Membrane topology of the Alg14 endoplasmic reticulum UDP-GlcNAc transferase subunit.
N-linked glycosylation begins in the endoplasmic reticulum with the synthesis of a highly conserved dolichol-linked oligosaccharide precursor. The UDP-GlcNAc glycosyltransferase catalyzing the second sugar addition of this precursor consists in most eukaryotes of at least two subunits, Alg14 and Alg13. Alg14 is a membrane protein that recruits the soluble Alg13 catalytic subunit from the cytosol to the face of the endoplasmic reticulum (ER) membrane where this reaction occurs. Here, we investigated the membrane topology of Saccharomyces cerevisiae Alg14 and its requirements for ER membrane association. Alg14 is predicted by most algorithms to contain one or more transmembrane spanning helices (transmembrane domains (TMDs)). We provide evidence that Alg14 contains a C-terminal cytosolic tail and an N terminus that resides within the ER lumen. However, we also demonstrate that Alg14 lacking this TMD is functional and remains peripherally associated with ER membranes, suggesting that additional domains can mediate ER association. These conclusions are based on the functional analysis of Alg13/Alg14 chimeras containing Alg13 fused at either end of Alg14 or truncated Alg14 variants lacking the predicted TMD; protease protection assays of Alg14 in intact ER membranes; and extraction of Alg14-containing ER membranes with high pH. These yeast Alg13-Alg14 chimeras recapitulate the phylogenetic diversity of Alg13-Alg14 domain arrangements that evolved in some protozoa. They encode single polypeptides containing an Alg13 domain fused to Alg14 domain in either orientation, including those lacking the Alg14 TMD. Thus, this Alg13-Alg14 UDP-GlcNAc transferase represents an unprecedented example of a bipartite glycosyltransferase that evolved by both fission and fusion.
Averbeck
Nicole
N
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, New York 11794-5215, USA.
Keppler-Ross
Sabine
S
Dean
Neta
N
D000217:Acyltransferases / Q000737:chemistry*; D000465:Algorithms; D020134:Catalytic Domain; D003600:Cytosol / Q000378:metabolism; D010450:Endopeptidases / Q000737:chemistry; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D016695:Glycosyltransferases / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism / Q000502:physiology*; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism / Q000502:physiology*; D013347:Subcellular Fractions / Q000378:metabolism
D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D000217:Acyltransferases; C050304:acyl-(acyl-carrier-protein)-UDP-N-acetylglucosamine acyltransferase; D016695:Glycosyltransferases; C506082:Alg13 protein, S cerevisiae; C506083:Alg14 protein, S cerevisiae; D017351:N-Acetylglucosaminyltransferases; D010450:Endopeptidases; C099488:TEV protease