Figure 2.

Three mutant forms of Ste6p are aberrantly ER localized by immunofluorescence. The localization of wild-type and mutant forms of Ste6-HAp was examined by coimmunofluorescence with the ER marker Kar2p. The pattern of Ste6p localization is revealed by staining with the anti-HA antibody (A–D, top panels), and the position of the ER in the same cells is detected by staining with an antibody to the ER marker Kar2p (E–H, bottom panels). The secondary antibodies, a Rhodamine-conjugated anti-mouse and FITC-conjugated anti-rabbit, were used to detect Ste6-HAp and Kar2p, respectively. Strains used were the same as in Figure 1. It should be noted that the wild-type and mutant ste6 gene products are expressed from a high-copy number (2μ) plasmid, resulting in variability of staining from cell to cell. Consistent with a significant decrease in the metabolic stability of Ste6–166p (see Figure 3), the proportion of cells harboring this mutant protein that exhibit detectable immunofluorescence was considerably lower than for wild-type Ste6p or for the Ste6–13p and Ste6–90p mutant proteins. For clarity, examples of the brightest staining cells are shown for Ste6–166p.