The UDP-GlcNAc/MurNAc family of eukaryotic and prokaryotic enzymes use UDP-GlcNAc or UDP-MurNAc-pentapeptide as donors, dolichol-P or polyprenol-P as acceptors, and generate sugar-P-P-polyisoprenols. A series of six conserved sequences, designated A through F and ranging from 5 to 13 amino acid residues, has been identified in this family. To determine whether these conserved sequences are required for enzyme function, various mutations were examined in hamster UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT). Scramble mutations of sequences B-F, generated by scrambling the residues within each sequence, demonstrated that each is important in GPT. While E and F scrambles appeared to prevent stable expression of GPT, scrambling of B-D resulted in GPT mutants that could be stably expressed and bound tunicamycin, but lacked enzymatic activity. Further, the C and D scramble mutants had an unexpected sorting defect. Replacement of sequences B-F with prokaryotic counterparts from either the B.subtilis mraY or E.coli rfe genes also affected GPT by preventing expression of the mutant protein (B, F) or inhibiting its enzymatic activity (C-E). For the C-E replacements, no acquisition of acceptor activity for polyprenol-P, the fully unsaturated natural bacterial acceptor, was detected. These studies show that the conserved sequences of the UDP-GlcNAc/MurNAc family are important, and that the eukaryotic and prokaryotic counterparts are not freely interchangeable. Since several mutants were efficiently expressed and bound tunicamycin, yet lacked enzymatic activity, the data are consistent with these sequences having a direct role in product formation.

UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) is an endoplasmic reticulum (ER) enzyme responsible for synthesis of GlcNAc-P-P-dolichol, the committed step of dolichol-P-P-oligosaccharide synthesis. The sequence of hamster GPT predicted multiple transmembrane segments (Zhu, X., and Lehrman, M. A. (1990) J. Biol. Chem. 265, 14250 -14255). GPT has also been predicted to act on the cytosolic face of the ER membrane, based on topological studies of its substrates and products. In this report we test these predictions by: (i) immunofluorescence microscopy with antibodies specific for native GPT sequences or epitope tags inserted into GPT, after selective permeabilization of the plasma membrane with digitonin; (ii) insertion of Factor Xa cleavage sites; (iii) in vitro translation of GPT; and (iv) site-directed mutagenesis.The loops between the 1st and 2nd and between the 9th and 10th predicted transmembrane spans of GPT were found to be cytosolic. In contrast, the loop between the 6th and 7th transmembrane spans, as well as the carboxyl terminus, were lumenal. Thus, hamster GPT must cross the ER membrane at least three times, consistent with previous computer-assisted predictions. There was no apparent N-glycosylation or signal sequence cleavage detected by in vitro translation. The cytosolic loop between the 9th and 10th transmembrane spans is the largest hydrophilic segment in GPT and, as judged by site-directed mutagenesis, has a number of conserved residues essential for activity. Hence, these results directly support the hypothesis that dolichol-P-P-oligosaccharide assembly is initiated in the cytosol and that a downstream intermediate must translocate to the lumenal face of the ER membrane.Many functions have been attributed to the asparaginelinked class of oligosaccharides on glycoproteins (1), such as directly mediating specific interactions with endoplasmic reticulum (ER) 1 chaperones (2, 3), and there is considerable interest in the mechanisms governing their synthesis. UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT) is responsible for the synthesis of GlcNAc-P-P-dolichol, the first intermediate in the synthesis of dolichol-P-P-oligosaccharides, which serve as precursors of asparagine-linked glycans (reviewed in Refs. 4 and 5). GPT is well known as a site of action for tunicamycin (Tn), although at least one other site of action exists (6). GPT is found in all eukaryotes and has been purified from bovine mammary gland (7) and cloned from hamster cells (8, 9), Saccharomyces cerevisiae (the ALG7 protein) (10, 11), Schizosaccharomyces pombe (12), Leishmania (13), and mouse (14). Although computer-assisted analysis of the hamster sequence predicted that GPT might contain as many as 10 transmembrane segments (8), there has been little direct information regarding the topological structure of this critical enzyme.GPT is of interest because it is one of a small group of glycosyltransferases that diverge from the common type II model typical of glycosyltransferases found in the Golgi apparatus. Type II glycos...

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.At least two well characterized families of eukaryotic glycoconjugates, the dolichol-linked oligosaccharides (1) and glycosylphosphatidylinositols (2) are synthesized by enzymes of the endoplasmic reticulum (ER).1 Each pathway requires the polyisoprene lipid dolichol-P, either as the carrier for glycoconjugate assembly in the case of dolichol-linked oligosaccharides or for synthesis of dolichol-P-monosaccharides that serve as sugar donors in both pathways. Dolichol-P is limiting in cells (3-5). Thus, there must be careful regulation of the enzymes that use dolichol-P as an acceptor. Failure to balance the flux of dolichol-P into various enzymatic reactions would be expected to cause defects in glycoconjugate synthesis. For example, excessive synthesis of GlcNAc-P-P-dolichol at the expense of mannose-P-dolichol and glucose-P-dolichol results in truncated dolichol-linked oligosaccharides (6, 7) lacking glucose residues necessary for interactions with the ER chaperones calnexin (8) and calreticulin (9).Our group and others have been interested in the structure and mechanism of action of UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase (GPT; EC 2.7.8.15) (10). This critical enzyme is responsible for the committed step of dolichol-linked oligosaccharide synthesis, i.e. the production of GlcNAc-P-P-dolichol, and catalyzes the following reversible reaction.