FIGURE 7:

Importance of the Hof1–Cyk3 interaction in the absence of Inn1, and roles of the Cyk3 SH3 domain. (A, B) Inability of Cyk3 overexpression to suppress inn1∆ in the absence of the Hof1–Cyk3 interaction or of a normal Cyk3 SH3 domain. (A) Lack of suppression of the inn1∆ growth defect. Strains LY1310 (inn1∆ [pUG36-INN1]; rows 1–4) and MWY1296 (inn1∆ hof1^WA [pUG36-INN1]; rows 5–7) were transformed with LEU2-marked, high-copy plasmids carrying no insert (pRS425, 2µ vector), CYK3 (pRS425-CYK3), or cyk3 mutants (pRS425GW-CYK3^PAPA and pRS425GW-CYK3^WA), or with pRS315GW-INN1 (CEN INN1). Transformants were grown, spotted, and examined as described in Figure 6A except that SC–Ura-Leu and SC–Leu + 5-FOA plates were used. (B) Lack of suppression of the inn1∆ defect in PS formation. Strains MWY938 (inn1∆ [pUG36-INN1] [pRS425-CYK3]) and MWY934 (inn1∆ [pUG36-INN1] [pRS425GW-CYK3^PAPA]) were grown on SC–Leu + 5-FOA plates at 24°C for 3 d to eliminate the URA3 INN1 plasmid and then grown to exponential phase at 24°C in SC–Leu medium and examined by electron microscopy. Cells shown are representative of more than 50 cells examined for MWY938 (see also Nishihama, Schreiter, Onishi, Vallen, et al., 2009) and of 50 cells examined for MWY934; of the latter, only two cells had recognizable PS-like structures. Scale bar, 0.5 µm. (C) Loss of Cyk3 localization in the absence of both Hof1–Cyk3 interaction and a functional Cyk3 SH3 domain. (Top panel) Merged GFP (green) and CFP (magenta) images are shown for strains MWY1436 (left; cyk3^WA-2GFP [YCp111-CDC3-CFP]) and MWY1438 (right; cyk3^WA,PAPA-2GFP [YCp111-CDC3-CFP]). Representative cells are shown; cell bodies are outlined. Scale bar, 2 µm. Bottom panel, protein levels of wild-type and mutant Cyk3. Strains MWY815 (CYK3-2GFP), MWY1414 (cyk3^WA-2GFP), MWY673 (cyk3^PAPA-2GFP), and MWY1418 (cyk3^WA,PAPA-2GFP) were grown to exponential phase in YM-P medium at 24°C, and protein extracts were analyzed by Western blotting (see Materials and Methods).