We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.

Kinesin-14s and microtubule dynamics define fission yeast mitotic and meiotic spindle assembly and elongation.

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Previous work has shown that purified double mutant A204C/C374A yeast actin is polymerization-deficient in vitro under physiological concentrations. To understand the importance of the 204 residue in subdomain 4, a series of actin proteins with a single mutation at this position were created with Cys-374 retained. Only yeast expressing A204G-, A204S-, or A204C-actin were viable. The A204G and A204S strains were sensitive to cold temperature and hyperosmolarity, whereas the A204C strain showed more profound effects on growth under these conditions. Cells expressing A204C-actin exhibited anomalies previously observed for A204C/C374A actin, including abnormal actin structures. A204G- and A204S-actin proteins had 12- and 13-fold increased critical concentrations, respectively, relative to wild-type. Only at very high concentrations could A204C actin polymerize when ATP was bound; when hydrolyzed, the ADP-containing A204C filaments depolymerized, demonstrating a profound difference in critical concentration between ATP and ADP states with A204C actin. A correlation between size of the residue substituted at position 204 and energy minimization of actin filament models was observed. We propose that the region surrounding residue 204 is involved in interactions that change depending on the phosphorylation state of the bound nucleotide that might reflect different conformations of F-actin subunits.