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Rho5p downregulates the yeast cell integrity pathway

Hans-Peter Schmitz, Stefanie Huppert, Anja Lorberg and Jürgen J. Heinisch^*,

Institut für Mikrobiologie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, Geb. 26. 12, D-40225 Düsseldorf, Germany
^* Present address: Universität Hohenheim, Institut für Lebensmitteltechnologie, Fachgebiet Gärungstechnologie (150f), Garbenstr. 25, D-70599 Stuttgart, Germany

View larger version (14K): [in a new window]   Fig. 1. A rho5 deletion shows increased resistance against caffeine. Serial dilutions of strains HSH1-2B (wt) and HD261-1A (rho5) were spotted onto the media indicated and incubated at 30°C for 2 days.

View larger version (58K): [in a new window]   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.

View larger version (53K): [in a new window]   Fig. 3. Phenotypes of bck1/rho5- and slt2/rho5-double mutants. (A) Strains HPY1-2A (bck1), HPY1-2D (rho5), HPY1-2C (bck1, rho5) 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 (rho5), HSH1-7B (slt2) and HSH1-1A (slt2, rho5) were spotted onto the media indicated and incubated at the given temperatures for 2 days.

View larger version (55K): [in a new window]   Fig. 4. Slt2p phosphorylation in wild-type and rho5 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.

View larger version (61K): [in a new window]   Fig. 5. Phenotypes of a bem2/rho5 double mutant. Serial dilutions (103, 102, 10, from left to right) of strains HSH2-1B (rho5), HSH2-5A (wt), HSH2-8B (bem2, rho5) and HSH2-2B (bem2) were incubated on full medium at the temperature and with the drugs indicated for 2 days.

View larger version (71K): [in a new window]   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.

View larger version (24K): [in a new window]   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.