Figure 3.

Cn Cho1 is an integral membrane protein that primarily localizes to the endoplasmic reticulum. A, Cho1 protein in C. neoformans localizes to the ER membrane in a similar manner to Cho1 from other yeasts. In this strain, the CHO1 gene is fused with an mCherry tag at its C terminus, and its expression is under the control of the C. neoformans actin promoter. ER tracker was used to visualize the ER membrane. Among over 100 cells analyzed, ∼99% showed co-localization of Cho1 protein with the ER tracker. Additional data were shown in Fig. S1. Scale bar, 10 μm. B, membrane protein separation assay confirms that Cho1 is an integral membrane protein. As described under “Experimental procedures,” a total membrane fraction obtained from cells expressing Cho1 was extracted with 1× PBS buffer, 1% Triton X-100, or salt (1 m NaCl). Following ultracentrifugation, equivalent amounts of the insoluble pellet (P) and the supernatant (S) were analyzed by Western blotting using RFP-tag antibody.