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

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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

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

View larger version (40K): [in a new window]   Fig. 1. PtdIns(3)P accumulation is lethal in PI 3-phosphatase-deficient cells. (A) Deletion of the Vps34p PI 3-kinase complex component VPS30 rescues the lethal phenotype of ymr1 sjl2 sjl3 cells. ymr1 sjl2ts sjl3 vps30 cells were transformed with either pRS415-VPS30 or an empty vector. Transformants were then streaked to 5-FOA plates lacking the amino acid leucine, and growth was scored after 5 days at 26°C. (B) Quantitative comparison of in vivo PtdIns(3)P levels in wild-type, ymr1 sjl2ts sjl3 triple-mutant, ymr1 sjl2ts sjl3 vps30 quadruple-mutant, and vps30 cells at 38°C. Levels of deacylated product corresponding to PtdIns(3)P are shown. Data represent the mean±s.d. of three independent experiments.

View larger version (52K): [in a new window]   Fig. 2. Depletion of GAL1-driven Ymr1-13xmyc in ymr1 sjl2 sjl3 cells on dextrose leads to inviability. (A) GAL1-YMR1-13xmyc sjl2 sjl3 cells were transformed either with pRS416-YMR1 or an empty vector, and transformants were grown overnight in selective media containing either galactose to stimulate Ymr1-myc expression, or dextrose to suppress expression. Aliquots of these cultures were subsequently streaked to selective plates containing the same sugar, and growth was scored after 3 days at 26°C. (B) Growth on dextrose causes depletion of GAL1-driven Ymr1-myc. GAL1-YMR1-13xmyc sjl2 sjl3 cells were maintained in log phase in YP dextrose media, and 1 OD600 equivalent of cells was harvested at each indicated time point into ice-cold TCA. Samples were processed as described in Materials and Methods. Results are representative of three independent experiments. The estimated half-life of Ymr1-myc was approximately 4 hours, which roughly corresponded to the doubling time of the cultures when grown under these conditions. (C) Depletion of Ymr1-myc in GAL1-YMR1-13xmyc sjl2 sjl3 cells on dextrose specifically affects cellular PtdIns(3)P levels. Cells were shifted into YP dextrose medium at the indicated time prior to labeling and were maintained in early log phase. Cells measuring 5OD600 units were harvested per time point, and labeling was carried out as in Fig. 1 except that there was no pre-incubation at 38°C. Results are representative of two independent experiments conducted in duplicate.

View larger version (93K): [in a new window]   Fig. 3. Gene-dosage-dependent suppressors of YMR1-13xmyc depletion. (A) GAL1-YMR1-13xmyc sjl2 sjl3 cells were grown overnight in YP dextrose medium to initiate depletion of Ymr1-myc and transformed with yEP352 high-copy-number S. cerevisiae genomic library plasmids (see Materials and Methods) and subsequently plated to selective media in the presence of dextrose. Colonies were picked 4 days later and re-streaked to selective media containing dextrose. Plates were incubated for 4 days at 26°C. A representative transformant for each unique SYD plasmid is shown. Isolates that also rescued ymr1ts sjl2 sjl3 cells at 35°C are underlined and set in bold print (also refer to Table 2). (B) Osmotic support rescues the lethal phenotype of ymr1 sjl2ts sjl3 cells at 38°C (upper panels). ymr1 sjl2ts sjl3 cells were transformed with either pRS415-YMR1 or an empty vector. Transformants were grown under selection to log phase and tenfold serial dilutions were plated to selective plates with or without 1M sorbitol and the plates were incubated for 3 days at the indicated temperature. Osmotic support rescues the lethal phenotype of sjl2 sjl3 depleted of Ymr1 (lower panels). ymr1 sjl2 sjl3 cells carrying pGAL1-YMR1-myc were grown on galactose and then changed to media containing glucose to deplete Ymr1. Tenfold serial dilutions were plated to selective plates (+dextrose) with or without 1M sorbitol and the plates were incubated for 3 days.

View larger version (35K): [in a new window]   Fig. 4. Proper PtdIns(3)P metabolism is important for appropriate regulation of Rho1p/Pkc1p-mediated cell-integrity pathway signaling. (A) Schematic representation of the Rho1p/Pkc1p-mediated cell-integrity pathway. Figure is adapted from Audhya and Emr (Audhya and Emr, 2002), and shows the level at which SYDs are expected to act on the cell-integrity pathway based on their known cellular functions (also refer to Table 2). (B) Slt2p activity is severely mis-regulated in ymr1ts sjl2 sjl3 cells. Wild-type and ymr1ts sjl2 sjl3 cells were grown to log phase at 26°C in YP dextrose media and subsequently shifted to 38°C. Cells were harvested at 1 OD600 unit for each time point into TCA at a final concentration of 10%, and samples were processed for western blot analysis as described in Materials and Methods. The equivalent of 0.2 OD600 units was loaded per lane. Note that results represent a 2-minute exposure using a super signal ECL detection system (Pierce) for the wild-type cells. Phospho-Slt2p signals are significantly stronger in ymr1ts sjl2 sjl3 samples, and represent a 10-second exposure under the same incubation conditions as the wild-type sample. Accordingly, Slt2p is constitutively activated in the basal state, and further hyperactivated upon heat stress in ymr1ts sjl2 sjl3 cells compared with wild-type cells. Blots were stripped and re-probed with antisera directed against the soluble glucose-6-phosphate dehydrogenase (G6PDH) enzyme as a control for protein loading. (C) vps34 cells are defective for regulation of heat-shock-induced cell-integrity pathway signaling. Wild-type and vps34 samples were resolved together by SDS-PAGE, transferred to the same nitrocellulose filter, which was probed as in Panel B. As an additional control, there was no detectable difference in the stability of 3xHA-tagged Slt2p between the wild-type and either ymr1ts sjl2 sjl3 or vps34 cells (our unpublished observations). (D) Slt2p is required to promote viability of ymr1 sjl2ts sjl3 cells. Cells were grown to log phase in YP dextrose media and tenfold serial dilutions were plated as in Fig. 1. Plates were incubated at the indicated temperature and growth was scored after 3 days. Synergism with slt2 implicates the Rho1p/Pkc1p signaling interface in PtdIns(3)P accumulation-mediated toxicity.

View larger version (40K): [in a new window]   Fig. 5. Pkc1p fragments that contain the HR1 domains but lack the protein kinase domain interfere with Rho1p/Pkc1p signal propagation. (A) Overexpression of the Pkc1-T615 fragment quenches hyperactivation of Slt2p in ymr1ts sjl2 sjl3 cells. ymr1ts sjl2 sjl3 cells were transformed with either an empty vector, or the SYD22 Pkc1-T615-containing plasmid, and samples were prepared as in Fig. 4. Samples were resolved together by SDS-PAGE, transferred to the same nitrocellulose filter, which was probed as in Fig. 4B. Results are representative of two independent transformants. (B) Overexpression of the Rho1p-interacting HR1 domains of Pkc1p is sufficient to promote viability of ymr1ts sjl2 sjl3 cells at restrictive temperature. The sequences corresponding to the domain architecture of Pkc1p as predicted by the SMART database is shown for each Pkc1p construct. ymr1ts sjl2 sjl3 cells were transformed with the indicated high-copy-number plasmid and transformants were grown to log phase under the appropriate selection. Tenfold serial dilutions were then spotted to selective media at the indicated temperature and growth was scored after 3 days. Note that overexpression of the full-length PKC1 is growth inhibitory in these cells.

View larger version (26K): [in a new window]   Fig. 6. Rom2p is required for the lethal effects of PtdIns(3)P accumulation in ymr1 sjl2 sjl3 cells. (A) ymr1 sjl2ts sjl3 rom2 cells were transformed with a pRS415-derived plasmid carrying either wild-type ROM2, YMR1 or no insert. Transformants were then streaked to 5-FOA plates lacking the amino acid leucine to select for maintenance of the pRS415 vector. Growth was scored after 5 days at 26°C. Results are representative of two independent transformants. (B) Cellular PtdIns(3)P levels are not significantly reduced in ymr1 sjl2ts sjl3 rom2 cells. Cells were grown to early log phase under appropriate selection, then pre-incubated at either 26°C or 38°C for 20 minutes, and deacylated [3H]-glycero-phosphoinositols were analyzed by HPLC as in Fig. 1. Data represent the mean±s.d. of two experiments performed in duplicate. (C) Deletion of ROM2 attenuates hyperactivation of the Rho1p/Pkc1p-mediated Slt2p MAPK pathway. Samples were resolved together by SDS-PAGE and the nitrocellulose filter was probed as in Fig. 4. Results represent a 20-second exposure under the same conditions described for Fig. 4A. Note that the basal level of active Slt2p in ymr1 sjl2ts sjl3 cells is greater than the maximal heat-stress-induced signal in wild-type cells, indicating constitutive Rho1p/Pkc1p signaling in PI 3-phosphatase-deficient cells.

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