The Yeast Proprotein Convertase Encoded by YAP3 Is a Glycophosphatidylinositol-anchored Protein That Localizes to the Plasma Membrane

Abstract

The yeast YAP3 gene encodes an aspartyl endoprotease that cleaves precursor proteins at selected pairs of basic amino acids and after single arginine residues. Biosynthetic studies of this proprotein processing enzyme indicate that Yap3 is predominantly cell-associated and migrates as a approximately 160-kDa protein on SDS-polyacrylamide gel electrophoresis. Nearly equal amounts of Yap3 are immunodetected in a-haploid, alpha-haploid, and a/alpha-diploid yeast, demonstrating that the expression of YAP3 is not mating type-specific. As shown by endoglycosidase H treatment, which drastically reduces both the estimated molecular mass and the heterogeneity of the protein on SDS-polyacrylamide gel electrophoresis (68 versus 160 kDa), the oligosaccharides N-linked to the protein are subjected to extensive outer chain mannosylation. Outer chain sugar mannosylation takes place in the Golgi apparatus and is commonly found on yeast secreted glycoproteins and/or cell wall mannoproteins. Treatment of the total yeast membranes with chemical agents known to disrupt protein-protein and protein-lipid interactions reveal that Yap3 is membrane-associated. Based upon the release of the membrane-bound form by bacterial phosphatidylinositol phospholipase C digestion and metabolic labeling of the protein with myo-[3H]inositol, Yap3 owes its association with the membrane to the addition of a glycophosphatidylinositol anchor. The cellular localization of Yap3 has been addressed by subcellular fractionation studies. In both differential centrifugation of intracellular organelles and sucrose density gradients, the bulk of Yap3 at steady state co-localizes with the plasma membrane azide-insensitive ATPase. Furthermore, consistent with the transport of Yap3 to the plasma membrane, the endoprotease sediments with secretory vesicles which accumulate at restrictive temperature in the late secretory mutant sec1-1. We therefore conclude that the endoprotease encoded by YAP3 is a glycophosphatidylinositol-anchored protein, which can process substrates both intracellularly and at the cell surface.

Amino Acid Sequence Requirement for Efficient Incorporation of Glycosylphosphatidylinositol-associated Proteins into the Cell Wall of Saccharomyces cerevisiae

“…Substitution of the site residue from Asn, an authentic site residue, with Ser, Gly, Ala, Asp, or Cys slightly decreased the efficiency of GPI attachment, while replacement of Asn with other amino acid residues including Gln, Glu, and Thr resulted in an almost complete loss of the GPI attachment efficiency (33). Based on this selectivity of the site residue, we determined the sites of two GPI-dependent cell wall proteins, Yjr151c (24) and Ydr077w/Sed1p (15), and two GPI-anchored plasma membrane proteins, Yir039c (24) and Ylr120c/Yap3p (43)(44)(45). The C-terminal sequences, shown in Fig.…”

“…We examined the requirement of these amino acid residues for the cell wall incorporation by using a model construct. It contains a stretch of 15 serines followed by the site and the -plus region of Ylr120c/Yap3p, a well characterized GPI-anchored plasma membrane protein (43)(44)(45), fused to a HA-tagged ␣-galactosidase (Fig. 4A, SCT01).…”

“…Cell Wall Incorporation of Mutated Yap3p GPI-anchored Plasma Membrane Protein-S. cerevisiae Yap3p, an aspartic protease, is a GPI-anchored plasma membrane protein (43)(44)(45). Using this protein, we tested whether the replacement of amino acid residues at the -4 or -5 and -2 sites directs the protein into the cell wall.…”

Protein O-Mannosyltransferases Associate with the Translocon to Modify Translocating Polypeptide Chains

“…To further verify these results, we analyzed one of the proteins affected in pmt⌬ mutants, Yps1, which is a glycosylphosphatidylinositol-anchored aspartic protease belonging to a family of fungal yapsins localizing to the plasma membrane (38). We expressed an N-terminal HA-tagged version of Yps1 ( HA Yps1) in WT and pmt2 mutant strains and analyzed its glycosylation by Western blot before and after deglycosylation using Endo H. In WT, HA Yps1 showed an apparent molecular mass of ϳ90 kDa that shifted to ϳ75 kDa upon Endo H treatment (Fig.…”

Membrane topology and post‐translational modification of the Saccharomyces cerevisiae essential protein Rot1

“…Thus, the membrane fraction of extracts from cells expressing Rot1-HA was treated with PI-PLC from two different sources, B. cereus and B. thuringiensis (two different enzymes were used, as some substrate specificity was described and not all GPI yeast proteins were equally recognized by the enzymes; Vossen et al, 1997). GST-Yap3, a well-known GPI-anchored protein (Ash et al, 1995), was used as a positive control. Figure 3 shows that in contrast to GST-Yap3, which was partially released in the soluble fraction upon membranes being treated with the PI-PLC enzymes, Rot1-HA entirely recovered in the membrane phase.…”