O-mannosylation precedes and potentially controls the N-glycosylation of a yeast cell wall glycoprotein

Abstract

Secretory proteins in yeast are N- and O-glycosylated while they enter the endoplasmic reticulum. N-glycosylation is initiated by the oligosaccharyl transferase complex and O-mannosylation is initiated by distinct O-mannosyltransferase complexes of the protein mannosyl transferase Pmt1/Pmt2 and Pmt4 families. Using covalently linked cell-wall protein 5 (Ccw5) as a model, we show that the Pmt4 and Pmt1/Pmt2 mannosyltransferases glycosylate different domains of the Ccw5 protein, thereby mannosylating several consecutive serine and threonine residues. In addition, it is shown that O-mannosylation by Pmt4 prevents N-glycosylation by blocking the hydroxy amino acid of the single N-glycosylation site present in Ccw5. These data prove that the O- and N-glycosylation machineries compete for Ccw5; therefore O-mannosylation by Pmt4 precedes N-glycosylation.

Figure 1

The amino-acid sequence of the Saccharomyces cerevisiae covalently linked cell-wall protein 5. The amino-acid sequence was deduced from the DNA sequence (Mrsa et al., 1997). The arrow indicates the Kex2 processing site; the potential N-glycosylation site is underlined. The first boxed sequence (solid lines) corresponds to the typical repeat motif of the proteins with internal repeats (Pir) family. Another such motif is also present (dashed box), although it is less conserved than the first motif.

Figure 2

Western blot of Ccw5–ProtA. The protein was isolated from Saccharomyces cerevisiae wild-type (WT) cells and from protein mannosyl transferase (pmt) mutants, as indicated. Ccw5–ProtA, a fusion of covalently linked cell-wall protein 5 (Ccw5) with two protein A (ProtA) epitopes.

Figure 3

Covalently linked cell-wall protein 5 is N-glycosylated in the pmt4Δ mutant. Ccw5–HA (covalently linked cell wall protein 5 tagged with haemagglutinin) obtained from cell walls of wild-type (WT) and pmt4Δ cells was immunodetected after SDS–polyacrylamide gel electrophoresis (see the Methods section). Endoglycosidase H (Endo H) treatment was carried out using the method of Sanders et al. (1999). pmt4, protein mannosyl transferase 4D.

Figure 4

N-glycosylation of covalently linked cell wall protein 5 is prevented in wild-type cells by the action of protein mannosyl transferase 4. M, mannosyl residue; OST, oligosaccharyl transferase; Pmt4, protein mannosyl transferase 4; WT, wild-type.

Figure 5

Analysis of the protein sequence of Ccw5–ProtA isolated from wild-type and pmt4Δ mutant cells. The ten hydroxy amino acids shown in red are mannosylated by Pmt4. The N-glycosylation site Asn–Ala–Thr (NAT) is shown in blue, except for the positions modified in pmt4Δ. CcW5, covalently linked cell wall protein 5; pmt4, protein mannosyl transferase 4; ProtA, protein A epitope; WT, wild type. The N-glycosylation sites are shown in the boxes.