The components of the Saccharomyces cerevisiae mannosyltransferase complex M-Pol I have distinct functions in mannan synthesis.
The yeast Saccharomyces cerevisiae processes N-linked glycans in the Golgi apparatus in two different ways. Whereas most of the proteins of internal membranes receive a simple core-type structure, a long branched polymer termed mannan is attached to the glycans of many of the proteins destined for the cell wall. The first step in mannan synthesis is the initiation and extension of an alpha-1,6-linked polymannose backbone. This requires the sequential action of two enzyme complexes, mannan polymerases (M-Pol) I and II. M-Pol I contains the proteins Mnn9p and Van1p, although the stoichiometry and individual contributions to enzyme action are unclear. We report here that the two proteins are each present as a single copy in the complex. Both proteins contain a DXD motif found in the active site of many glycosyltransferases, and mutations in this motif in Mnn9p or Van1p reveal that both proteins contribute to mannose polymerization. However, the effects of these mutations on both the in vivo and in vitro activity are distinct, suggesting that the two proteins may have different roles in the complex. Finally, we show that a simple glycoprotein based on hen egg lysozyme can be used as a substrate for modification by purified M-Pol I in vitro.
Stolz
Jurgen
J
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.
Munro
Sean
S
D020816:Amino Acid Motifs; D000818:Animals; D020134:Catalytic Domain; D002645:Chickens; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D008351:Mannans / Q000096:biosynthesis* / Q000737:chemistry; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009113:Muramidase / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D021122:Protein Subunits; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D006023:Glycoproteins; C086605:MNN9 protein, S cerevisiae; D008351:Mannans; D008562:Membrane Glycoproteins; D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; D008364:Mannosyltransferases; D009113:Muramidase