Characterization and purification of the 94-kDa glucose-regulated protein.
Increased synthesis of so-called glucose-regulated proteins (grp) of 78 and 94 kDa occurs in mammalian cells exposed to a variety of agents, including 2-mercaptoethanol, tunicamycin, agents which perturb calcium homeostasis, and amino acid analogs. Herein we describe a number of properties of 94-kDa grp (grp 94) and present a method for its purification to homogeneity. The protein, within the endoplasmic reticulum (ER), is modified by the addition of high mannose-containing oligosaccharides. The predicted amino acid sequence of grp 94, as determined by others, has revealed the protein to contain a putative transmembrane domain near its amino terminus, but in addition, a potential endoplasmic reticulum retention sequence (KDEL) at its COOH terminus. Consequently, the question of whether grp 94 exists as a transmembrane or luminal protein of the ER remains controversial. Results using isolated microsomes subjected to either limited proteolysis or lactoperoxidase-mediated iodination were consistent with the idea that the grp is a transmembrane protein. On the other hand, using the method of sodium carbonate extraction, grp 94 exhibited properties of both a luminal and integral membrane protein. These results raise the question of whether there exist two different forms of grp 94 within the ER.
Kang
H S
HS
Department of Medicine, University of California, San Francisco 94143.
Welch
W J
WJ
D000818:Animals; D015153:Blotting, Western; D000001:Calcimycin / Q000494:pharmacology; D004533:Egtazic Acid / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D018840:HSP70 Heat-Shock Proteins; D006367:HeLa Cells; D006360:Heat-Shock Proteins / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008970:Molecular Weight; D051381:Rats
D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D008565:Membrane Proteins; C018551:glucose-regulated proteins; D000001:Calcimycin; D004533:Egtazic Acid