loci during mating-type switching was evaluated using novel genetic and molecular methods . These experiments demonstrate that engineered interphase chromosome loops are biologically active-capable of influencing the course of intrachromosomal recombination . They also provide insight into the mechanism of mating-type switching by revealing a causal relationship between defined chromosomal topologies and the choice of donor locus. EMBO J, 2001 Jun 1, 20(11), 2896 - 906 Silent repair accounts for cell cycle specificity in the signaling of oxidative DNA lesions; Leroy C et al.; Reactive oxygen species are the most important source of DNA lesions in aerobic organisms, but little is known about the activation of the DNA checkpoints in response to oxidative stress . We show that treatment of yeast cells with sublethal concentrations of hydrogen peroxide induces a Mec1-dependent phosphorylation of Rad53 and a Rad53-dependent cell cycle delay specifically during S phase . The lack of Rad53 phosphorylation after hydrogen peroxide treatment in the G1 and G2 phases is due to the silent repair of oxidative DNA lesions produced at these stages by the base excision repair (BER) pathway . Only the disruption of the BER pathway and the accumulation and/or treatment of DNA intermediates by alternative repair pathways reveal the existence of primary DNA lesions induced at all phases of the cell cycle by hydrogen peroxide . Our data illustrate both the concept of silent repair of DNA damage and the high sensitivity of S-phase cells to hydrogen peroxide. EMBO J, 2001 Jun 1, 20(11), 2823 - 34 The RNA polymerase III transcription initiation factor TFIIIB participates in two steps of promoter opening; Kassavetis GA et al.; Evidence for post-recruitment functions of yeast transcription factor (TF)IIIB in initiation of transcription was first provided by the properties of TFIIIB-RNA polymerase III-promoter complexes assembled with deletion mutants of its Brf and B" subunits that are transcriptionally inactive because they fail to open the promoter . The experiments presented here show that these defects can be repaired by unpairing short (3 or 5 bp) DNA segments spanning the transcription bubble of the open promoter complex . Analysis of this suppression phenomenon indicates that TFIIIB participates in two steps of promoter opening by RNA polymerase III that are comparable to the successive steps of promoter opening by bacterial RNA polymerase holoenzyme . B" deletions between amino acids 355 and 421 interfere with the initiating step of DNA strand separation at the upstream end of the transcription bubble . Removing an N-terminal domain of Brf interferes with downstream propagation of the transcription bubble to and beyond the transcriptional start site. EMBO J, 2001 Jun 1, 20(11), 2742 - 56 SKP1-SnRK protein kinase interactions mediate proteasomal binding of a plant SCF ubiquitin ligase; Farras R et al.; Arabidopsis Snf1-related protein kinases (SnRKs) are implicated in pleiotropic regulation of metabolic, hormonal and stress responses through their interaction with the kinase inhibitor PRL1 WD-protein . Here we show that SKP1/ASK1, a conserved SCF (Skp1-cullin-F-box) ubiquitin ligase subunit, which suppresses the skp1-4 mitotic defect in yeast, interacts with the PRL1-binding C-terminal domains of SnRKs . The same SnRK domains recruit an SKP1/ASK1-binding proteasomal protein, alpha4/PAD1, which enhances the formation of a trimeric SnRK complex with SKP1/ASK1 in vitro . By contrast, PRL1 reduces the interaction of SKP1/ASK1 with SnRKs . SKP1/ASK1 is co-immunoprecipitated with a cullin SCF subunit (AtCUL1) and an SnRK kinase, but not with PRL1 from Arabidopsis cell extracts . SKP1/ASK1, cullin and proteasomal alpha-subunits show nuclear co-localization in differentiated Arabidopsis cells, and are observed in association with mitotic spindles and phragmoplasts during cell division . Detection of SnRK in purified 26S proteasomes and co-purification of epitope- tagged SKP1/ASK1 with SnRK, cullin and proteasomal alpha-subunits indicate that the observed protein interactions between SnRK, SKP1/ASK1 and alpha4/PAD1 are involved in proteasomal binding of an SCF ubiquitin ligase in Arabidopsis. EMBO J, 2001 Jun 1, 20(11), 2702 - 14 The death substrate Gas2 binds m-calpain and increases susceptibility to p53-dependent apoptosis; Benetti R et al.; Gas2 is a caspase-3 substrate that plays a role in regulating microfilament and cell shape changes during apoptosis . Here we provide evidence that overexpression of Gas2 efficiently increases cell susceptibility to apoptosis following UV irradiation, etoposide and methyl methanesulfonate treatments, and that these effects are dependent on increased p53 stability and transcription activity . To investigate possible pathways linking Gas2 to p53, a yeast two-hybrid screen swas performed, indicating m-calpain as a strong Gas2- interacting protein . Moreover, we demonstrate that Gas2 physically interacts with m-calpain in vivo and that recombinant Gas2 inhibits calpain-dependent processing of p53 . Importantly, the Gas2 dominant-negative form (Gas2171-314) that binds calpain but is unable to inhibit its activity abrogates Gas2's ability to stabilize p53, to enhance p53 transcriptional activity and to induce p53-dependent apoptosis . Finally, we show that Gas2 is able to regulate the levels of p53 independently of Mdm2 status, suggesting that, like calpastatin, it may enhance p53 stability by inhibiting calpain activity. Mol Genet Metab, 2001 Jun, 73(2), 138 - 48 A novel inborn error in the ligand-binding domain of the vitamin D receptor causes hereditary vitamin D-resistant rickets; Malloy PJ et al.; Mutations in the vitamin D receptor (VDR) cause hereditary vitamin D-resistant rickets (HVDRR), an autosomal recessive disease resulting in target organ resistance to 1,25-dihydroxyvitamin D(3) {1,25(OH)(2)D(3)} . In this report, we describe the clinical case and molecular basis of HVDRR in an Asian boy exhibiting the typical clinical features of the disease including alopecia . Using cultured dermal fibroblasts from the patient, 1,25(OH)(2)D(3) resistance was demonstrated by a shift in the dose response required for 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) mRNA induction . Western blot showed that the cells express a normal size VDR but contained reduced levels of receptor compared to normal cells . At 24 degrees C, the affinity of the patient's VDR for {(3)H}1,25(OH)(2)D(3) was 50-fold lower than the VDR in normal fibroblasts . Sequence analysis identified a unique T to G missense mutation in exon 6 that changed phenylalanine to cysteine at amino acid 251 (F251C) . The recreated F251C mutant VDR showed reduced transactivation activity using a 24-hydroxylase promoter-luciferase reporter . Maximal transactivation activity exhibited by the WT VDR was not achieved by the mutant VDR even when the cells were treated with up to 10(-6) M 1,25(OH)(2)D(3) . However, the transactivation activity was partially rescued by addition of RXRalpha . In the yeast two-hybrid system and GST-pull-down assays, high concentrations of 1,25(OH)(2)D(3) were needed to promote F251C mutant VDR binding to RXRalpha, indicating defective heterodimerization . In conclusion, a novel mutation was identified in the VDR LBD that reduces VDR abundance and its affinity for 1,25(OH)(2)D(3)