Hamster UDP-N-Acetylglucosamine:Dolichol-P N-Acetylglucosamine-1-P Transferase Has Multiple Transmembrane Spans and a Critical Cytosolic Loop

Abstract

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...

Physical Interactions among Human Glycosyltransferases Involved in Dolichol-Linked Oligosaccharide Biosynthesis

“…3A). These results agreed well with the membrane topological features that had previously been demonstrated for hamster (11) and yeast (12)(13)(14) enzymes. Therefore, for these enzymes, pBT-STE-based bait constructs were used for subsequent analyses of physical interactions with prey constructs (Fig.…”

Transmembrane Topology of Pmt1p, a Member of an Evolutionarily Conserved Family of Protein O-Mannosyltransferases

“…These include the glycosyltransferases involved in the synthesis of the lipid oligosaccharide precursor for N-glycans (3,42) and the glycosylphosphatidylinositol (GPI) anchor (3,43). The topological organization of these transferases is not well characterized, but for the ones investigated so far the putative transmembrane topology does not resemble the structure of Pmt1p.…”

“…This enzyme initiates N-linked glycosylation by catalyzing the synthesis of GlcNAc-P-P-dolichol. Lehrman and coworkers (42,46) demonstrated that GPT is a multimeric enzyme with multiple, most likely 10 transmembrane spans. The largest hydrophilic segment, located between TM 9 and TM 10, is facing the cytoplasm.…”

Conserved amino acid residues found in a predicted cytosolic domain of the lipopolysaccharide biosynthetic protein WecA are implicated in the recognition of UDP-N-acetylglucosamine

“…The topology of two members of this family, the bacterial protein MraY that is involved in peptidoglycan synthesis (Bouhss et al, 1999) and the eukaryotic GPT that is involved in protein glycosylation (Dan et al, 1996), has been characterized experimentally. Bacterial and eukaryotic proteins share discrete regions of conserved amino acid sequence, especially those located in hydrophilic segments of the protein that are exposed to the cytosolic face of the plasma membrane or the membrane of the endoplasmic reticulum (Bouhss et al, 1999 ;Dal Nogare & Lehrman, 1988).…”