PtdIns(3)P accumulation in triple lipid-phosphatase-deletion mutants triggers lethal hyperactivation of the Rho1p/Pkc1p cell-integrity MAP kinase pathway.
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 ymr1Delta sjl2Delta sjl3Delta mutant cells. We conducted a genetic screen to isolate genes that, when overexpressed, would rescue the conditional lethality of ymr1Delta sjl2Delta sjl3Delta 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 sjl2Delta sjl3Delta 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 ymr1Delta sjl2Delta sjl3Delta 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.
Parrish
William R
WR
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.
Stefan
Christopher J
CJ
Emr
Scott D
SD
D002470:Cell Survival / Q000502:physiology; D004789:Enzyme Activation / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism; D020935:MAP Kinase Signaling System / Q000502:physiology*; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008954:Models, Biological; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism* / Q000633:toxicity; D010744:Phosphoric Monoester Hydrolases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000187:drug effects; D029701:Saccharomyces cerevisiae Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013696:Temperature; D013997:Time Factors; D020741:rho GTP-Binding Proteins / Q000378:metabolism*
D020662:Guanine Nucleotide Exchange Factors; D018129:Phosphatidylinositol Phosphates; C490929:ROM2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C055525:phosphatidylinositol 3-phosphate; C490702:PKC1 protein, S cerevisiae; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; C072724:SLT2 protein, S cerevisiae; C496521:Ymr1 protein, S cerevisiae; D010744:Phosphoric Monoester Hydrolases; C051302:RHO1 protein, S cerevisiae; D020741:rho GTP-Binding Proteins