Analysis of the structure and synthesis of GRP94, an abundant stress protein of the endoplasmic reticulum.
The synthesis and cotranslational modification of GRP94, an abundant, resident protein of the endoplasmic reticulum belonging to the hsp90 family of stress proteins, has been studied in transient expression studies in COS cells. A fraction of the expressed murine GRP94 was more highly glycosylated than normal, having a greater number of endoglycosidase H (Endo H)-sensitive oligosaccharide moieties than authentic GRP94. To understand better the basis for the appearance of the hyperglycosylated form and determine the acceptor sites that were used for this extra glycosylation, we have used in vitro mutagenesis techniques to construct a set of point mutants and deletion mutants of GRP94. Analysis of the expression of wild-type GRP94 and the mutant proteins has revealed that Asn-196 is the acceptor site used in normal glycosylation of GRP94 and that hyperglycosylation is dependent upon the level of expression of the GRP94 and is occurring at acceptor sites in the carboxy-terminal region of the protein. We have shown, in addition, that Cys-117 is involved in the formation of a disulfide-bonded homodimer of GRP94. Finally, analysis of deletions made from the amino terminus of the mature protein has demonstrated that these alterations change the pattern of usage of the remaining N-glycosylation sites in the mutants.
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Department of Molecular Microbiology and Immunology, Saint Louis University School of Medicine, MO 63104.
Mazzarella
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism; D004247:DNA; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006031:Glycosylation; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D017384:Sequence Deletion
D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; C018551:glucose-regulated proteins; D004247:DNA; D003545:Cysteine