Oligomerization of Hamster UDP-GlcNAc:Dolichol-P GlcNAc-1-P Transferase, an Enzyme with Multiple Transmembrane Spans*
- From the Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, Texas 75235-9041
Abstract
Hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT), which initiates N-linked glycosylation by catalyzing the synthesis of GlcNAc-P-P-dolichol, has multiple transmembrane spans and a catalytic site that probably exists on the cytosolic face of the endoplasmic reticulum membrane (Dan, N., Middleton, R. M., and Lehrman, M. A. (1996) J. Biol. Chem. 271, 30717–30725). In this report, we demonstrate that GPT forms functional oligomers, probably dimers. Oligomers were detected by chemical cross-linking of GPT and by a dominant-negative effect caused by co-expression of enzymatically inactive (but properly folded) GPT mutants. The GPT mutants had no effect on two other dolichol-P-dependent endoplasmic reticulum enzymes. Mixing experiments indicated that mature GPT was competent for oligomerization. Oligomerization appeared to be favored in detergent extracts compared with intact microsomes. Detergent treatments were found to prevent, rather than promote, nonspecific aggregation of GPT.
These results demonstrate that GPT subunits can physically interact and influence each other. The implications of oligomerization for enzyme function are discussed. From these results, we conclude that GPT is one of a very small number of multitransmembrane span enzymes that can form multimers.
Footnotes
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↵* This work was supported by National Institutes of Health Grant GM38545 and Robert Welch Foundation Grant I-1168.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‡ To whom correspondence should be addressed: Dept. of Pharmacology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9041. Tel.: 214-648-2323; Fax: 214-648-2971; E-mail: LEHRMAN{at}UTSW.SWMED.EDU.
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↵1 The abbreviations used are: ER, endoplasmic reticulum; BS3, bis(sulfosuccinimidyl)suberate; GPT, GlcNAc-1-P transferase; GPDS, glucose-P-dolichol synthase; Tn, tunicamycin; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonate.
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↵2 A. Dal Nogare, N. Dan, and M. A. Lehrman, unpublished results.
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- Received November 8, 1996.
- Revision received March 24, 1997.
