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- Table 1.
Plasmids
pUC19 Yanisch-Perron et al., 1985. pUG6 Used as a source for the G418 resistance marker located between direct repeats of the loxP site ( Güldener et al., 1996). pFA6-HIS3MX6 Used as a source for the heterologous Sphis5+ marker ( Longtine et al., 1998). pHPS100 Vector carrying both the information for a lexA-Rlmlp_N fusion protein and the corresponding lacZ reporter gene ( Kirchrath et al., 2000). pJJH447 The GAL1/10 promoter was amplified by PCR using the oligonucleotide pair GAL 1atg and GAL 10atg ( Table 2) from genomic DNA prepared from strain VW1A ( Lorberg et al., 1999) and subcloned as an EcoRI/BamHI fragment into the polylinker site of YEp352 ( Hill et al., 1986). pUC19RHO5her RHO5 was amplified by PCR from genomic S. cerevisiae DNA using RHO5-3neu and RHO5-5neu as primers and digested with BglII and BclI. The resulting 2.0 kb fragment was ligated into pUC19 digested with BamHI. Transcription of RHO5 proceeds in opposite direction to that of lacZ. pUC19RHO5hin The same PCR generated fragment as in pUC19RHO5her was inserted in the opposite orientation into pUC19. pSH4 RHO5 was amplified by PCR from pUC19RHO5hin using RHO5Bam and RHO5-3′Sph as primers and then digested with BamHI and SphI. The resulting 1.2 kb fragment was ligated into pJJH447 digested with BamHI and SphI. pHPS116 The 2.1 kb product of the PCR mutagenesis generating the activated RHO5 allele (see `In vitro mutagenesis' in Materials and Methods) was digested with SphI and ligated into pUC19 digested with the same enzyme. Transcription of the modified RHO5 gene proceeds in the same direction as that of the lacZ gene. pSH8 Vector carrying the activated RHO5 allele under the control of the GAL1/10 promoter. Obtained from an in vivo recombination of pSH4 (6.8 kb PstI/Asp7181 fragment) and pHPS116 (2.1 kb SphI fragment). - Table 2.
Oligonucleotides used in this study
delRHO5-5 5′-CGC CGA AAT ATT TAT TAA ATA CAT ATA GTA TAC TAA TAA GTC TGC TTC GTA CGC TGC AGG TCG AC-3′ delRHO5-3 5′-CGC CGA AAT ATT TAT TAA ATA CAT ATA GTA TAC TAA TAA GTC TGC TTC GTA CGC TGC AGG TCG AC-3′ RHO5-5neu 5′-CCA CAT CGG TAA GTG ATG ACC-3′ RHO5-3neu 5′-CCA TTA CTA TGG TTC AAC CTG G-3′ RHO5Q91Hneu 5′-CAT AAC GGT CTT AAA CGA TCG TAA TCT TCG TGT CCT GCG GTG TCC CAT AAA TTG-3′ RHO5Bam 5′-GGA TGG ATC CAT GAG GTC TAT TAA ATG TGT G-3′ RHO5-3¢Sph 5′-GGA TGC ATG CTT GGA AAA GAA AGT CGC GG-3′ delBEM2-5 5′-GGA TTG TAT ACA TTT ACC ACG AAA ATT GTT TAT TGC TTG AAA TAATCC ATA GGC CAC TAG TGG ATC TG-3′ delBEM2-3 5′-GGA CAA TTC ATC TCT TTT CCT GCG GTT ATG AGC GAA ACC AAC GTT GCT TCG TAC GCT GCA GGT CGA C-3′ GAL1atg 5′-GGC GAA TTC TAT AGT TTT TTC TCC TTG ACG-3′ GAL10atg 5′-CTC GGA TCC TTA TAT TGA ATT TTC-3′ M13 reverse 5′-CAG GAA ACA GCT ATG ACC ATG-3′ Restriction sites used for subcloning are underlined.
- Table 3.
Yeast strains
DHD5 MATa/α ura3-52/ura3-52 leu2-3,112/leu2-3,112 his3-11,15/his3-11,15 MAL/MAL SUC/SUC GAL/GAL Arvanitidis and Heinisch, 1994 HD261-1A MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP MAL GAL SUC This study HSH1-1A MATa ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP slt2::LEU2 MAL GAL SUC This study HD252 MATa/α ura3-52/ura3-52 his3-11,15/his3-11,15 leu2-3,112/leu2-3,112 trp1::loxP/trp1::loxP rho5::loxP-KanMX-loxP/RHO5 MAL/MAL GAL/GAL SUC/SUC This study HSH1-1C MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP MAL GAL SUC This study HSH1-4A MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP MAL GAL SUC This study HSH1-1A MATa ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP slt2::LEU2 MAL GAL SUC This study HSH1-3A MATa ura3-52 his3-11,15 leu2-3,112 trp1::loxP slt2::LEU2 MAL GAL SUC This study HSH1-2B MATa ura3-52 his3-11,15 leu2-3,112 trp1::loxP MAL GAL SUC This study HSH1-7B MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP slt2::LEU2 MAL GAL SUC This study HSH1-5A MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP MAL GAL SUC This study HSH1-2A MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP slt2::LEU2 MAL GAL SUC This study MALY5-3B MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP slt2::LEU2 MAL GAL SUC Lorberg et al., 2001 HSH2-2B MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP bem2::Sphis5+ MAL GAL SUC This study HSH2-1B MATα ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP MAL GAL SUC This study HSH2-5A MATa ura3-52 his3-11,15 leu2-3,112 trp1::loxP MAL GAL SUC This study HSH2-8B MATa ura3-52 his3-11,15 leu2-3,112 trp1::loxP rho5::loxP-KanMX-loxP bem2::Sphis5+ MAL GAL SUC This study PA109-1C MATa ura3 his3 leu2 trp1 rme1 GAL bck1::HIS3MX6 Delley and Hall, 1999 HPY1-2A MATa ura3 his3 leu2 trp1 GAL bck1::HIS3MX6 This study HPY1-2B MATα ura3 his3 leu2 trp1 GAL This study HPY1-2C MATa ura3 his3 leu2 trp1 GAL bck1::HIS3MX6 rho5::loxP-KanMX-loxP This study HPY1-2D MATα ura3 his3 leu2 trp1 GAL rho5::loxP-KanMX-loxP This study - Table 4.
Indirect determination of Slt2p MAP-kinase activity in a rho5 deletion background
Growth temperature Wildtype Δrho5 25°C 0.7±0.3 2.3±1.2 37°C 8.4±5.3 50.5±22.7 The activity of the Slt2p MAP-kinase cascade was determined using the plasmid-based reporter system described previously ( Kirchrath et al., 2000); for details, see Materials and Methods. The mean and standard deviation values are shown for four different transformants.
- Fig. 1.
A rho5 deletion shows increased resistance against caffeine. Serial dilutions of strains HSH1-2B (wt) and HD261-1A (Δrho5) were spotted onto the media indicated and incubated at 30°C for 2 days.
- Fig. 2.
Phenotypes of RHO5Q91H overexpression. Cells were grown on glucose-containing medium and streaked onto plates containing 2% galactose to induce transcription of the mutated protein. Plates were incubated at the temperatures indicated for 3-4 days. Strain DHD5 was used as a host in this experiment. The construction of the mutant is described in Materials and Methods. pGALrho5*, strain carrying the activated RHO5Q91H allele; wt, wildtype.
- Fig. 3.
Phenotypes of bck1/rho5- and slt2/rho5-double mutants. (A) Strains HPY1-2A (Δbck1), HPY1-2D (Δrho5), HPY1-2C (Δbck1, Δrho5) and HPY1-2B (wt) were streaked out on rich medium and incubated at the temperatures shown and with the drugs indicated for 3-4 days. (B) Serial dilutions (103, 102, 10, from left to right) of strains HSH1-2B (wt), HSH1-4A (Δrho5), HSH1-7B (Δslt2) and HSH1-1A (Δslt2, Δrho5) were spotted onto the media indicated and incubated at the given temperatures for 2 days.
- Fig. 4.
Slt2p phosphorylation in wild-type and Δrho5 cells under inducing conditions at 37°C. 50 μg of total protein was loaded in each lane and immunological detection was performed as described previously ( Lorberg et al., 2001). Phosphospecific antibodies were used to detect the dually phosphorylated kinase in the upper lane. In the lower lane a polyclonal antiserum was employed as a loading control.
- Fig. 5.
Phenotypes of a bem2/rho5 double mutant. Serial dilutions (103, 102, 10, from left to right) of strains HSH2-1B (Δrho5), HSH2-5A (wt), HSH2-8B (Δbem2,Δ rho5) and HSH2-2B (Δbem2) were incubated on full medium at the temperature and with the drugs indicated for 2 days.
- Fig. 6.
Influence of Rho5p on actin dynamics. (A) Effect of a rho5 deletion on the distribution of actin upon heat shock. Cells were investigated at the times indicated (for details, see Materials and Methods). Strains employed were HSH1-1C and HSH1-4A. (B) Effect of RHO5Q91H overexpression on actin kinetics. Strain DHD5 was transformed with pJJH447 (vector not carrying any RHO5 sequences) or pSH8 (pGALrho5*; vector carrying the activated RHO5Q91H allele). Yeast cells were grown in glucose-containing minimal medium overnight. Then cells were inoculated into 50 ml of fresh minimal medium containing the carbon source indicated and grown for about 4 hours to an optical density of between 0.3 and 0.6 at 30°C. To observe actin dynamics under heat shock the cultures were shifted to 37°C, and actin distribution after heat shock was investigated at the times indicated using the staining procedure described in Materials and Methods.
- Fig. 7.
Model for Rho5p action. The possible action of Rho5p in the cellular integrity pathway consistent with the results reported in this work is indicated. For details, see Discussion.
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