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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mouse lymphoma cell line W7MG1 is stably infected with mouse mammary tumor virus and produces the viral envelope glycoprotein precursor Pr74, but the mature envelope proteins gp52 and gp33, which are derived from Pr74 by posttranslational processing, are produced only when the cells are cultured with a glucocorticoid agonist. The current study demonstrated that even when W7MG1 cells are grown with hormone, the conversion of Pr74 to gp52 and gp33 is an inefficient process in this cell line. At least 2 h of exposure to glucocorticoid were required to induce the appearance of gp52 and gp33; furthermore, Pr74 labeled in the absence of hormone was not converted to gp52 and gp33 upon subsequent addition of hormone. RNA synthesis inhibitors blocked the hormonal induction of gp52 and gp33, indicating that the hormone acts by promoting the expression of a new gene(s) required for the production of gp52 and gp33, rather than by inhibiting the expression of a gene(s) that prevents processing of Pr74. Subcellular fractionation studies demonstrated that Pr74 produced in either the presence or absence of hormone was associated primarily with the ER, whereas gp52 and gp33 were found in the Golgi and plasma membrane fractions. The Pr74 molecules from W7MG1 cells grown either with or without glucocorticoid coimmunoprecipitated with BiP/GRP78 and sedimented as aggregates of heterogeneous size. In contrast, Pr74 from virus-producing GR3A mouse mammary tumor cells, which process Pr74 more efficiently, sedimented as apparent monomers, dimers, and trimers.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Mar
PMID:The effect of glucocorticoid on the subcellular localization, oligomerization, and processing of mouse mammary tumor virus envelope protein precursor Pr74. 131 42

In the mouse mammary tumor virus (MMTV)-infected mouse T-lymphoma cell line W7MG1, glucocorticoid hormone regulates two aspects of MMTV gene expression: hormone stimulates MMTV gene transcription and increases the ratio of mature envelope proteins to envelope precursor protein produced. To separate these two effects and determine the mechanism by which hormone regulates the conversion of the envelope precursor Pr74 to the mature cleaved products gp52 and gp33, we constructed expression vectors in which the envelope gene is constitutively transcribed. Surprisingly, the envelope precursor protein Pr74 encoded by two independently isolated, allelic envelope genes behaved differently. Pr74-P (encoded by the ENV/P gene) was processed efficiently to the mature products gp52 and gp33, independently of the level of expression, hormonal induction of cellular genes, or the presence of other MMTV proteins. In contrast, under the same conditions, Pr74-N (encoded by the ENV/N gene) was not processed further despite being relatively stable. In sucrose gradient analyses, Pr74-P sedimented as monomers, whereas Pr74-N was found in high mol wt aggregates of heterogeneous size. Coimmunoprecipitation analysis determined that Pr74-N associated with BiP, whereas Pr74-P did not. This is indicative of improper folding of Pr74-N in the endoplasmic reticulum.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1992 Mar
PMID:Two different genes coding for processable and nonprocessable forms of a viral envelope protein can account for the apparent hormonal stimulation of protein processing in W7MG1 lymphoma cells. 131 43

Hemin induces nonterminal differentiation of human K562 erythroleukemia cells, which is accompanied by the expression of certain erythroid cell-specific genes, such as the embryonic and fetal globins, and elevated expression of the stress genes hsp70, hsp90, and grp78/BiP. Previous studies revealed that, as during heat shock, transcriptional induction of hsp70 in hemin-treated cells is mediated by activation of heat shock transcription factor (HSF), which binds to the heat shock element (HSE). We report here that hemin activates the DNA-binding activity of HSF2, whereas heat shock induces predominantly the DNA-binding activity of a distinct factor, HSF1. This constitutes the first example of HSF2 activation in vivo. Both hemin and heat shock treatments resulted in equivalent levels of HSF-HSE complexes as analyzed in vitro by gel mobility shift assay, yet transcription of the hsp70 gene was stimulated much less by hemin-induced HSF than by heat shock-induced HSF. Genomic footprinting experiments revealed that hemin-induced HSF and heat shock-induced HSF, HSF2, and HSF1, respectively, occupy the HSE of the human hsp70 promoter in a similar yet not identical manner. We speculate that the difference in occupancy and/or in the transcriptional abilities of HSF1 and HSF2 accounts for the observed differences in the stimulation of hsp70 gene transcription.
Mol Cell Biol 1992 Sep
PMID:Activation of heat shock factor 2 during hemin-induced differentiation of human erythroleukemia cells. 150 7

The mouse Wnt-1 gene, a target for insertional activation in mouse mammary tumor virus-induced mammary tumors, encodes poorly secreted, cysteine-rich glycoproteins required for proper central nervous system development. We have been analyzing the biosynthesis of Wnt-1 proteins in several cell lines that express Wnt-1 cDNA from heterologous promoters. A protein of 78 kDa was found to be associated with the intracellular forms of Wnt-1 proteins in mammalian and avian cells by using multiple antisera against Wnt-1 proteins. We have identified p78 as the binding protein BiP with anti-BiP antisera and by its release from Wnt-1 immunoprecipitates upon incubation with MgCl2 and ATP. Experiments with a Wnt-1 mutant that lacks the sequence encoding the signal peptide indicates that Wnt-1 proteins must enter the secretory pathway in order to interact with BiP. We demonstrate that Wnt-1 proteins are associated with BiP in cells in which active Wnt-1 proteins are produced, such as a cultured mammary epithelial cell line and Wnt-1 transgenic mouse mammary tumor cells. The association of Wnt-1 proteins with BiP may be a factor in determining the efficiency of secretion of Wnt-1 gene products.
Mol Cell Biol 1992 Feb
PMID:Interaction of Wnt-1 proteins with the binding protein BiP. 153 Oct 88

The endoplasmic reticulum (ER)-localized chaperone protein, GRP78-BiP, is involved in the folding and oligomerization of secreted and membrane proteins, including the simian virus 5 hemagglutinin-neuraminidase (HN) glycoprotein. To understand this interaction better, we have constructed a series of HN mutants in which specific portions of the extracytoplasmic domain have been deleted. Analysis of these mutant polypeptides expressed in CV-1 cells have indicated that GRP78-BiP binds to selective sequences in HN and that there exists more than a single site of interaction. Mutant polypeptides have been characterized that are competent and incompetent for association with GRP78-BiP. These mutants have been used to show that the induction of GRP78-BiP synthesis due to the presence of nonnative protein molecules in the ER is dependent on GRP78-BiP complex formation with its substrates. These studies have implications for the function of the GRP78-BiP protein and the mechanism by which the gene is regulated.
Mol Biol Cell 1992 Feb
PMID:Analysis in vivo of GRP78-BiP/substrate interactions and their role in induction of the GRP78-BiP gene. 155 Sep 58

SEC63 encodes a protein required for secretory protein translocation into the endoplasmic reticulum (ER) of Saccharomyces cerevisiae (J. A. Rothblatt, R. J. Deshaies, S. L. Sanders, G. Daum, and R. Schekman, J. Cell Biol. 109:2641-2652, 1989). Antibody directed against a recombinant form of the protein detects a 73-kDa polypeptide which, by immunofluorescence microscopy, is localized to the nuclear envelope-ER network. Cell fractionation and protease protection experiments confirm the prediction that Sec63p is an integral membrane protein. A series of SEC63-SUC2 fusion genes was created to assess the topology of Sec63p within the ER membrane. The largest hybrid proteins are unglycosylated, suggesting that the carboxyl terminus of Sec63p faces the cytosol. Invertase fusion to a loop in Sec63p that is flanked by two putative transmembrane domains produces an extensively glycosylated hybrid protein. This loop, which is homologous to the amino terminus of the Escherichia coli heat shock protein, DnaJ, is likely to face the ER lumen. By analogy to the interaction of the DnaJ and Hsp70-like DnaK proteins in E. coli, the DnaJ loop of Sec63p may recruit luminal Hsp70 (BiP/GRP78/Kar2p) to the translocation apparatus. Mutations in two highly conserved positions of the DnaJ loop and short deletions of the carboxyl terminus inactivate Sec63p activity. Sec63p associates with several other proteins, including Sec61p, a 31.5-kDa glycoprotein, and a 23-kDa protein, and together with these proteins may constitute part of the polypeptide translocation apparatus. A nonfunctional DnaJ domain mutant allele does not interfere with the formation of the Sec63p/Sec61p/gp31.5/p23 complex.
Mol Cell Biol 1992 Jul
PMID:Topology and functional domains of Sec63p, an endoplasmic reticulum membrane protein required for secretory protein translocation. 162 Jan 30

A hybrid protein consisting of the Escherichia coli lipoprotein signal sequence attached to the mature sequence of the B subunit of heat-labile enterotoxin (Lipo-EtxB) was expressed in yeast and E. coli. Analyses of cell lysates from Saccharomyces cerevisiae and E. coli expressing the protein revealed that both organisms were able to assemble Lipo-EtxB into oligomers that were (i) stable in the presence of sodium dodecyl sulphate, (ii) resistant to proteinase K degradation, and (iii) able to bind to GM1-ganglioside receptors. Each of these properties are characteristic of the wild-type B subunit pentamer produced in E. coli. Assembly of Lipo-EtxB was found to be unaffected in a sec18 mutant of S. cerevisiae, which possesses a temperature-sensitive defect in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus, but was found not to assemble in a sec53 mutant, which causes the misfolding of proteins targeted to the ER. A kar2-1 mutation with a defect in the yeast homologue of BiP caused an 18-fold reduction in Lipo-EtxB assembly at the non-permissive temperature in S. cerevisiae. However, introduction of the wild-type KAR2 gene on a plasmid into the kar2-1 mutant completely suppressed the inhibition of Lipo-EtxB assembly. This provides the first evidence that KAR2 facilitates the assembly of an oligomeric protein in yeast and thus implicates KAR2 as a 'molecular chaperone'. The possible mechanisms of enterotoxoid assembly in E. coli and S. cerevisiae are discussed.
Mol Microbiol 1991 Nov
PMID:Targeting and assembly of an oligomeric bacterial enterotoxoid in the endoplasmic reticulum of Saccharomyces cerevisiae. 177 57

Immunoglobulin light chains are usually secreted from cells when they are synthesized alone or in molar excess of heavy chains, but, there have been reports of nonsecreted light chains. We wished to determine whether immunoglobulin heavy chain binding protein (BiP), which blocks the transport of free heavy chains, might be responsible for the lack of secretion of some light chains. In two murine lymphoid cell lines that synthesize but do not secrete immunoglobulin light chains, the free light chain polymers were found bound to BiP. Examination of 20 other cell lines and hybridomas failed to disclose any cells synthesizing free or excess light chains that associated with BiP, in all cases the free light chains were secreted as dimers. Despite their association with BiP and their blocked secretion, the aberrant light chains could combine with heavy chains and could be secreted as intact Ig molecules. Thus, while light chains do not usually express signals which allow them to bind to BiP, it appears that such signals can be expressed on certain light chains, resulting in their combination with BiP and blocked secretion. When single chain mutant cell lines are isolated from parental lines producing both heavy and light chains, they are almost always light chain producers suggesting that free heavy chains are much more toxic than free light chains. In both PC700 and P3X63Ag cells, however, clones that have lost either heavy chains or transport-defective light chains are present at the same frequency. Our findings that the light chains in both of these lines are associated with BiP raise the possibility that BiP actually contributes to heavy chain toxicity instead of preventing it.
Mol Immunol 1990 Jul
PMID:Association of transport-defective light chains with immunoglobulin heavy chain binding protein. 211 93

The role of N-linked glycosylation in protein maturation and transport has been studied by using the simian virus 5 hemagglutinin-neuraminidase (HN) protein, a model class II integral membrane glycoprotein. The sites of N-linked glycosylation on HN were identified by eliminating each of the potential sites for N-linked glycosylation by oligonucleotide-directed mutagenesis on a cDNA clone. Expression of the mutant HN proteins in eucaryotic cells indicated that four sites are used in the HN glycoprotein for the addition of N-linked oligosaccharide chains. These functional glycosylation sites were systematically eliminated in various combinations from HN to form a panel of mutants in which the roles of individual carbohydrate chains and groups of carbohydrate chains could be analyzed. Alterations in the normal glycosylation pattern resulted in the impairment of HN protein folding and assembly which, in turn, affected the intracellular transport of HN. The severity of the consequences on HN maturation depended on both the number of deleted carbohydrate sites and their position in the HN molecule. Analysis of the reactivity pattern of HN conformation-specific monoclonal antibodies with the mutant HN proteins indicated that one specific carbohydrate chain plays a major role in promoting the correct folding of HN. Another carbohydrate chain, which is not essential for the initial folding of HN was found to play a role in preventing the aggregation of HN oligomers. The HN molecules which were misfolded, owing to their altered glycosylation pattern, were retained in the endoplasmic reticulum. Double-label immunofluorescence experiments indicate that misfolded HN and folded HN are segregated in the same cell. Misfolded HN forms disulfide-linked aggregates and is stably associated with the resident endoplasmic reticulum protein, GRP78-BiP, whereas wild-type HN forms a specific and transient complex with GRP78-BiP during its folding process.
Mol Cell Biol 1990 May
PMID:Different roles of individual N-linked oligosaccharide chains in folding, assembly, and transport of the simian virus 5 hemagglutinin-neuraminidase. 218 15

Cell lines established from the Lepidopteran insect Spodoptera frugiperda (e.g., Sf9) are used routinely as hosts for the expression of foreign proteins by baculovirus vectors. Previously, we showed that human tissue plasminogen activator (t-PA) was expressed, N-glycosylated, and secreted by Sf9 cells infected with a recombinant baculovirus (Jarvis DL, Summers MD: Mol Cell Biol 9:214-223, 1989). We also showed that t-PA secretion was blocked by tunicamycin (TM), an inhibitor of N-glycosylation, but not by castanospermine (CS) or N-methyldeoxynojirimycin, inhibitors of the initial steps in N-linked oligosaccharide processing. This suggested that the addition, but not the processing, of N-linked oligosaccharides is required for the secretion of recombinant t-PA from baculovirus-infected Sf9 cells. In this study, we present a more generalized evaluation of the role of N-glycosylation in the transport of recombinant glycoproteins through the Sf9 cell secretory pathway. Several different secretory or membrane-bound glycoproteins were expressed in control, TM-treated, or CS-treated Sf9 cells, and their appearance in the medium or on the cell surface was measured. The results showed that TM blocked the transport of some, but not all, of these proteins, whereas CS did not block the transport of any. This suggests that N-glycosylation is sometimes required for the transport of recombinant glycoproteins through the Sf9 secretory pathway, while processing of the oligosaccharides is not. At least two other proteins, p80 and p31, consistently coimmunoprecipitated with the nonglycosylated precursors of recombinant glycoproteins expressed in TM-treated Sf9 cells. Neither was antigenically related to any of the recombinant proteins. Relatively larger amounts of p80 and p31 were coprecipitated when transport was completely blocked by TM compared to when transport was only reduced or was unaffected. These results suggest that p80 and p31 block the transport of some nonglycosylated glycoprotein precursors in TM-treated Sf9 cells by binding to them and producing transport-incompetent heterooligomeric complexes. If this speculation is correct, then p80 and p31 are functionally analogous to the mammalian immunoglobulin heavy chain binding/glucose-regulated 78 kilodalton protein (BiP/GRP78).
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PMID:Role of glycosylation in the transport of recombinant glycoproteins through the secretory pathway of lepidopteran insect cells. 234 87


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