Fig. 4. Characterization of For3. (A) The N-terminus of For3 binds directly to constitutively active Rho3. L40 cells expressing the indicated proteins were cultured on an SD plate with (+) or without (-) histidine at 30°C for 3 days. `Control' means empty vector. (B,C) Localization of HA-For3. for3 null cells containing pREP41HA-for3 were grown in EMM without thiamine at 30°C for 18 hours. Before (-) and after (+) treatment with 10 µM Lat-A or 100 µM MBC for 10 minutes, the cells were fixed and stained with Bodipy-phallacidin or TAT-1 (green in merged images), anti-HA antibody Y-11 (red in merged images), and DAPI (blue in merged images). Deconvoluted 3D images are shown. Arrowheads in B indicate colocalization of HA-For3 with F-actin. No HA-For3 signal was detected in the cells grown with thiamine (data not shown). (D) Localization of HA-For3 in rho3 cdc42 double-deficient cells. rho3 null cells containing pREP41HA-for3 and pREP1cdc42 or pREP1cdc42T17N were grown in EMM without thiamine at 25°C for 20 hours and were fixed and stained with Bodipy-phallacidin (green in merged images), Y-11 (red in merged images), and DAPI (blue in merged images). Deconvoluted 3D images are shown. (E) Summary of the functional analysis of For3 and truncated proteins. (F) Localization of For3 and truncated proteins using their YFP fusion proteins. Wild-type cells containing pREP1YFP-for3, REP1YFP-for3NM, pREP1YFP-for3N2, or pREP1YFP-for3MC were grown in EMM containing thiamine until early log phase at 25°C. Arrowheads and arrows indicate the signal of YFP at the cell ends and the division site, respectively. Bars, 10 µm.

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