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Fig. 2. The putative GTPase mutants of vps1 affect actin cytoskeleton. (A) Alignment of the GTPase domain and the GED domain from mammalian dynamin-1 with three yeast dynamin-related proteins. Numbers on the top indicate the amino acid positions of Vps1p. Underlined in the GTPase domain are the residues that were mutated. (B) Temperature sensitivities of the vps1 ${Delta}$ cells containing various GTPase mutants of vps1. Strains YMC457 (vector/vps1 ${Delta}$ ), YMC458 (Vps1^WT/vps1 ${Delta}$ ), YMC459 (vps1^K42E/vps1 ${Delta}$ ), YMC460 (vps1^S43N/vps1 ${Delta}$ ) and YMC461 (vps1^G315D/vps1 ${Delta}$ ) cultured at 24°C were first diluted to similar density from which further serial dilutions were made. We spotted 5 µl of each dilution onto the YEPD plates and incubated at indicated temperature for one day. (C) LAT-A sensitivity of the vps1 ${Delta}$ and GTPase mutants. Cells shown in (B) were cultured in YEPD at 30°C and subjected to LAT-A halo assay as described in Materials and Methods. The bar chart in the right shows the quantified result. (D) The uracil-uptake assays in the YMC458 (VPS1) ( ${bullet}$ ), YMC457 (vps1 ${Delta}$ ) ( ${blacksquare}$ ) and YMC459-461 (vps1^K42E, vps1^S43N and vps1^G315D) ( ${blacktriangleup}$ , ${blacktriangledown}$ , ${diamondsuit}$ ) at both 30°C (left) and 37°C (right).

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