Proteolytic processing of the Saccharomyces cerevisiae cell wall protein Scw4 regulates its activity and influences its covalent binding to glucan.

Yeast cell wall contains a number of proteins that are either non-covalently (Scw-proteins), or covalently (Ccw-proteins) bound to ß-1,3-glucan, the latter either through GPI-anchors and ß-1,6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). It was shown that a part of Scw4, previously identified among the non-covalently bound cell wall proteins, was covalently attached to wall polysaccharides by a so far unknown alkali sensitive linkage. Thus Scw4 could be released from cell walls by treatments with hot SDS, mild alkali, or ß-1,3-glucanases, respectively. It was further shown that non-covalently bound Scw4 (SDS released) underwent the Kex2 proteolytic processing. In this paper it was demonstrated that Scw4 was also processed by yapsins at a position 9 amino acids downstream of the Kex2 cleavage site. Scw4 cleaved at the yapsin site had a markedly lower potential for covalent attachment to glucan. The overproduction of the fully processed form of Scw4 lead to high mortality, particularly in the stationary phase of growth, and to markedly increased cell size. On the other hand, the overproduction of Scw4 processed only by Kex2 or not processed at all had no apparent change in mortality indicating that only the smallest, completely mature form of Scw4 had the activity leading to observed phenotype changes.

Functional implications of MIR domains in protein O-mannosylation.

Protein O-mannosyltransferases (PMTs) represent a conserved family of multispanning endoplasmic reticulum membrane proteins involved in glycosylation of S/T-rich protein substrates and unfolded proteins. PMTs work as dimers and contain a luminal MIR domain with a ß-trefoil fold, which is susceptive for missense mutations causing α-dystroglycanopathies in humans. Here, we analyze PMT-MIR domains by an integrated structural biology approach using X-ray crystallography and NMR spectroscopy and evaluate their role in PMT function in vivo. We determine Pmt2- and Pmt3-MIR domain structures and identify two conserved mannose-binding sites, which are consistent with general ß-trefoil carbohydrate-binding sites (α, ß), and also a unique PMT2-subfamily exposed FKR motif. We show that conserved residues in site α influence enzyme processivity of the Pmt1-Pmt2 heterodimer in vivo. Integration of the data into the context of a Pmt1-Pmt2 structure and comparison with homologous ß-trefoil - carbohydrate complexes allows for a functional description of MIR domains in protein O-mannosylation.