Rho1p and Cdc42p act after Ypt7p to regulate vacuole docking

Abstract

Rho GTPases, which control polarized cell growth through cytoskeletal reorganization, have recently been implicated in the control of endo‐ and exocytosis. We now report that both Rho1p and Cdc42p have a direct role in mediating the docking stage of homotypic vacuole fusion. Vacuoles prepared from strains with temperature‐sensitive alleles of either Rho1p or Cdc42p are thermolabile for fusion. RhoGDI (Rdi1p), which extracts Rho1p and Cdc42p from the vacuole membrane, blocks vacuole fusion. The Rho GTPases can not fulfill their function as long as priming and Ypt7p‐dependent tethering are inhibited. However, reactions that are reversibly blocked after docking by the calcium chelator BAPTA have passed the point of sensitivity to Rdi1p. Extraction and removal of Ypt7p, Rho1p and Cdc42p from docked vacuoles (by Gdi1p, Gyp7p and Rdi1p) does not impede subsequent membrane fusion, which is still sensitive to GTPγS. Thus, multiple GTPases act in a defined sequence to regulate the docking steps of vacuole fusion.

Membrane Organization and Dynamics in Cell Polarity

“…In the fungus Cryptococcus neoformans, Cdc42 is mislocalized to the cytoplasm when the Rho GDI homolog is overexpressed (Price et al 2008). This is consistent with published data that recombinant Rdi1 or overexpressed Rdi1 in yeast can extract Rho proteins from the plasma membrane or vacuole membrane (Eitzen et al 2001;Tcheperegine et al 2005;Tiedje et al 2008). Therefore, recycling through GDIs may, like endocytosis, play an important role in the establishment and maintenance of polarity.…”

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

“…S2). As an additional test of the requirement for Ypt7p for fusion, an anti-Ypt7p antibody (42) reduces fusion nearly to background levels (Fig. 1B).…”

Enhanced Membrane Fusion in Sterol-enriched Vacuoles Bypasses the Vrp1p Requirement

“…To learn more about the actin-regulated steps of vacuole fusion, we devised a multicopy suppressor screen using mutant strains within the pathway from Rho proteins to actin (Eitzen, 2003). We would have preferred to perform this screen with the Rho mutants cdc42-1 and rho1-104, which are defective for vacuole fusion (Eitzen et al, 2001;Mü ller et al, 2001). However, these strains were prone to genetic reversion, likely due to the presence of semi-functional Rho proteins.…”

“…Vacuoles were then diluted 10-fold in PS buffer, reisolated by centrifugation at 20,000 ϫ g for 5 min at 4°C, and resuspended in 30 l FRB-1ϫ ATP with 0.5 mg/ml cytosol and subjected to standard fusion reaction and assay conditions. Stimulating factors (pure components ϭ IB2, Sec18p, and calmodulin) and inhibiting factors were added as indicated and prepared as previously described (Eitzen et al, 2001. Cytosol was prepared from strains K91, BY4742, and KTY10 grown to OD 600 of 4, lysed by vortexing in ice-cold FRB, 2 mM ATP, 1 mM DTT, and 1 volume glass beads for eight 1-min bursts followed by centrifugation at 20,000 ϫ g, at 4°C for 20 min.…”