Functional and physical interactions between Inn1 and Cyk3. (A) Suppression of inn1Δ growth defect by overexpression of Cyk3. Strain LY1310 (inn1Δ [pUG36-INN1]) was transformed with a vector control (Vect; YEplac181) or with LEU2-marked high copy plasmids carrying IQG1 (YEp181-IQG1), HOF1 (pTSV30A-HOF1), CYK3 (P1, pBK132; P2, pBK133), MLC1 (pBK65), or INN1 (pGP564-INN1). Transformants were streaked on SC-Leu and SC-Leu+FOA plates and incubated at 25°C for 3 d to ask whether any of the candidate plasmids could replace the URA3-marked pUG36-INN1. (B) Restoration of PS formation in inn1Δ cells by overexpression of Cyk3. Strain LY1310 (inn1Δ [pUG36-INN1]) was transformed with pRS425-CYK3, incubated on an SC-Leu+FOA plate at 24°C for 3 d to eliminate plasmid pUG36-INN1, grown to exponential phase in SC-Leu medium at 24°C, and examined by TEM. (C–E) Interaction of the SH3 domain of Cyk3 with the PIPPLP motif (amino acids 159–165) of Inn1 as determined by two-hybrid analysis (C and D) and in vitro protein-binding assays (E). Experiments were performed as described for Fig. 4 (C–E) using a Cyk3 SH3 domain fragment (amino acids 1–70) instead of Hof1. In C, the diagram shows the motifs of Cyk3 (SH3 and TGc [putative transglutaminase domain]). Asterisk indicates that Inn1(130–180) failed to interact with Cyk3-SH3 for unknown reasons. (F) Localization of Cyk3 in inn1Δ cells. Strain YEF5216 (inn1Δ) was transformed with plasmid pRS315GW-CYK3-2GFP, grown overnight on an SC-Leu plate at 25°C, and imaged by DIC and fluorescence microscopy.