Rhodamine-phalloidin (Rd-phalloidin) staining of actin in yeast was performed as described by Pringle et al. (1989) (link). Rd-phalloidin staining of actin filaments in vitro was performed according to Drubin et al. (1993) (link). For analyzing protein localization in cells emerging from stationary phase, 1-ml samples of yeast cells were removed at 1-h intervals following release from G0. Cells were processed for immunofluorescence essentially as described by Pringle et al. (1991) (link) and Ayscough and Drubin (1997) . The cold methanol/acetone step (Pringle et al., 1991 (link)) was required to observe actin, Aip3p/ Bud6p, Abp1p, Arp2p, Cdc10p, Cdc11p, cofilin, calmodulin, Gin4p, Myo2p, Sla2p, and Spa2. To observe Sla1p, cells were treated as in Pringle (1991) except that they were fixed in formaldehyde for 10 min rather than 60 min. To visualize Cdc42p, Bem1p, Sec4p, Sec8p, and Smy1p, we followed the protocol described by Ziman et al. (1993) (link) in which the methanol/acetone step was replaced with an incubation of the cells in 0.5% SDS for 5 min. Table II lists the primary antibodies used in this study and the dilutions used. Secondary antibodies used were fluorescein-isothiocyanate (FITC) conjugated goat anti–mouse and FITC goat anti–rabbit (Cappell/ Organon Technika, Malvern, PA) at a dilution of 1:1,000 and CY3-conjugated sheep anti–rabbit (Sigma Chem. Co.) at a dilution of 1:200. Cells were viewed with a Zeiss Axioskop fluorescence microscope with a 100 W mercury lamp and a Zeiss 100X Plan-NeoFluar oil immersion objective. Images were recorded using T-MAX 400 film (Kodak). In addition, images were captured electronically using a 200-E CCD camera (Sony Electronics Inc. San José, CA) and displayed on a Micron 133 computer (Micron Electronics Inc., Nampa, ID) using Northern Exposure software (Phase 3 Imaging Systems, Milford, MA). All images show cells after 4 h release from the G0 arrest.