FIGURE 2:

(A) Sec3p with an N-terminal TM anchor was largely absent from the 50,000 × g supernatant. Total cell lysates (T) from WT (NY2128; left panel) and the tos2TM-sec3-4xHA mutant (NY3238; right panel) were centrifuged at 50,000 × g for 20 min, separated into supernatant (S) and pellet (P) fractions, and then analyzed by Western blot analysis on a 6% SDS–PAGE gel with anti-HA antibody, anti-Sso1/2, or anti-Snc1/2 serum. (B, C) All exocyst subunits fused at their N-termini to tos2-TM were expressed well. The expression of all exocyst subunits fused at their N-termini to tos2-TM and tagged at their C-termini to either HA (Sec3p, Exo70p, Sec6p, Exo84p) or myc (Sec5p, Sec8p, Sec10p, Sec15p) tail were examined by western blot using either anti-HA or anti-myc antibody. All strains are heterozygous diploids except for tos2TM-sec3-4xHA, which is viable as a homozygous haploid, and tos2TM-exo70-3xHA, which contains a 2µ SEC1 plasmid to bypass the requirement of the essential EXO70 gene. All WT counterparts are haploids expressing an exocyst subunit tagged with either HA or myc tag as indicated. Adh1p was used as a loading control.