Mechanisms of Inn1 bud neck localization. (A and B) Strains LY1321 (INN1-GFP cyk3Δ; A) and LY1325 (INN1-GFP cyk3Δ hof1Δ [pRS316-HOF1]; B) were transformed with plasmid YCp111-CDC3-CFP, and the LY1325 transformants were incubated on an FOA plate to eliminate the HOF1 plasmid. Cells were examined as described in Fig. 4 F. 1DC, one central dot; 1DA, one asymmetric dot (Table I and Video 7). (C) Localization of Inn1 lacking its Hof1- and Cyk3-binding sites. Strain LY1310 (inn1Δ [pUG36-INN1]) was transformed with HIS3-marked plasmids carrying RFP-tagged wild-type (WT) or mutant INN1 alleles. After growth on an SC-His+FOA plate at 25°C to eliminate pUG36-INN1, DIC and fluorescence images were captured. (D) Loss of Inn1 localization in latA-treated hof1Δ cells. Wild-type (LY1324), cyk3Δ (LY1321), and hof1Δ (LY1328 after eliminating plasmid pRS316-HOF1 by growth on an FOA plate) strains were grown to exponential phase in YM-P medium at 25°C. Portions of each culture were treated with latA for 20 min, and cells were imaged by DIC and fluorescence microscopy. Images of representative latA-treated cells (left) and percentages of large-budded cells with localized Inn1-GFP (right) are shown. The experiments were performed twice with similar results. Bars, 2 µm.