Abstract
In the yeast Saccharomyces cerevisiae many of the N-linked glycans on cell wall and periplasmic proteins are modified by the addition of mannan, a large mannose-containing polysaccharide. Mannan comprises a backbone of approximately 50 alpha-1,6-linked mannoses to which are attached many branches consisting of alpha-1,2-linked and alpha-1,3-linked mannoses. The initiation and subsequent elongation of the mannan backbone is performed by two complexes of proteins in the cis Golgi. In this study we show that the product of the MNN10/BED1 gene is a component of one of these complexes, that which elongates the backbone. Analysis of interactions between the proteins in this complex shows that Mnn10p, and four previously characterized proteins (Anp1p, Mnn9p, Mnn11p, and Hoc1p) are indeed all components of the same large structure. Deletion of either Mnn10p, or its homologue Mnn11p, results in defects in mannan synthesis in vivo, and analysis of the enzymatic activity of the complexes isolated from mutant strains suggests that Mnn10p and Mnn11p are responsible for the majority of the alpha-1, 6-polymerizing activity of the complex.
Highlights
The glycoproteins of the cell wall of the yeast Saccharomyces cerevisiae are modified with both N-linked and O-linked glycans
Mnn10p Is a Component of the Anp1p-Mnn9p Complex of the cis Golgi—To determine whether Mnn10p was a component of one of the two Mnn9p-containing complexes, the same co-immunoprecipitation approach was followed that was originally used to reveal the interactions between Mnn9p and the other members of the complexes [12]
Deletion of Mnn10p or Mnn11p Alters the in Vitro Mannosyltransferase Activity of mannan polymerases (M-Pol) II—Because the level of Anp1p is reduced in the ⌬mnn10 and ⌬mnn11 strains, it is possible that the defect in mannan synthesis seen is not because the proteins are directly involved in mannan synthesis, but rather because they are required for the stability or integrity of other proteins in the complex, which are responsible for mannan synthesis
Summary
The glycoproteins of the cell wall of the yeast Saccharomyces cerevisiae are modified with both N-linked and O-linked glycans. Blotting of the digitonin lysate of the Hoc1p-Myc-expressing cells revealed two bands, of which only the upper is present in blots of cellular proteins solubilized directly in SDS-containing sample buffer (Fig. 2B and data not shown).
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