QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 1 September 2005
doi: 10.1242/jcs.02535

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Related articles in JCS Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mizunuma, M. Articles by Miyakawa, T. Search for Related Content PubMed PubMed Citation Articles by Mizunuma, M. Articles by Miyakawa, T. Social Bookmarking                         What's this?

Implication of Pkc1p protein kinase C in sustaining Cln2p level and polarized bud growth in response to calcium signaling in Saccharomyces cerevisiae

Department of Molecular Biotechnology, Graduate School of Advanced Sciences of Matter, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739-8530, Japan

View larger version (50K): [in a new window]   Fig. 1. The scz6 mutation, a mutant allele of the PKC1 gene, suppresses various phenotypes of the zds1 strain. (A) Effect of scz6/pkc1-834 and stt1-1, mutant alleles of PKC1, on the growth of the zds1 mutant strain on solid medium. Wild-type (DHT22-1b), zds1 (YAT1), zds1 scz6/pkc1-834 (YMM4020), scz6/pkc1-834 (YMM28), zds1 stt1-1 (YMM134), and stt1-1 (YMM114) cells were spotted on YPD plates and grown at 25°C (2 days), or at 33°C or 37°C with or without 300 mM CaCl2 (2 days). (B,C) Cell morphology (B) and flow cytometry analysis of PI-stained cells (C) of various strains after 6 hours incubation with 100 mM CaCl2 at 25°C (1C, haploid; 2C, diploid).

View larger version (50K): [in a new window]   Fig. 2. Pkc1p is not involved in the regulation of oscillation of SWE1 and CLN2 mRNAs by calcium during cell cycle. (A) SWE1 or CLN2 mRNA levels in synchronized cell cultures of various strains were determined by Northern blotting, using the SWE1, CLN2, and ACT1 probes. Cells of wild-type (YMM180), pkc1-834 (YMM179) and stt1-1 (YMM196) at early log-phase (OD600 of 0.20.3) were synchronized with -factor in G1, and resuspended in YPD or YPD plus 100 mM CaCl2. Samples were taken at an interval of 20 minutes after release from arrest. (B) Changes in the relative intensity of the SWE1, CLN2 and ACT1 mRNA. The amount of the SWE1 and CLN2 mRNA in A was normalized to a constant level of ACT1 mRNA. In each case, the maximum value for the first cell cycle was referred to as 1. Time 0 is the point of release from the G1-phase arrest.

View larger version (91K): [in a new window]   Fig. 3. Pkc1p is required for the maintenance of Cln2p. (A) Oscillation of Swe1 or Cln2 protein levels during cell-cycle progression in various strains that were synchronized with -factor was determined by western blot analysis. Cells in early log-phase (OD600 of 0.20.3) of wild-type (YMM180), pkc1-834 (YMM179), zds1 (YMM187), zds1 pkc1-834 (YMM178) and stt1-1 (YMM196) strains, with a chromosomally integrated construct encoding genomic copies of Swe1p-9xMyc and Cln2p-3xHA, were synchronized with -factor in G1, and resuspended in YPD or YPD plus 100 mM CaCl2 at 25°C. Samples were taken at of 20-minute intervals after release from arrest. Cdc28 protein was used as an internal loading control. (B) Stability of Cln2p. Wild-type (YMM180), pkc1-834 (YMM179) and stt1-1 (YMM196) cells were cultured at 37°C for 2 hours; and then samples were taken at the indicated times after the addition of 100 µg/ml cycloheximide, and subjected to western bolt analysis. The asterisk indicates a non-specific band. (C) DNA content for the same samples as in A. (D) Effect of over-expression of CLN2 on the bud growth of the zds1 pkc1-834 strain. A strain containing a chromosomally integrated construct for GAL-regulated Cln2p-3xHA (YMM170-1; cln2::GAL1–CLN2HA–LEU2) were grown in the medium containing 2% raffinose (GAL1 promoter off) or 2% galactose (GAL1 promoter on) for 8 hours at 25°C.

View larger version (39K): [in a new window]   Fig. 4. Pkc1p is required for the maintenance of Ca2+-induced polarized bud growth. The zds1 (YMM187) and zds1 pkc1-834 (YMM178) strains was grown in YPD medium to early-log phase at 25°C. The cells were synchronized in G1 with -factor and then released into YPD medium with or without 100 mM CaCl2 at 25°C. Samples were taken at 20-minute intervals after removal of -factor and fixed with formaldehyde. (A) F-actin staining with Rhodamine-phalloidin. (B) The cumulative percentage of cells that initiated bud formation (left) or the percentage of F-actin patches localized at the incipient bud site (right; actin patches localized at the second-cycle buds were excluded) is plotted for the same samples shown in A.

View larger version (24K): [in a new window]   Fig. 5. Mutation sites in Pkc1p. (A) The pkc1-834 mutant allele carries a single nucleotide change (A2501G) that causes replacement of asparagine 834 with aspartic acid. The stt1-1 mutant allele carries a single nucleotide change (C3304T) that causes replacement of proline 1102 with serine. (B) The amino acids P1102 and N834 of Pkc1p are important for its function. pkc1 or zds1 pkc1 cells with low-copy plasmids, carrying either PKC1 (YCp50-PKC1), stt1-1 (YCp50-PKC1P1102S) or pkc1-834 (YCp50-PKC1N834D), were spotted on YPD plate with or without 300 mM CaCl2 and grown at the indicated temperatures for 23 days.

View larger version (39K): [in a new window]   Fig. 6. Intragenic complementation between the pkc1-834 and stt1-1 mutations. (A) Diploid strains of ZDS1/ZDS1 (W303-1D), zds1 PKC1/zds1 PKC1 (YMM125), zds1 pkc1-834/zds1 pkc1-834 (YMM219), zds1 stt1-1/zds1 stt1-1 (YMM218), and zds1 pkc1-834/zds1 stt1-1 (YMM220) were spotted onto YPD plates with the indicated concentration of CaCl2 and incubated at the indicated temperatures. (B) Alkaline phosphatase colony assay of the various strains. Cells were spotted on YPD plates and cultured at 25°C for 2 days. The plates were further incubated overnight at 25°C or 35°C and alkaline phosphatase released from the cells as a result of cell lysis was detected using BCIP. The blue color (seen here as darker disks) indicates defective cell walls. The mpk1 (TNP46) strain was used as a positive control. (C) Mpk1 activation by heat-shock in various strains. Wild-type (DHT22-1b), pkc1-834 (YMM28) and stt1-1 (YMM114) cells were shifted from 25°C to 37°C, and incubated for 2 hours. The Mpk1p phosphorylation was monitored by western blotting. Immunoblot analysis was carried out using anti-phospho-p44/42 MAPK antibody (top panel) or anti-Mpk1p antibody to detect Mpk1p (bottom panel). (D) The temperature sensitivity of stt1-1 is partially suppressed by overexpression of MPK1. scz6 (YMM28) or stt1-1 (YMM114) cells with high-copy plasmids: control (YEp24), PKC1 (YEp24-PKC1) or MPK1 (YEp24-MPK1), were spotted on YPD plates and grown at the indicated temperatures for 2 days.

View larger version (25K): [in a new window]   Fig. 7. pkc1-834 and stt1-1 are not required for the regulation of Hsl1p abundance. Comparison of Hsl1p abundance in various strains. Wild-type (YMM53), pkc1-834 (YMM143), stt1-1 (YMM144) and mpk1 (YMM70) strains with a chromosomally integrated construct encoding genomic copies of 3xHA-Hsl1p were grown in YPD at 25°C until early log phase. CaCl2 was added to the cell cultures at the indicated time, to a final concentration of 100 mM, and samples were taken for western blot analysis.

View larger version (80K): [in a new window]   Fig. 8. Rho1p GTPase is involved in the Ca2+-induced polarized bud growth. Effect of several rho1 mutant alleles on the cell morphology of the zds1 mutant strain. Wild-type (YOC729), zds1 (YMM230), zds1 rho1-2 (YMM232), zds1 rho1-3 (YMM147), zds1 rho1-4 (YMM148) and zds1 rho1-5 (YMM149) cells were used. Cell morphology after 6 hours of incubation with 300 mM CaCl2 at 25°C was examined.

View larger version (25K): [in a new window]   Fig. 9. Summary and model of novel Pkc1p pathway for polarized bud growth in budding yeast. The arrows and the blunt-ended line indicate positive and negative controls, respectively.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter