Biosynthesis of the side chain of yeast glycosylphosphatidylinositol anchors is operated by novel mannosyltransferases located in the endoplasmic reticulum and the Golgi apparatus

Abstract

Glycosylphosphatidylinositol (GPI) anchors of the yeast Saccharomyces cerevisiae have been reported to contain throe different types of side chains attached to the α1,2-linked mannose of the conserved protein-ethanolamine- PO4-Manα1,2Manα1,6Manα1,4GlcNH2-inositol glycan core. The possible side chains are Manα1,2- or Manα1,2Manα1,2- or Manα1,3Manα1,2- (Fankhauser, C., Homan, S. W., Thomas Oates, J. E., McConville, M. J., Desponds, C., Conzelmann, A., and Ferguson, M. A. (1993) J. Biol. Chem. 268, 26365-26374). To determine in what subcellular compartment these side chains are made, we metabolically labeled GPI-anchored membrane proteins with myo-[2-3H]inositol in secretion mutants blocked at various stages of the secretory pathway and analyzed the anchor structure of the labeled glycoproteins. When the exit of vesicles from the endoplasmic reticulum or entry into the cis-Golgi were blocked in sec12 or sec18 cells, all anchors contained a side chain consisting of a single α1,2-linked mannose. GPI proteins trapped in the cis- Golgi of sec7 contained Manα1,3Manα1,2- but no Manα1,2Manα1,2- side chains. Mutants blocked at later stages of the secretory pathway made increased amounts of side chains containing two mannoses. Manα1,2Manα1,2- and Manα1,3Manα1,2-side chains were preferentially associated with ceramide- and diacylglycerol-containing GPI anchors, respectively. Mnn1, mnn2, mnn3, mnn5, and mnt1(=kre2), i.e. mutants which lack or down-regulate 1,2- and 1,3-mannosyltransferases used in the elongation of N- and O-glycans in the Golgi, add the fifth mannose to GPI anchors normally. The same conclusion was reached through the analysis of deletion mutants in KTR1, KTR2, KTR3, KTR4, and YUR1 which all are open reading frames with high homology to MNT1. Mutants deficient in the Golgi elongation of N-glycans such as anp1, van1, mnn9 are deficient in the maturation of the N-glycans of GPI- anchored glycoproteins, but process the GPI anchor side chain normally. Data are consistent with the idea that the fourth mannose is added to proteins as part of the anchor precursor glycolipid in the endoplasmic reticulum, whereas the fifth mannose is added by not yet identified α1,3- and α1,2- mannosyltransferases located in the Golgi apparatus.