Saccharomyces cerevisiae mid2p is a potential cell wall stress sensor and upstream activator of the PKC1-MPK1 cell integrity pathway

Abstract

The MID2 gene of Saccharomyces cerevisiae encodes a protein with structural features indicative of a plasma membrane-associated cell wall sensor. MID2 was isolated as a multicopy activator of the Skn7p transcription factor. Deletion of MID2 causes resistance to calcofluor white, diminished production of stress-induced cell wall chitin under a variety of conditions, and changes in growth rate and viability in a number of different cell wall biosynthesis mutants. Overexpression of MID2 causes hyperaccumulation of chitin and increased sensitivity to calcofluor white. alpha-Factor hypersensitivity of mid2Delta mutants can be suppressed by overexpression of upstream elements of the cell integrity pathway, including PKC1, RHO1, WSC1, and WSC2. Mid2p and Wsc1p appear to have overlapping roles in maintaining cell integrity since mid2Delta wsc1Delta mutants are inviable on medium that does not contain osmotic support. A role for MID2 in the cell integrity pathway is further supported by the finding that MID2 is required for induction of Mpk1p tyrosine phosphorylation during exposure to alpha-factor, calcofluor white, or high temperature. Our data are consistent with a role for Mid2p in sensing cell wall stress and in activation of a response that includes both increased chitin synthesis and the Mpk1p mitogen-activated protein kinase cell integrity pathway. In addition, we have identified an open reading frame, MTL1, which encodes a protein with both structural and functional similarity to Mid2p.

FIG. 1

Multicopy MID2 activates Skn7p-LexA-dependent transcription of HIS3, which allows growth on medium lacking histidine. Reporter strains containing Skn7p-LexA and either pRS425 (TK60) or pRS425-MID2 (TK61) were grown on selective medium lacking histidine and containing 30 mM 3AT.

FIG. 2

Cell biology of Mid2p. (A) Immunoblot analysis of cell extracts from TK82 (vector only) (lane a), TK84 (MID2-HA) (lane b), pmt1Δ pmt2Δ (vector only) (lane c), and pmt1Δ pmt2Δ (MID2-HA). (B) Immunoblot analysis of cell extracts from TK82 (vector only) (lane a) and TK85 (ΔS/T-Mid2p-HA) (lane b). (C) Immunoblot analysis of cell fractions from TK84 to demonstrate membrane association of Mid2p. LSS, low-speed-spin pellet fraction. (D) In cells expressing pRS426-MID2-GFP (TK98), Mid2p-GFP is localized to the cell periphery.

FIG. 3

Deletion of MID2 has effects on growth of different cell wall mutants. (A) Representative tetratype tetrad from TK101 (kre6Δ mid2Δ heterozygous diploid). (B) Representative tetratype tetrad from TK102 (kre9Δ mid2Δ heterozygous diploid). (C) Single cells containing the indicated plasmids were placed on selective agar and grown at 30°C for 4 days. Photo is representative of effect seen in three isolates each of three transformations. (D) Representative tetratype tetrad from TK103 (fks1Δ mid2Δ heterozygous diploid).

FIG. 4

Dosage of MID2 affects sensitivity to calcofluor white. Mid-log-phase cells were diluted to a concentration of 3 × 10⁶ cells/ml; 5 μl of this suspension and three subsequent 10-fold serial dilutions were each spotted onto the indicated medium. (A) SEY6210a (wild type) (row a) and TK88 (mid2Δ) (row b) cells were spotted onto YEPD containing 0 and 20 μg of calcofluor white (CFW) per ml. (B) TK82 [wild type (pRS426)] (row a), TK83 [wild type (pRS426-MID2)] (row b), TK86 [wild type (pVT101U)] (row c), and TK87 [wild type (pVT101U-MID2)] (row d) were spotted on uracil dropout medium containing 0 and 2.5 μg of calcofluor white per ml. (C) TK86 (row a), TK87 (row b), TK99 [chs3Δ (pVT101U)] (row c), and TK100 [chs3Δ (pVT101U-MID2)] (row d) were spotted onto uracil dropout medium containing 0, 2.5, and 15 μg of calcofluor white per ml.

FIG. 5

Genetic interactions between MID2 and members of the cell integrity pathway. (A) Representative tetratype tetrads of TK104 (mid2Δ wsc1Δ heterozygous diploid) dissected onto either YEPD or YEPD plus 1 M sorbitol. YEPD plates were incubated for 60 h at 30°C, YEPD–1 M sorbitol plates were incubated for 80 h at 30°C. WT, wild type. (B) Members of the cell integrity pathway suppress α-factor-induced death in mid2Δ mutants. Wild-type cells containing pRS426 vector only and mid2Δ mutants carrying pRS426, YEP13-PKC1, pBM743-PKC1^R398A (GAL-driven, hyperactive PKC1), pRS426-RHO1, pRS426-WSC1, pRS426-WSC2, pRS316-BCK1-20 (hyperactive BCK1), YEP352-MPK1, and pRS425-MTL1 in liquid medium were exposed to α-factor. Percentage of survival was measured by spreading liquid medium before and after α-factor exposure (330 mn) on petri dishes and counting colonies derived from single cells.

FIG. 6

Immunoblot analysis of Mpk1p-HA tyrosine phosphorylation. Lanes are loaded with equal amounts of extracts from strains TK96 [wild type (pFL44)] (lanes a), TK97 [wild type (pFL44-MPK1-HA)] (lanes b), TK93 [mid2Δ (pFL44)] (lanes c), and TK94 [mid2Δ(pFL44-MPK1-HA)] (lanes d). Cultures exposed to α-factor (A), calcofluor white (B), or high-temperature growth (C) were harvested at the indicated times, and total cell proteins were subject to SDS-PAGE and Western blotting. In the top panel of each pair, tyrosine phosphorylation of Mpk1p-HA is detected by antiphosphotyrosine antibody 4G10. In the second panel of each pair, equal loading of Mpk1p-HA is demonstrated by anti-HA antibody HA11.

FIG. 7

Model of Mid2p activity. Mid2p responds to cell wall stress by activating the cell integrity pathway and increasing chitin synthesis.