Figure 3.
The PH domain and phosphoinositide binding activity are necessary for plasma membrane association of Slm1 and Slm2. (A) Subcellular localization of HA-tagged wild-type and mutant variants of Slm1 and Slm2 expressed under the control of the GAL1 promoter and visualized by indirect immunofluorescence using antibodies against the HA-tag followed by Cy2-conjugated secondary antibodies. Shown are single z-sections of W303a cells expressing wild-type HA-tagged Slm1 and Slm2 (Slm1 and Slm2); C-terminally truncation mutants lacking the PH domain (Slm1ΔC and Slm2ΔC) and full-length Slm1 point mutants containing substitutions in the PH domain (Slm1M1 and Slm1M1). (B) Quantification of plasma membrane association of wild-type and mutant Slm1 and Slm2 variants. A density profile plot was generated using NIH Image 1.62. Pixel intensity was determined by a “raw average plot” of a cross section through the center of the cell from single confocal slices obtained from four to six cells.