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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 ymr1^ts sjl2 sjl3 cells. ymr1^ts 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 ymr1^ts 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. ymr1^ts 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.

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