Yapsins are a family of aspartyl proteases required for cell wall integrity in Saccharomyces cerevisiae

Abstract

The yeast cell wall is a crucial extracellular organelle that protects the cell from lysis during environmental stress and morphogenesis. Here, we demonstrate that the yapsin family of five glycosylphosphatidylinositol-linked aspartyl proteases is required for cell wall integrity in Saccharomyces cerevisiae. Yapsin null mutants show hypersensitivity to cell wall perturbation, and both the yps1Delta2Delta mutant and the quintuple yapsin mutant (5ypsDelta) undergo osmoremedial cell lysis at 37 degrees C. The cell walls of both 5ypsDelta and yps1Delta2Delta mutants have decreased amounts of 1,3- and 1,6-beta-glucan. Although there is decreased incorporation of both 1,3- and 1,6-beta-glucan in the 5ypsDelta mutant in vivo, in vitro specific activity of both 1,3- and 1,6-beta-glucan synthesis is similar to wild type, indicating that the yapsins affect processes downstream of glucan synthesis and that the yapsins may be involved in the incorporation or retention of cell wall glucan. Presumably as a response to the significant alterations in cell wall composition, the cell wall integrity mitogen-activated kinase signaling cascade (PKC1-MPK pathway) is basally active in 5ypsDelta. YPS1 expression is induced during cell wall stress and remodeling in a PKC1-MPK1-dependent manner, indicating that Yps1p is a direct, and important, output of the cell wall integrity response. The Candida albicans (SAP9) and Candida glabrata (CgYPS1) homologues of YPS1 complement the phenotypes of the yps1Delta mutant. Taken together, these data indicate that the yapsins play an important role in glucan homeostasis in S. cerevisiae and that yapsin homologues may play a similar role in the pathogenic yeasts C. albicans and C. glabrata.

FIG. 1.

Yapsin deletion mutants undergo osmoremedial cell lysis at 37°C. (A) Strains with multiple yapsin deletions show a profound growth defect on YPD at 37°C that is suppressed by the addition of 1.0 M sorbitol to the medium. (B) Reintroduction of either YPS1 or YPS2 (CEN plasmids under endogenous promoters) to the 5ypsΔ background complements temperature sensitivity.

FIG. 2.

Yapsin deletion mutants show differential susceptibility to cell wall-active drugs. Cells (5 μl of a 1-OD₆₀₀ suspension) were pronged onto YPD plates supplemented with the indicated drug. Drug concentrations: calcofluor white, 20 μg/ml; caspofungin, 200 ng/ml; Congo Red, 200 μg/ml; caffeine, 6 mM. Plates were incubated at 30°C (unless otherwise indicated) for 3 to 5 days.

FIG. 3.

PKC1-MPK1 is basally activated in 5ypsΔ. The phosphorylation of MAP kinases Mpk1p and Kss1p was assayed by immunoblot analysis of protein extracts from wild-type and 5ypsΔ cells harboring MPK1-HA (pDL922) or KSS1-HA (pXZ358) using the phosphospecific antibody anti-active MAPK (Promega). Anti-HA (12CA5) was used to demonstrate equal protein loading. (A) Mpk1p is not phosphorylated at 30°C in wild type (WT) but is phosphorylated at 30°C in the 5ypsΔ mutant. (B) Kss1p, the MAPK of the SVG pathway, is not phosphorylated at 30°C in either wild-type or 5ypsΔ strains but is responsive to α-factor.

FIG. 4.

Incorporation of 1,3- and 1,6-β-glucan is decreased in the 5ypsΔ mutant, but the specific activity of 1,3- and 1,6-β-glucan synthesis is not. (A) Log-phase cultures of wild-type (WT) and 5ypsΔ cells were pulse-labeled with [¹⁴C]glucose for 5 min. The cell walls were isolated and processed as described in Materials and Methods to determine the amount of label uptake. (B) The specific activity of 1,3- and 1,6-β-glucan synthase was determined as described in Materials and Methods. Extracts were prepared from cells harvested in log phase at 30°C. Data are expressed as percentage of wild-type incorporation/specific activity and are the means of two independent experiments performed in duplicate.

FIG. 5.

Synthetic genetic interaction between yps1Δ and cbk1Δ. Serial 10-fold dilutions of a 1-OD₆₀₀ suspension of the indicated strains were pronged onto plates containing YPD plus 12.5 μg/ml calcofluor white, grown for 4 days at 30°C, and photographed. WT, wild type.

FIG. 6.

Overexpression of YPS1 induces caspofungin tolerance in wild-type cells and suppresses phenotypes of cell wall mutants. (A) A suspension of wild-type (WT) cells harboring either a 2μ-plasmid or YPS1/SAP9 in the same plasmid under a constitutive promoter was spotted on YPD supplemented with 500 ng/ml of caspofungin. (B) Serial 10-fold dilutions of either wild type or the indicated cell wall mutants containing pG5 or pGYPS1 were pronged on YPD and incubated at 30°C for 2 days. (C) Serial 10-fold dilutions of wild-type or smi1Δ strains containing pG5 or pGYPS1 were spotted on YPD and incubated at 30°C for 2 days. Yapsin homologues from Candida albicans (SAP9) and Candida glabrata (CgYPS1) complement the caspofungin sensitivity of yps1Δ. (D) Serial 10-fold dilutions of wild-type and yps1Δ cells harboring pRS426-GPD, pRS426-GPD-SAP9, or pRS416-CgYPS1 were pronged on YPD plates supplemented with 100 ng/ml of caspofungin and incubated at 30°C for 3 days.