Biochemical characterization and membrane topology of Alg2 from Saccharomyces cerevisiae as a bifunctional alpha1,3- and 1,6-mannosyltransferase involved in lipid-linked oligosaccharide biosynthesis.
N-Linked glycosylation involves the ordered, stepwise synthesis of the unique lipid-linked oligosaccharide precursor Glc(3)Man(9) GlcNAc(2)-PP-Dol on the endoplasmic reticulum (ER), catalyzed by a series of glycosyltransferases. Here we characterize Alg2 as a bifunctional enzyme that is required for both the transfer of the alpha1,3- and the alpha1,6-mannose-linked residue from GDP-mannose to Man(1)GlcNAc(2)-PP-Dol forming the Man(3)GlcNAc(2)-PP-Dol intermediate on the cytosolic side of the ER. Alg2 has a calculated mass of 58 kDa and is predicted to contain four transmembrane-spanning helices, two at the N terminus and two at the C terminus. Contradictory to topology predictions, we prove that only the two N-terminal domains fulfill this criterion, whereas the C-terminal hydrophobic sequences contribute to ER localization in a nontransmembrane manner. Surprisingly, none of the four domains is essential for transferase activity because truncated Alg2 variants can exert their function as long as Alg2 is associated with the ER by either its N- or C-terminal hydrophobic regions. By site-directed mutagenesis we demonstrate that an EX(7)E motif, conserved in a variety of glycosyltransferases, is not important for Alg2 function in vivo and in vitro. Instead, we identify a conserved lysine residue, Lys(230), as being essential for activity, which could be involved in the binding of the phosphate of the glycosyl donor.
Kämpf
Michael
M
Lehrstuhl für Zellbiologie und Pflanzenphysiologie, Universität Regensburg, 93053 Regensburg, Germany.
Absmanner
Birgit
B
Schwarz
Markus
M
Lehle
Ludwig
L
D020816:Amino Acid Motifs / Q000502:physiology; D002462:Cell Membrane / Q000201:enzymology* / Q000235:genetics; D003600:Cytosol / Q000378:metabolism; D004286:Dolichols / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D006031:Glycosylation; D006155:Guanosine Diphosphate Mannose / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D008563:Membrane Lipids / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D004286:Dolichols; C520165:Glc3Man9GlcNAc2-PP-Dol; D008563:Membrane Lipids; D008565:Membrane Proteins; D009844:Oligosaccharides; D029701:Saccharomyces cerevisiae Proteins; D006155:Guanosine Diphosphate Mannose; C083733:ALG2 protein, S cerevisiae; D008364:Mannosyltransferases