Glycosylation and secretion of acid phosphatase in Schizosaccharomyces pombe

Abstract

We have purified secreted acid phosphatase of Schizosaccharomyces pombe. The enzyme is N-glycosylated, the associated carbohydrate accounts for 90% of the total molecular mass and the protein moiety has a molecular mass of 54 kDa. The deglycosylated enzyme still exhibits enzymatic activity. Using antibodies recognizing the protein moiety of the enzyme we have identified two intracellular precursors of acid phosphatase: an unglycosylated membrane-bound 54-kDa form that accumulates in the presence of tunicamycin and a partially glycosylated 72-kDa form that accumulates mostly in membranes of cells grown in rich medium. We further showed that the conversion of the 54-kDa and 72-kDa forms to partially glycosylated and fully glycosylated acid phosphatase is a regulated process. Growth conditions determine how much of translated 54-kDa acid phosphatase is glycosylated to the 72-kDa form and how much remains unglycosylated in membranes. When cells are grown in a rich medium, 5% of the total acid phosphatase protein remains as unglycosylated enzyme and 8% as partially glycosylated 72-kDa form. In cells grown in the minimal medium, however, all of the 54-kDa and 72-kDa forms of acid phosphatase are rapidly processed to fully glycosylated enzyme. The 72-kDa form and the unglycosylated form of acid phosphatase are not secreted or transported to the plasma membrane.Eucaryotic secretory proteins including those of Saccharomyces cerevisiae are synthesized at the endoplasmic reticulum, are transported to the Golgi apparatus and via vesicles to the cell surface. Many of them are N-glycosylated on their secretory pathway. In yeast the pathway of Asn-linked glycosylation starts with the transfer of the oligosaccharide precursor, (GlcNAc)2(Man)9(Glc)3, from the dolichyl diphosphate to appropriate asparagine residues [l, 21. This oligosaccharide is further processed by removal of the three glucose units and a mannose unit [l, 31. Glycoproteins that are secreted have large polymannose chains added to the oligosaccharide core units, whereas glycoproteins destined to remain in the cell have the core oligosaccharide increased by 1 -4 mannose residues [4 -61.Mainly by the use of temperature-sensitive mutants that are blocked at distinct steps in the export of cell-surface glycoproteins, it has been shown that the transfer of the (Glc)3(Man)9(GlcNAc)z core oligosaccharides to the protein occurs in the endoplasmic reticulum and that extension through the addition of outer polymannose chains takes place in Golgi-like organelles [7]. Little is known concerning the regulation of the Asn-linked glycosylation pathway in yeast. Data of Chu and Malley indicate that glucose impairs Nglycosylation of invertase and that the nonglycosylated polypeptide is rapidly degraded by an endogenous protease PI.Correspondence to M. E. Schweingruber, Institut fur Allgemeine Mikrobiologie der Universitat Bern, Baltzerstrasse 4, CH-3012 Bern, SwitzerlandAbbreviations. Endoglycosidase H, endo-N-acetylglucosaminidase H ; Tris/NaCl, 20 mM Tris/HCl, 150 mM NaC1, pH ...

Molecular and Phenotypic Analysis ofCaVRG4, Encoding an Essential Golgi Apparatus GDP-Mannose Transporter

“…Native gel electrophoresis and activity staining of acid phosphatase from C. albicans were carried out as described by Schweingruber et al (45) for the homologous Schizosaccharomyces pombe enzyme, with the following modifications. Cells were grown in YPAD(ϩUri) to the log phase (OD 600 , 5), and acid phosphatase activity was induced by overnight cultivation at 30°C in 20 ml of phosphate-reduced YPAD(ϩUri) depleted of phosphate as described previously (59).…”

“…The lysate was centrifuged for 10 min, and the resulting supernatant was transferred and centrifuged for 10 min to remove insoluble precipitates. The supernatant was mixed with 0.5 volume of sample buffer (59), and 5 l was applied to a gel and subjected to 5% native gel electrophoresis at 4°C as described previously (45). After incubation for 10 min in 100 mM sodium acetate (pH 4.0) (SA), the gel was incubated at 37°C in SA containing 0.5 mg of ␣-naphthyl acid phosphate (Sigma) per ml for 1 h. Color was developed by adding 50 ml of SA (prewarmed to 60°C) containing 0.7 mg of o-dianisidine (tetrazotized; Sigma) per ml and incubating the preparation at 60°C for 10 min.…”

Candida albicans Pmr1p, a Secretory Pathway P-type Ca2+/Mn2+-ATPase, Is Required for Glycosylation and Virulence

“…Acid Phosphatase Zymogram-In situ acid phosphatase activity assays were performed following a modified method of Schweingruber et al (46). Briefly, cells were grown overnight in phosphate-depleted Sabouraud glucose medium to induce acid phosphatase expression.…”

The Srp54 GTPase is essential for protein export in the fission yeast Schizosaccharomyces pombe.

“…To determine whether S. pombe cells depleted of Srp54 display a translocation defect, we used pulse-labeling followed by immunoprecipitation to examine the fates of two ER-targeted proteins, acid phosphatase, which is secreted, and BiP, which remains in the lumen of this organelle. Acid phosphatase exists in three previously characterized forms: a 50.5-kDa cytoplasmic precursor, a 70-kDa core-glycosylated form produced upon translocation into the ER, and a diffuse high-molecular-weight band corresponding to the mature, secreted form (85). Figure 2B shows the results of immunoprecipitating extracts of the cells for which growth data are shown in Fig.…”