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Fig. 3. The actin defects caused by over-expression of the GTPase domain mutants of vps1. (A) Over-expression of the GTPase domain mutants caused abnormal actin structures. Strains integrated with the following GAL1-driven vps1 constructs, YMC463 (Gal-Vps1^WT), YMC464 (Gal-vps1^K42E), YMC465 (Gal-vps1^S43N) and YMC466 (Gal-vps1^G315D), as well as YMC462 (vector), were cultured to mid-log phase in the presence of 2% raffinose (strain name in brackets, see Table 1). Galactose was added to 2% to induce the expression of the vps1 mutants for 4 hours at 30°C. Cells were then collected for actin staining (Bar, 5 µm). (B) Over-expression of the GTPase mutants caused lethality in wild-type cells. The strains as described in (A) were grown on glucose-containing plates and replica-plated onto galactose plates and incubated at 37°C for 1 day before being photographed. (C) Cells over-expressing the vps1 GTPase mutants are hypersensitive to LAT-A. The strains as described in (A) were plated on the galactose-containing medium at 30°C and the LAT-A with the indicated concentration was applied (left panel). A summary of the halo assay is shown in the graph at the right.

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