Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking

Rho GTPases, which control polarized cell growth through cytoskeletal reorg anization, have recently been implicated in the control of endo- and exocyt osis. We now report that both Rho1p and Cdc42p have a direct role in mediat ing the docking stage of homotypic vacuole fusion. Vacuoles prepared from s trains with temperature-sensitive alleles of either Rho1p or Cdc42p are the rmolabile for fusion. RhoGDI (Rdi1p), which extracts Rho1p and Cdc42p from the vacuole membrane, blocks vacuole fusion. The Rho GTPases can not fulfil l their function as long as priming and Ypt7p-dependent tethering are inhib ited. However, reactions that are reversibly blocked after docking by the c alcium chelator BAPTA have passed the point of sensitivity to Rdi1p. Extrac tion and removal of Ypt7p, Rho1p and Cdc42p from docked vacuoles (by Gdi1p, Gyp7p and Rdi1p) does not impede subsequent membrane fusion, which is stil l sensitive to GTP gammaS. Thus, multiple GTPases act in a defined sequence to regulate the docking steps of vacuole fusion.