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First published online November 23, 2005
doi: 10.1242/10.1242/jcs.02649

Journal of Cell Science 118, 5589-5601 (2005)
Published by The Company of Biologists 2005
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Research Article

PtdIns(3)P accumulation in triple lipid-phosphatase-deletion mutants triggers lethal hyperactivation of the Rho1p/Pkc1p cell-integrity MAP kinase pathway

William R. Parrish*, Christopher J. Stefan and Scott D. Emr{ddagger}

Department of Cellular and Molecular Medicine and the Howard Hughes Medical Institute, University of California at San Diego, School of Medicine, La Jolla, California 92093-0668, USA

{ddagger} Author for correspondence (e-mail: semr{at}ucsd.edu)

Accepted 16 August 2005

In the budding yeast Saccharomyces cerevisiae, the regulation of phosphatidylinositol 3-phosphate [PtdIns(3)P] is an essential function shared by the myotubularin-related phosphatase Ymr1p and the synaptojanin-like phosphatases Sjl2p and Sjl3p. The aim of this study was to gain further insight into the mechanisms underlying the toxicity of PtdIns(3)P accumulation in ymr1{Delta} sjl2{Delta} sjl3{Delta} mutant cells. We conducted a genetic screen to isolate genes that, when overexpressed, would rescue the conditional lethality of ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-mutant cells expressing YMR1 from the dextrose-repressible GAL1 promoter. This approach identified 17 genes that promoted growth of the triple mutant on media containing dextrose. Interestingly, the most frequently isolated gene product was a truncated form of PKC1 (Pkc1-T615) that lacked the C-terminal kinase domain. This Pkc1-T615 fragment also rescued the lethality of ymr1ts sjl2{Delta} sjl3{Delta} cells at restrictive temperature, and further mapping of the rescuing activity showed that the N-terminal Rho1-GTP-interacting HR1 domains (Pkc1-T242) were sufficient. This indicated that the PKC1 fragments might act by interfering with Rho1-GTP signal propagation. Consistent with this, deletion of the ROM2 gene, which encodes a major Rho1p guanine-nucleotide exchange factor, bypassed the lethal effect of PtdIns(3)P accumulation in ymr1{Delta} sjl2{Delta} sjl3{Delta} triple-mutant cells. Furthermore, cells deficient in phosphoinositide 3-phosphatase (PI 3-phosphatase) activity were defective for Rho1p/Pkc1p pathway regulation, which included an inability of these cells to adapt to heat stress. Taken together, the results of this study indicated that aberrant Rho1p/Pkc1p signaling contributes to the lethal effects of PtdIns(3)P accumulation in cells deficient in PI 3-phosphatase activity.

Key words: Rho1p, PtdIns 3-kinase, Myotubularin, Cell-integrity MAP kinase pathway, Pkc1p


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© The Company of Biologists Ltd 2005