3 C) by high levels of either the WCA domain of Las17p or by calmodulin, which are known to interact directly with Arp2/3 complex. traffic (Jahn and Sudhof, 1999). The membrane fusion stage of trafficking is unexpectedly complex. It requires the coordinated action of chaperones (such as NSF and -SNAP), SNAREs, GTPases (Rab and Rho families), lipids (steroid and phosphoinositides), and regulated calcium fluxes. Actin has a central role in several trafficking events (Qualmann et Dutogliptin al., 2000; Foti et al., 2001; Goode and Rodal, 2001; Lechler et al., 2001). Actin filaments are required for the transport and spatial targeting of secretory proteins (Pruyne et al., 1998; Guo et al., 2001), organelle inheritance during cell division (Catlett and Weisman, 2000), and the maintenance of Golgi structure (Mullholland et Dutogliptin al., 1997; Valderrama et al., 1998). In different systems, membrane fusion has been shown to be promoted by stabilization of F-actin (Koffer et al., 1990; Jahraus et al., 2001), F-actin disassembly (Vitale et al., 1991; Muallem et al., 1995), or actin remodeling, i.e., disassembly plus reassembly (Bernstein et al., 1998; Lang et al., 2000). Actin remodeling also accompanies synaptic stimulation (Colicos et al., 2001). Actin ligands can modulate phagosomeCendosome fusion in vitro (Jahraus et al., 2001), and actin binds to purified endosomal and lysosomal vesicles, an association which can nucleate actin polymerization (Mehrabian et al., 1984; Taunton, 2001). Actin and myosin V are involved directly in vacuole movement into the bud during cell division (Hill et al., 1996; Catlett et al., 2000). Rho-GTPases regulate actin rearrangement (Hall, 1998) by signaling to multiple downstream effector complexes such as the Wiskott-Aldrich syndrome protein (WASp)* and the Arp2/3 complex (Higgs and Pollard, 1999). In each of these studies, it was assumed that the relevant actin molecules are cytosolic or cytoskeletal, though none of the data precludes a role for organelle-bound actin. We study membrane fusion with vacuoles from (Wickner and Haas, 2000). Purified yeast vacuoles undergo homotypic fusion in simple buffers containing ATP. All of the proteins and lipids needed for fusion are bound to the vacuole membrane. The reaction occurs in three stages termed priming, docking, and fusion. Priming, initiated by the ATPase Sec18p, releases Sec17p (Mayer et Dutogliptin al., 1996) and disassembles a cis complex of SNAREs (Ungermann et al., 1998a). Priming liberates the HOPS complex (for homotypic fusion and vacuole Rabbit Polyclonal to DGKB protein sorting)/VPS class C complex (Sato et al., 2000; Seals et al., 2000), which then associates with GTP-bound Ypt7p to initiate docking (Price et al., 2000). Completion of docking requires SNAREs (Ungermann et al., 1998b), the vacuole membrane potential (Ungermann et al., 1999), phosphoinositides (Mayer et al., 2000), and the Rho-GTPases Cdc42p and Rho1p (Eitzen et al., 2001; Mller et al., 2001). Docking culminates in a transient release of vacuole lumenal calcium (Peters and Mayer, 1998). Calcium activates calmodulin, which binds to the V0 domain of the vacuolar ATPase, triggering the formation of trans-pairs of V0 plus the t-SNARE Vam3p, leading to organelle fusion (Peters et al., 2001). Two Rho-GTPases which are required for vacuole fusion, Cdc42p and Rho1p (Eitzen et al., 2001; Mller et al., 2001), can regulate actin structure (Pringle et al., 1995; Helliwell et al., 1998) through a well-studied cascade which includes Las17p/Bee1p (yeast WASp) and the Arp2/3 complex (Fig. 1) . A recent screen of a library of yeast strains with defined gene deletions (Seeley et al., 2002) suggested that this cascade of actin regulatory genes is needed to maintain normal vacuole structure. We now report that the proteins of.