FIG 3.

Bem4p interacts with Sho1p and is an effector of Sho1p function. (A) P∼Kss1p levels in wild-type cells and the bem4Δ mutant containing the indicated alleles. The total levels of Kss1p and protein (assessed by Pgk1p) were similar between samples. (B) Wild-type cells and the ste20Δ mutant containing an empty plasmid (YEp351) or high-copy-number Bem4p-GFP were grown to mid-log phase in YEP-GAL and evaluated by immunoblot analysis. (C) High-copy-number BEM4 stimulates filamentous growth under nutrient-limiting conditions. Cells containing a control plasmid (YEp351) or high-copy-number Bem4p-GFP were grown to mid-log phase in YEPD (GLU) or YEP-Gal (GAL) medium and examined by microscopy at a magnification of ×100 (bar, 25 μm). (D) Bem4p-HA and Sho1p-GFP associate, as determined by co-IP analysis. Band intensity relative to input levels after background subtraction was determined by ImageJ. Input levels are 10% for WCE. (E) Dependency of Cdc24p, Cdc42p, Bem4p, and Ste12p on the hyperpolarized growth phenotype of Sho1p. Cells were grown for 24 h on YEP-Gal medium. Bar, 25 μm. (F) Quantitation of cell length for the strains used for panel E. More than 100 cells were examined in separate trials for each experiment. With the exception of GAL-SHO1 and GAL-SHO1 ste12 strains, which show a diversity of cell lengths, <10% deviation is seen between trials. (G) Role of Sho1p in regulating the association of Bem4p with the pellet fractions. The experiment was performed in triplicate; the asterisk denotes a P value of <0.05.