Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

OSW2 is a meiotically-induced gene required for spore wall formation. osw2Δspores are sensitive to ether treatment. Except for this phenotype, the mutants do not show obvious sporulation defects
thus, its function remains elusive. We found that deletion of both OSW2 and CHS3 results in a synthetic sporulation defect. The spore wall is composed of four layers, and chs3Δspores lack the outer two (chitosan and dityrosine) layers. Thus, Osw2 is involved in the assembly of the inner (glucan and mannan) layers. In agreement with this notion, a glycosylphosphatidylinositol-anchored protein reporter mislocalizes in osw2Δspores. The osw2Δmutation also exhibited a severe synthetic sporulation defect when combined with the deletion of a ß-1,6-glucan synthesis-related gene, BIG1. Osw2 is localized to the prospore membrane during sporulation. However, it disappears in mature spores, indicating that it is not a structural component of the spore wall. Given that Osw2 contains a probable 2-dehydropantoate 2-reductase domain, it may mediate an enzymatic reaction. Osw2 shows a weak similarity to other 2-dehydropantoate 2-reductase domain-containing proteins, Svl3 and Pam1. A pam1"svl3Δmutant exhibits vegetative cell and spore wall defects. Thus, the 2-dehydropantoate 2-reductase domain-containing proteins may have a similar function in glucan and/or mannan layer assembly.

DOI： 10.1093/jb/mvx082

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Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press (OUP)  

DOI： 10.1093/femsyr/fox095

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

During the developmental process of sporulation in Saccharomyces cerevisiae, membrane structures called prospore membranes are formed de novo, expand, extend, acquire a round shape, and finally become plasma membranes of the spores. GIP1 encodes a regulatory/targeting subunit of protein phosphatase type 1 that is required for sporulation. Gip1 recruits the catalytic subunit Glc7 to septin structures that form along the prospore membrane; however, the molecular basis of its localization and function is not fully understood. Here we show that Gip1 changes its localization dynamically and is required for prospore membrane extension. Gip1 first associates with the spindle pole body as the prospore membrane forms, moves onto the prospore membrane and then to the septins as the membrane extends, distributes around the prospore membrane after closure, and finally translocates into the nucleus in the maturing spore. Deletion and mutation analyses reveal distinct sequences in Gip1 that are required for different localizations and for association with Glc7. Binding to Glc7 is also required for proper localization. Strikingly, localization to the prospore membrane, but not association with septins, is important for Gip1 function. Further, our genetic analysis suggests that a Gip1-Glc7 phosphatase complex regulates prospore membrane extension in parallel to the previously reported Vps13, Spo71, Spo73 pathway.

DOI： 10.1091/mbc.E17-08-0521

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

The yeast spore wall is an excellent model to study the assembly of an extracellular macromolecule structure. In the present study, mutants defective in -1,6-glucan synthesis, including kre1, kre6, kre9 and big1, were sporulated to analyse the effect of -1,6-glucan defects on the spore wall. Except for kre6, these mutant spores were sensitive to treatment with ether, suggesting that the mutations perturb the integrity of the spore wall. Morphologically, the mutant spores were indistinguishable from wild-type spores. They lacked significant sporulation defects partly because the chitosan layer, which covers the glucan layer, compensated for the damage. The proof for this model was obtained from the effect of the additional deletion of CHS3 that resulted in the absence of the chitosan layer. Among the double mutants, the most severe spore wall deficiency was observed in big1 spores. The majority of the big1chs3 mutants failed to form visible spores at a higher temperature. Given that the big1 mutation caused a failure to attach a GPI-anchored reporter, Cwp2-GFP, to the spore wall, -1,6-glucan is involved in tethering of GPI-anchored proteins in the spore wall as well as in the vegetative cell wall. Thus, -1,6-glucan is required for proper organization of the spore wall. Copyright (c) 2017 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.3244

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

All asparagine-linked glycans (N-glycans) on the eukaryotic glycoproteins are primarily derived from dolichol-linked oligosaccharides (DLO), synthesized on the rough endoplasmic reticulum membrane. We have previously reported cloning and identification of the human gene, HMT-1, which encodes chitobiosyldiphosphodolichol beta-mannosyltransferase (beta 1,4-MT) involved in the early assembly of DLO. Considering that N-glycosylation is one of the most ubiquitous post-translational modifications for many eukaryotic proteins, the HMT-1 could be postulated as one of the housekeeping genes, but its transcriptional regulation remains to be investigated. Here we screened a 1 kb region upstream from HMT-1 open reading frame (ORF) for transcriptionally regulatory sequences by using chloramphenicol acetyl transferase (CAT) assay, and found that the region from -33 to -1 positions might act in HMT-1 transcription at basal level and that the region from -200 to -42 should regulate its transcription either positively or negatively. In addition, results with CAT assays suggested the possibility that two GATA-1 motifs and an Sp1 motif within a 200 bp region upstream from HMT-1 ORF might significantly upregulate HMT-1 transcription. On the contrary, the observations obtained from site-directed mutational analyses revealed that an NF-1/AP-2 overlapping motif located at -148 to -134 positions should serve as a strong silencer. The control of the HMT-1 transcription by these motifs resided within the 200 bp region could partially explain the variation of expression level among various human tissues, suggesting availability and importance of this region for regulatory role in HMT-1 expression.

DOI： 10.1007/s10616-015-9929-y

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Sporulation of Saccharomyces cerevisiae is a developmental process in which an ascus containing four haploid spores forms from a diploid cell. During this process, newly formed membrane structures called prospore membranes extend along the nuclear envelope and engulf and package daughter nuclei along with cytosol and organelles to form precursors of spores. Proteins involved in prospore membrane extension, Vps13 and Spo71, have recently been reported; however, the overall mechanism of membrane extension remains unclear. Here, we identified Spo73 as an additional factor involved in prospore membrane extension. Analysis of a spo73 Delta mutant revealed that it shows defects similar to those of a spo71 Delta mutant during prospore membrane formation. Spo73 localizes to the prospore membrane, and this localization is independent of Spo71 and Vps13. In contrast, a Spo73 protein carrying mutations in a surface basic patch mislocalizes to the cytoplasm and overexpression of Spo71 can partially rescue localization to the prospore membrane. Similar to spo71 Delta mutants, spo73 Delta mutants display genetic interactions with the mutations in the SMA2 and SPO1 genes involved in prospore membrane bending. Further, our bioinformatic analysis revealed that Spo73 is a dysferlin domain-only protein. Thus, these results suggest that a dysferlin domain-only protein, Spo73, functions with a dual pleckstrin homology domain protein, Spo71, in prospore membrane extension. Analysis of Spo73 will provide insights into the conserved function of dysferlin domains, which is related to dysferlinopathy.
IMPORTANCE Prospore membrane formation consists of de novo double-membrane formation, which occurs during the developmental process of sporulation in Saccharomyces cerevisiae. Membranes are formed into their proper size and shape, and thus, prospore membrane formation has been studied as a general model of membrane formation. We identified SPO73, previously shown to be required for spore wall formation, as an additional gene involved in prospore membrane extension. Genetic and cell biological analyses suggested that Spo73 functions on the prospore membrane with other factors in prospore membrane extension, counteracting the bending force of the prospore membrane. Spo73 is the first dysferlin domain-only protein ever analyzed. The dysferlin domain is conserved from yeast to mammals and is found in dysferlin proteins, which are involved in dysferlinopathy, although the precise function of the domain is unknown. Continued analysis of Spo73 will contribute to our understanding of the function of dysferlin domains and dysferlinopathy.

DOI： 10.1128/mSphere.00038-15

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae, with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc/LDH strains are expected to be of use for versatile analyses of human LDH.

DOI： 10.1093/jb/mvv061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Here, we report a novel method to produce microencapsulated enzymes using Saccharomyces cerevisiae spores. In sporulating cells, soluble secreted proteins are transported to the spore wall. Previous work has shown that the spore wall is capable of retaining soluble proteins because its outer layers work as a diffusion barrier. Accordingly, a red fluorescent protein (RFP) fusion of the alpha-galactosidase, Mel1, expressed in spores was observed in the spore wall even after spores were subjected to a high-salt wash in the presence of detergent. In vegetative cells, however, the cell wall cannot retain the RFP fusion. Although the spore wall prevents diffusion of proteins, it is likely that smaller molecules, such as sugars, pass through it. In fact, spores can contain much higher alpha-galactosidase activity to digest melibiose than vegetative cells. When present in the spore wall, the enzyme acquires resistance to environmental stresses including enzymatic digestion and high temperatures. The outer layers of the spore wall are required to retain enzymes but also decrease accessibility of the substrates. However, mutants with mild spore wall defects can retain and stabilize the enzyme while still permitting access to the substrate. In addition to Mel1, we also show that spores can retain the invertase. Interestingly the encapsulated invertase has significantly lower activity toward raffinose than toward sucrose. This suggests that substrate selectivity could be altered by the encapsulation.

DOI： 10.1128/AEM.00153-14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

In this study, we present a nonhazardous biological method of producing chitosan beads using the budding yeast Saccharomyces cerevisiae. Yeast cells cultured under conditions of nutritional starvation cease vegetative growth and instead form spores. The spore wall has a multilaminar structure with the chitosan layer as the second outermost layer. Thus, removal of the outermost dityrosine layer by disruption of the DIT1 gene, which is required for dityrosine synthesis, leads to exposure of the chitosan layer at the spore surface. In this way, spores can be made to resemble chitosan beads. Chitosan has adsorptive features and can be used to remove heavy metals and negatively charged molecules from solution. Consistent with this practical application, we find that spores are capable of adsorbing heavy metals such as Cu2+, Cr3+, and Cd2+, and removal of the dityrosine layer further improves the adsorption. Removal of the chitosan layer decreases the adsorption, indicating that chitosan works as an adsorbent in the spores. Besides heavy metals, spores can also adsorb a negatively charged cholesterol derivative, taurocholic acid. Furthermore, chitosan is amenable to chemical modifications, and, consistent with this property, dit1 Delta spores can serve as a carrier for immobilization of enzymes. Given that yeast spores are a natural product, our results demonstrate that they, and especially dit1 Delta mutants, can be used as chitosan beads and used for multiple purposes.

DOI： 10.1128/AEM.00677-14

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

The effect of natural IgG antibody recognizing beta-galactosyl epitope on hepatoma cell invasion was investigated. Anti-beta-galactosyl antibody dose-dependently suppressed hepatoma invasion underneath primarily cultured mesothelial cells monolayer without affecting the proliferation, to the same extent as natural IgG antibody with anti-alpha-galactosyl specificity, which had already been reported to have an anti-metastatic activity. The inhibitory effect of anti-beta-galactosyl antibody was completely canceled by adding lactose (galactose-beta-1, 4-glucose) to the medium, indicating that this antibody recognized some antigens with beta-galactosyl epitope. Hepatoma cells pretreated with this antibody for 48 h showed reduced invasive activity, while the pretreatment of mesothelial cells with the antibody did not affect hepatoma cells invasion. Anti-beta-galactosyl antibody also suppressed hepatoma cells adhesion to mesothelial cells monolayer. These results suggest that natural antibody with anti-beta-galactosyl specificity may recognize the beta-galactosyl epitope in some adhesion-related molecules on hepatoma cells, thus suppressing adhesion and invasion to mesothelial cells monolayer. These results suggest possible therapeutic uses of this antibody in the treatment of metastatic tumors.

DOI： 10.1007/s10616-012-9523-5

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The creation of haploid gametes in yeast, termed spores, requires the de novo formation of membranes within the cytoplasm. These membranes, called prospore membranes, enclose the daughter nuclei generated by meiosis. Proper growth and closure of prospore membranes require the highly conserved Vps13 protein. Mutation of SPO71, a meiosis-specific gene first identified as defective in spore formation, was found to display defects in membrane morphogenesis very similar to those seen in vps13Δ cells. Specifically, prospore membranes are smaller than in the wild type, they fail to close, and membrane vesicles are present within the prospore membrane lumen. As in vps13Δ cells, the levels of phophatidylinositol-4-phosphate are reduced in the prospore membranes of spo71 cells. SPO71Δ is required for the translocation of Vps13 from the endosome to the prospore membrane, and ectopic expression of SPO71 in vegetative cells results in mislocalization of Vps13. Finally, the two proteins can be coprecipitated from sporulating cells. We propose that Spo71 is a sporulation-specific partner for Vps13 and that they act in concert to regulate prospore membrane morphogenesis. © 2013, American Society for Microbiology. All Rights Reserved.

DOI： 10.1128/EC.00239-13

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS INC  

Protein N-glycosylation begins with the assembly of a lipid-linked oligosaccharide (LLO) on the endoplasmic reticulum (ER) membrane. The first two steps of LLO biosynthesis are catalyzed by a functional multienzyme complex comprised of the Alg7 GlcNAc phosphotransferase and the heterodimeric Alg13/Alg14 UDP-GlcNAc transferase on the cytosolic face of the ER. In the Alg13/14 glycosyltransferase, Alg14 recruits cytosolic Alg13 to the ER membrane through interaction between their C-termini. Bioinformatic analysis revealed that eukaryotic Alg14 contains an evolved N-terminal region that is missing in bacterial orthologs. Here, we show that this N-terminal region of Saccharomyces cerevisiae Alg14 localize its green fluorescent protein fusion to the ER membrane. Deletion of this region causes defective growth at 38.5 degrees C that can be partially complemented by overexpression of Alg7. Coimmunoprecipitation demonstrated that the N-terminal region of Alg14 is required for direct interaction with Alg7. Our data also show that Alg14 lacking the N-terminal region remains on the ER membrane through a nonperipheral association, suggesting the existence of another membrane-binding site. Mutational studies guided by the 3D structure of Alg14 identified a conserved alpha-helix involved in the second membrane association site that contributes to an integral interaction and protein stability. We propose a model in which the N- and C-termini of Alg14 coordinate recruitment of catalytic Alg7 and Alg13 to the ER membrane for initiating LLO biosynthesis.

DOI： 10.1093/glycob/cwr162

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Ribosome biogenesis starts with transcription of the large ribosomal RNA precursor (47S pre-rRNA), which soon combines with numerous factors to form the 90S preribosome in the nucleolus. Although the subsequent separation of the pre-90S particle into pre-40S and pre-60S particles is critical for the production process of mature small and large ribosomal subunits, its molecular mechanisms remain undetermined. Here, we present evidence that p32, fibrillarin (FBL), and Nop52 play key roles in this separation step. Mass-based analyses combined with immunoblotting showed that p32 associated with 155 proteins including 31 rRNA-processing factors (of which nine were components of small subunit processome, and six were those of RIX1 complex), 13 chromatin remodeling components, and six general transcription factors required for RNA polymerase III-mediated transcription. Of these, a late rRNA-processing factor Nop52 interacted directly with p32. Immunocytochemical analyses demonstrated that p32 colocalized with an early rRNA-processing factor FBL or Nop52 in the nucleolus and Cajal bodies, but was excluded from the nucleolus after actinomycin D treatment. p32 was present in the pre-ribosomal fractions prepared by cell fractionation or separated by ultracentrifugation of the nuclear extract. p32 also associated with pre-rRNAs including 47S/45S and 32S pre-rRNAs. Furthermore, knockdown of p32 with a small interfering RNA slowed the early processing from 47S/45S pre-rRNAs to 18S rRNA and 32S pre-rRNA. Finally, Nop52 was found to compete with FBL for binding to p32 probably in the nucleolus. Given the fact that FBL and Nop52 are associated with pre-ribosome particles distinctly different from each other, we suggest that p32 is a new rRNA maturation factor involved in the remodeling from pre-90S particles to pre-40S and pre-60S particles that requires the exchange of FBL for Nop52. Molecular & Cellular Proteomics 10: 10.1074/mcp.M110.006148, 1-23, 2011.

DOI： 10.1074/mcp.M110.006148

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DOI： 10.1128/MCB.01529-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Sporulation of Saccharomyces cerevisiae is a developmental process in which four haploid spores are generated inside a diploid cell. Gip1, a sporulation-specific targeting subunit of protein phosphatase type 1, together with its catalytic subunit, Glc7, colocalizes with septins along the extending prospore membrane and is required for septin organization and spore wall formation. However, the mechanism by which Gip1-Glc7 phosphatase promotes these events is unclear. We show here that Ysw1, a sporulation-specific coiled-coil protein, has a functional relationship to Gip1-Glc7 phosphatase. Overexpression of YSW1 partially suppresses the sporulation defect of a temperature-sensitive allele of gip1. Ysw1 interacts with Gip1 in a two-hybrid assay, and this interaction is required for suppression. Ysw1 tagged with green fluorescent protein colocalizes with septins and Gip1 along the extending prospore membrane during spore formation. Sporulation is partially defective in ysw1 Delta mutant, and cytological analysis revealed that septin structures are perturbed and prospore membrane extension is aberrant in ysw1 Delta cells. These results suggest that Ysw1 functions with the Gip1-Glc7 phosphatase to promote proper septin organization and prospore membrane formation.

DOI： 10.1128/EC.00095-09

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC CELL BIOLOGY  

Ascospore formation in yeast is accomplished through a cell division in which daughter nuclei are engulfed by newly formed plasma membranes, termed prospore membranes. Closure of the prospore membrane must be coordinated with the end of meiosis II to ensure proper cell division. AMA1 encodes a meiosis-specific activator of the anaphase promoting complex (APC). The activity of APC(Ama1) is inhibited before meiosis II, but the substrates specifically targeted for degradation by Ama1 at the end of meiosis are unknown. We show here that ama1 Delta mutants are defective in prospore membrane closure. Ssp1, a protein found at the leading edge of the prospore membrane, is stabilized in ama1 Delta mutants. Inactivation of a conditional form of Ssp1 can partially rescue the sporulation defect of the ama1 Delta mutant, indicating that an essential function of Ama1 is to lead to the removal of Ssp1. Depletion of Cdc15 causes a defect in meiotic exit. We find that prospore membrane closure is also defective in Cdc15 and that this defect can be overcome by expression of a form of Ama1 in which multiple consensus cyclin-dependent kinase phosphorylation sites have been mutated. These results demonstrate that APC(Ama1) functions to coordinate the exit from meiosis II with cytokinesis.

DOI： 10.1091/mbc.E08-06-0615

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED CENTRAL LTD  

Background: In Saccharomyces cerevisiae, nutrient limitation stimulates diploid cells to undergo DNA replication and meiosis, followed by the formation of four haploid spores. Septins are a family of proteins that assemble a ring structure at the mother-daughter neck during vegetative growth, where they control cytokinesis. In sporulating cells, the septin ring disassembles and septins relocalize to the prospore membrane.
Results: Here, we demonstrate that nutrient limitation triggers a change in the localization of at least two vegetative septins (Cdc10 and Cdc11) from the bud neck to the microtubules. The association of Cdc10 and Cdc11 with microtubules persists into meiosis, and they are found associated with the meiotic spindle until the end of meiosis II. In addition, the meiosis-specific septin Spr28 displays similar behavior, suggesting that this is a common feature of septins. Septin association to microtubules is a consequence of the nutrient limitation signal, since it is also observed when haploid cells are incubated in sporulation medium and when haploid or diploid cells are grown in medium containing non-fermentable carbon sources. Moreover, during meiosis II, when the nascent prospore membrane is formed, septins moved from the microtubules to this membrane. Proper organization of the septins on the membrane requires the sporulation-specific septins Spr3 and Spr28.
Conclusion: Nutrient limitation in S. cerevisiae triggers the sporulation process, but it also induces the disassembly of the septin bud neck ring and relocalization of the septin subunits to the nucleus. Septins remain associated with microtubules during the meiotic divisions and later, during spore morphogenesis, they are detected associated to the nascent prospore membranes surrounding each nuclear lobe. Septin association to microtubules also occurs during growth in non-fermentable carbon sources.

DOI： 10.1186/1471-2121-9-55

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Although RecQ5 beta is a ssDNA (single-stranded DNA)-stimulated ATPase and an ATP-dependent DNA helicase with strand-annealing activities, its cellular function remains to be explored. In the present paper, we used immunopurification and MS-based analyses to show that human DNA helicase RecQ5 beta is associated with at least four RNAP II (RNA polymerase II) subunits. RecQ5 beta was also present in complexes immunoprecipitated using three different antibodies against the large subunit of RNAP II, or in complexes immunoprecipitated using an anti-FLAG antibody against either FLAG-RNAP II 33 kDa subunit or FLAG-Pin 1. Different regions of the non-helicase domain of the RecQ5 beta molecule were associated with hypophosphorylated and hyperphosphorylated forms of the RNAP II large subunit independently of DNA and RNA. RecQ5 beta was also found in nuclear chromatin fractions and associated with the coding regions of the LDL (low-density lipoprotem) receptor and beta-actin genes. Knockdown of the RecQ5 beta transcript increased the transcription of those genes. The results of the present study suggest that RecQ5 beta has suppressive roles in events associated with RNAP II-dependent transcription.

DOI： 10.1042/BJ20071392

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Sporulation of the fission yeast Schizosaccharomyces pombe is a developmental process that generates gametes and that includes the formation of spore envelope precursors called the forespore membranes. Assembly and development of forespore membranes require vesicular trafficking from other intracellular membrane compartments. We have shown that phosphatidylinositol 3-kinase (PtdIns 3-kinase) is required for efficient and proper development of forespore membranes. The role of a FYVE domain protein, Sst4p, a homolog of Vps27p/Hrs, as a downstream factor for PtdIns 3-kinase in sporulation was investigated. sst4 Delta asci formed spores with oval-shaped morphology and with reduced viability compared to that of the wild-type spores. The extension of forespore membranes was inefficient, and bubble-like structures emerged from the leading edges of the forespore membranes. Sst4p localization was examined using fluorescent protein fusions and was found to be adjacent to the forespore membranes during sporulation. The localization and function of Sst4p were dependent on its FYVE domain and on PtdIns 3-kinase. Sst4p colocalized and interacted with Hse1p, a homolog of Saccharomyces cerevisiae Hse1p and of mammalian STAM. Mutations in all three UIM domains of the Sst4p/Hse1p complex resulted in formation of spores with abnormal morphology. These results suggest that Sst4p is a downstream factor of PtdIns 3-kinase and functions in forespore membrane formation.

DOI： 10.1128/EC.00211-07

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Pleckstrin-2 (PLEK2) has been implicated to be regulated by phosphatidylinositol (PI) 3-kinase, while pleckstrin1 (PLEK1) has been suggested to be a major PKC substrate in platelets. In this paper, we confirmed that PLEK2 specifically bound to the PI 3-kinase products in vitro and explored its behavior. PLEK2 was found to be expressed in various adherent cell lines, while PLEK1 expression was restricted to non-adherent cells in the protein level. Expression of PLEK2 in COS1 cells induced formation of protrusive F-actin structure and enhanced the actin rearrangements induced on collagen- or fibronectin-coated plates. A PLEK2 mutant incapable of binding to the PI 3-kinase products did not show any effect on actin rearrangement. Knockdown of PLEK2 by shRNA inhibited spreading of HCC2998 adenocarcinoma cells. PLEK2 colocalized with Rac and was suggested to be oligomerized. These results suggest that PLEK2 is involved in actin rearrangement in a PI 3-kinase dependent manner. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.06.132

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P-3) binding protein, which acts in F-actin rearrangement. The role of SWAP-70 in oncogenic transformation of mouse embryo fibroblasts (MEFs) by v-Src was examined by use of MEFs defective in SWAP-70. v-Src morphologically transformed MEFs lacking SWAP-70, but growth of the transformed cells in culture was slower than that of cells supplemented with exogenous SWAP-70. The v-Src-transformed MEFs deficient in SWAP-70 were unable to grow in soft agar while those expressing SWAP70 readily formed colonies, suggesting that SWAP-70 is required for anchorage independent growth of v-Src transformed MEFs. When transplanted in nude mice, tumors formed by the v-Src transformed SWAP-70(-/-) MEFs were smaller than those formed by cells expressing exogenous SWAP-70. These results suggest that SWAP-70 may be required for oncogenic transformation and contributes to cell growth in MEFs transformed by v-Src. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.03.011

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Signet-ring cell carcinoma is one of the most malignant tumors, classified histologically as a poorly differentiated adenocarcinoma. The ErbB2/ErbB3 complex is often constitutively activated, which suggests that the ErbB2/ErbB3 signaling pathway may be important for malignancy of this tumor. However, the mechanism underlying this activation has not been understood. Here, we show that ErbB2 and Muc4 bind in signet ring carcinoma cells, which was not seen in highly differentiated adenocarcinorna cell lines. ErbB3 was suggested to be a substrate of ErbB2 because knockdown of ErbB2 resulted in less phosphorylation of ErbB3. Inhibition of expression of Muc4 at the cell surface by the treatment of the cells with benzyl-GaINac, an inhibitor of mucin secretion, blocked phosphorylation of ErbB3, suggesting that activity of Erb132 depends on the expression of Muc4. These results supply the biochemical backgrounds in recent studies suggesting the contribution of Muc4 in the tumorigenesis. (c) 2007 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.bbrc.2007.01.133

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

SWAP-70 translocates to the plasma membrane in a phosphoinositide 3-kinase (PI 3-kinase)-dependent manner and contributes to membrane ruffling. It binds to phosphatidylinositol trisphosphate (PtdIns(3,4,5)P-3) through its PH domain, which is essential for the membrane translocation after EGF stimulation. We examined the behavior of the SWAP-70s which have mutations in the beta 3/beta 4 loop of the PH domain. The two mutants fused to green fluorescent protein (GFP) carrying the mutations failed to translocate to the plasma membrane. The sole PH domains carrying the same mutations behaved similarly. The PtdIns(3,4,5)P-3 binding activity of two mutants was comparable to that of the wild-type protein. These results suggest that translocation of SWAP-70 largely depends on the activity of the PH domain, and that not only PtdIns(3,4,5)P-3 binding activity, but also some additional activity of the PH domain is required for the translocation.

DOI： 10.1080/15216540701222880

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

TRIM11 is a member of the tripartite-motif-containing protein family and is known to destabilize humanin, an inhibitor of Alzheimer-like neuronal insults. In this study, we demonstrate that TRIM11 interacts with activator-recruited cofactor 105-kDa component (ARC105) that mediates chromatin-directed transcription activation and is a key regulatory factor for transforming growth factor P (TGFO) signaling. Coexpression of TRIM11 increased ARC105 degradation but a proteasome inhibitor suppressed this. Co-expression of TRIM11 and ARC105 also increased ubiquitination of ARC105. In addition, TRIM11 suppressed ARC105-mediated transcriptional activation induced with TGFO in a reporter assay. These results suggest that TRIM11, with the ubiquitin-proteasome pathway, regulates ARC105 function in TGF beta signaling. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

DOI： 10.1016/j.febslet.2006.07.066

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

N-linked glycosylation requires the synthesis of an evolutionarily conserved lipid-linked oligosaccharide (LLO) precursor that is essential for glycoprotein folding and stability. Despite intense research, several of the enzymes required for LLO synthesis have not yet been identified. Here we show that two poorly characterized yeast proteins known to be required for the synthesis of the LLO precursor, GlcNAc(2)-PP-dolichol, interact to form an unusual hetero-oligomeric UDP-GlcNAc transferase. Alg13 contains a predicted catalytic domain, but lacks any membrane-spanning domains. Alg14 spans the membrane but lacks any sequences predicted to play a direct role in sugar catalysis. We show that Alg14 functions as a membrane anchor that recruits Alg13 to the cytosolic face of the ER, where catalysis of GlcNAc(2)-PP-dol occurs. Alg13 and Alg14 physically interact and under normal conditions, are associated with the ER membrane. Overexpression of Alg13 leads to its cytosolic partitioning, as does reduction of Alg14 levels. Concomitant Alg14 overproduction suppresses this cytosolic partitioning of Alg13, demonstrating that Alg14 is both necessary and sufficient for the ER localization of Alg13. Further evidence for the functional relevance of this interaction comes from our demonstration that the human ALG13 and ALG14 orthologues fail to pair with their yeast partners, but when co-expressed in yeast can functionally complement the loss of either ALG13 or ALG14. These results demonstrate that this novel UDP-GlcNAc transferase is a unique eukaryotic ER glycosyltransferase that is comprised of at least two functional polypeptides, one that functions in catalysis and the other as a membrane anchor.

DOI： 10.1074/jbc.M507569200

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BLACKWELL PUBLISHING ASIA  

Calcineurin, a calcium/calmodulin-dependent serine/threonine protein phosphatase, is a key constituent of signaling pathways involved in antigen-dependent T-cell activation and development of the mammalian heart. In addition, calcineurin constitutes a part of the Wnt/calcium-signaling pathway that regulates early stages of dorsoventral axis formation in Xenopus embryos. Although some of the Wnt family members are involved in organ formation at relatively late stages of Xenopus development, the involvement of calcineurin in the development of those organs remains unclear. In the present study, we demonstrate that calcineurin inhibitors (cyclosporine A, FK506, and FK520), but not non-calcineurin inhibitors (rapamycin and GPI1046) that bind the same intracellular receptor as that for FK506, induce edema and gut coiling disruption and exhibit teratogenesis in the kidney, heart, gut, liver, and somitic tissue during Xenopus development. The same effects were observed by injecting the calcineurin inhibitors into the dorsal side, but not ventral side, of blastomeres at the 4-cell stage, although the inhibitors did not affect dorsoventral axis formation. These results suggest that calcineurin is involved in dorsal-side signaling that leads to the formation of the heart, kidney, liver, gut and somitic tissue during Xenopus embryogenesis.

DOI： 10.1111/j.1440-169X.2004.00733.x

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JOHN WILEY & SONS LTD  

NAD(+), an essential molecule involved in a variety of cellular processes, is synthesized through de novo and salvage pathways. NAD(+) synthetase catalyses the final step in both pathways. Here we show that this enzyme is encoded by the QNS1 gene in Saccharomyces cerevisiae. Expression of Escherichia coli or Bacillus subtilis NAD(+) synthetases was able to suppress the lethality of a qns1 deletion, while a B. subtilis NAD(+) synthetase mutant with lowered catalytic activity was not. Overexpression of QNS1 tagged with HA led to elevated levels of NAD(+) synthetase activity in yeast extracts, and this activity can be recovered by immunoprecipitation using anti-HA antibody. An allele of QNS1 was constructed that carries a point mutation predicted to reduce the catalytic activity. Overexpression of this allele, qns1(G521E), failed to elevate NAD(+) synthetase levels and qns1(G521E) could not rescue the lethality caused by the depletion of Qns1p. These results demonstrate that NAD(+) synthetase activity is essential for cell viability. A GFP-tagged version of Qns1p displayed a diffuse localization in both the nucleus and the cytosol. Finally, the rat homologue of QNS1 was cloned and shown to functionally replace yeast QNS1, indicating that NAD(+) synthetase is functionally conserved from bacteria to yeast and mammals. Copyright (C) 2003 John Wiley Sons, Ltd.

DOI： 10.1002/yea.1008

PubMed

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ROCKEFELLER UNIV PRESS  

Sporulation of Saccharomyces cerevisiae is a developmental process in which a single cell is converted into four haploid spores. GIP1, encoding a developmentally regulated protein phosphatase 1 interacting protein, is required for spore formation. Here we show that GIP1 and the protein phosphatase 1 encoded by GLC7 play essential roles in spore development. The gip1 Delta mutant undergoes meiosis and prospore membrane formation normally, but is specifically defective in spore wall synthesis. We demonstrate that in wild-type cells, distinct layers of the spore wall are deposited in a specific temporal order, and that gip1 Delta cells display a discrete arrest at the onset of spore wall deposition. Localization studies revealed that Gip1p and Glc7p colocalize with the septins in structures underlying the growing prospore membranes. Interestingly, in the gip1 Delta mutant, not only is Glc7p localization altered, but septins are also delocalized. Similar phenotypes were observed in a glc7-136 mutant, which expresses a Glc7p defective in interacting with Gip1p. These results indicate that a Gip1p-Glc7p phosphatase complex is required for proper septin organization and initiation of spore wall formation during sporulation.

DOI： 10.1083/jcb.200107008

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

The Saccharomyces cerevisiae HUT1 gene (scHUT1) and the Schizosaccharomyces pombe hut1(+) gene (sphut1(+)) encode hydrophobic proteins with approximately 30% identity to a human UDP-galactose transporter-related gene (UGTrel1) product. These proteins show a significant similarity to the nucleotide sugar transporter and are conserved in many eukaryotic species, but their physiological functions are not known. Both scHUT1 and sphut1(+) genes are non-essential for cell growth under normal conditions, and their disruptants show no defects in the modification of O- and N-linked oligosaccharides, but are sensitive to a membrane-permeable reducing agent, dithiothreitol (DTT). Consistent with this phenotype, scHUT1 has genetic interaction with ERO1, which plays an essential role in the oxidation of secretory proteins at the endoplasmic reticulum (ER). Overexpression of the MPD1 or MPD2 genes, which were isolated as multicopy suppressors of protein disulphide isomerase (PDI) depletion, could not replace the essential function of PDI in Delta hut1 S. cerevisiae cells. Our results indicate that scHut1p and spHut1p are functional homologues, and their physiological function is to maintain the optimal environment for the folding of secretory pathway proteins in the ER. Copyright (C) 2001 John Wiley & Sons, Ltd.

DOI： 10.1002/yea.707

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：SPRINGER  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC  

A novel ficolin-related gene was isolated from the mouse liver lambda ZAPII cDNA library. The protein encoded by this gene consists of both collagen-and fibrinogen-like domains, which are common features of the ficolin family, and was named mouse ficolin-A. The amino acid sequence of mouse ficolin-A is 60.2, 59.8, 59.8, and 59.6% identical to those of porcine ficolin-alpha, -beta, human ficolin-1, and EBP-37/P35, respectively. Northern blot analysis showed that mRNA of mouse ficolin-A is highly expressed in liver and spleen. Immunoblot analysis using an anti-mouse ficolin-A antiserum showed that mouse ficolin-A is a plasma protein with binding activity to elastin and GlcNAc. (C) 1998 Academic Press.

DOI： 10.1006/bbrc.1998.8344

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC JNL-COMP SUBSCRIPTIONS  

The third multicopy suppressor gene of the PDI1 deletion from Saccharomyces cerevisiae, MPD2, was isolated and characterized, The MPD2 gene encodes a protein with a putative signal sequence, ER retention signal, and a disulfide isomerase active site Like sequence, The amino acid sequence around the active site like sequence is similar to the thioredoxin-like domains of PDI and PDI related proteins, although the similarity is comparatively low. A Delta-pdi1 strain overproducing Mpd2p showed slow growth and was sensitive to 1 mM dithiothreitol. Mpd2p can be detected in wild type cells and is a glycoprotein. Although the MPD2 gene was not essential for growth, overexpression of the gene partially restored the maturation defect of carboxypeptidase Y caused by the PDI1 deletion. Mutagenesis analysis revealed that Mpd2p can compensate for the loss of PDI with its CXXC sequence. (C) 1997 Academic Press.

DOI： 10.1006/bbrc.1997.7426

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED RES FOUND  

We compared biochemically and immunohistochemically rkCBP17.5, a rat kidney beta-galactoside-binding protein (Mr 17,500), with galectin-5, a beta-galactoside-binding protein (Mr 18,000), initially isolated from the rat lung. The result suggests that rkCBP17.5 is highly similar to galectin-5.

DOI： 10.2220/biomedres.17.491

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

A novel elastin-binding protein, EBP-37, was recently identified and purified from human plasma, Its partial amino acid sequences showed significant homology to porcine ficolins, which were originally purified from porcine uterus membranes as multimeric proteins with fibrinogen- and collagen-like domains. Here we report the presence of ficolins in an elastin-binding fraction of porcine plasma and the direct binding of recombinant porcine filcolin-alpha to elastin, In addition, a cDNA encoding a human counterpart of porcine ficolins that is composed of 319 amino acids and is different from EBP-37 was cloned and named human ficolin-1. Northern blotting of various human tissues revealed that human ficolin-1 mRNA is highly expressed in peripheral blood leukocytes. These data suggested that there are at least two kinds of ficolin-related proteins in both pig and human, and they may function as plasma proteins with elastin-binding activities.

DOI： 10.1093/oxfordjournals.jbchem.a021474

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Oxford University Press  

Using affinity chromatography on lactosyl-Sepharose, a β-galactoside-binding protein of 38 kDa was detected in mouse L1210 lymphocytic leukemia cells. Immunoblotting analysis revealed that it is distinct from any known larger molecular weight galectin, The partial amino acid sequences of the 38 kDa protein indicated that it is a novel member of the galectin family. This 38 kDa lectin is expressed in lymphocytic cell lines but not macrophage-like cell lines.

DOI： 10.1093/oxfordjournals.jbchem.a021436

Scopus

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

MPD1, a yeast gene the overexpression of which suppresses the inviability caused by the loss of protein disulfide isomerase (PDI) was isolated and characterized. The MPD1 gene product retained a single disulfide isomerase active site sequence (APWCGHCK), an N-terminal putative signal sequence, and a C-terminal endoplasmic reticulum (ER) retention signal, and was a novel member of the PDI family, The gene product, identified in yeast extract, contained core size carbohydrates. MPD1 was not essential for growth, but overexpression of the gene suppressed the maturation defect of carboxypeptidase Y caused by PDI1 deletion, indicative of the related function to PDI in the yeast ER.

DOI： 10.1016/0014-5793(95)00750-4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

In order to study the elastin-binding factors in blood, human plasma was applied to an alpha-elastin-Sepharose column. The column-binding fraction contained a 37-kDa protein, which was tentatively named EBP-37. Partial amino acid sequences of EBP-37 were determined. It had collagenous and non-collagenous domains, Homology searches of the sequences revealed that the protein is very similar but not identical to ficolins, transforming growth factor-beta 1 (TGF-beta 1)-binding proteins from porcine uterus membranes, Direct interaction of EBP-37 with elastin was confirmed by demonstrating the binding of the isolated EBP-37 to alpha-elastin on a nitrocellulose membrane using the EBP-37-specific antiserum, The existence of oligomers and multimers crosslinked by disulfide bonds was demonstrated by immunoblot analysis, Possible functions of EBP-37 are discussed.

DOI： 10.1093/oxfordjournals.jbchem.a124802

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：BIOMED RES FOUND  

It was demonstrated that the IgG with anti-alpha-galactosyl specificity (anti-alpha-Gal IgG) occurs in normal human serum. Subsequently, we isolated the IgG with anti-beta-galactosyl specificity (anti-beta-Gal IgG) from normal human serum by affinity chromatography using lactose-Sepharose as affinity adsorbent (15). In this study, the carbohydrate specificity of anti-beta-Gal IgG was further examined. Purified human anti-beta-Gal IgG showed distinct specificity with the structure of Gal beta 1-4Glc in oligosaccharide inhibition experiments. However, it did not react with Gal beta 1-4Glc beta 1-1'Cer nor NeuAc alpha 2-3Gal beta 1-4Glc beta 1-1'Cer(GM3) in thin-layer chromatography (TLC)*, suggesting that besides Gal beta 1-4Glc, more inner structure may be important. The ELISA* for anti-beta-Gal IgG and anti-alpha-Gal IgG using synthetic lactose or melibiose-BSA* conjugates was developed, and the levels of these antibodies in normal and patient sera were measured. Their concentrations were found to vary in the range of 10-280 mu g/ml and 10-220 mu g/ml, respectively, in normal subjects. Distinctly higher levels over these ranges were found in two terminal patients with malignant tumor. Anti-beta-Gal antibodies were also found in the sera from various non-primates. Purified calf anti-beta-Gal antibodies showed the same specificity as that of humans.

DOI： 10.2220/biomedres.15.17

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN BIOCHEMICAL SOC  

A genomic DNA clone for protein disulfide isomerase (PDI) of Saccharomyces cerevisiae was isolated by hybridization with synthesized oligonucleotide probes based on a partial amino acid sequence of yeast PDI. The introduction of a multiple copy plasmid carrying this fragment into yeast caused a tenfold increase in PDI specific activity and in the amount of PDI antigen in the extract. The gene on this fragment was named PDI1. The nucleotide sequence of the gene predicts a polypeptide of 522 amino acids with about 30% identity to mammalian PDIs. The predicted amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum retention signal of yeast (HDEL), and two putative active site sequences of PDI (WCGHCK). The predicted polypeptide is acidic and contains five putative glycosylation sites, consistent with the molecular properties of the purified yeast PDI [T. Mizunaga et al. (1990) J. Biochem. 108, 846-851]. The PDI1 gene was mapped on chromosome III. A gene disruption experiment revealed that the PDI1 gene is essential for cell growth.

DOI： 10.1093/oxfordjournals.jbchem.a123576

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATL ACAD SCIENCES  

Glycosylation site binding protein (GSBP) has been shown to be identical to protein disulfide isomerase (PDI; EC 5.3.4.1) in a variety of multicellular organisms. We have utilized immunological and biochemical techniques to determine if GSBP and PDI are identical in yeast. Antiserum prepared against yeast GSBP identified in microsomes by its ability to be labeled with a peptide photoaffinity probe was found to recognize PDI purified from yeast. Moreover, this purified yeast PDI was found to be specifically labeled by the photoaffinity probe originally used to identify GSBP in a variety of eukaryotes. On the basis of these observations, we conclude that yeast GSBP and PDI are the same protein. The structure of the yeast PDI gene revealed a product with sequence similarity to higher eukaryotic PDI/GSBP. Disruption of this gene in yeast resulted in a recessive lethal mutation, indicating that PDI/GSBP is required for cell viability.

DOI： 10.1073/pnas.88.10.4453

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：FEDERATION AMER SOC EXP BIOL  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：GUSTAV FISCHER VERLAG  

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