Figure 5.

The exocyst failed to associate with the plasma membrane in the exo70-45 sec3ΔN double mutant. Wild type, sec3ΔN, exo70-45 and exo70-45 sec3ΔN cells were grown to mid-log phase at 34°C, and then shifted to 18°C for 6 h. Cell lysates were prepared and centrifuged successively at 500 g and 13 000 g to generate S1, P1 and S2, P2, respectively. Equal volumes of samples were subjected to SDS–PAGE and analyzed by Western blot, using antibodies against the exocyst components Sec3 and Sec8. For Exo70, the endogenous copy of Exo70 or exo70-45 was tagged by GFP and an anti-GFP antibody was used to detect the Exo70-GFP or Exo70-45-GFP fusion protein. As controls, the anti-Sso1 (the plasma membrane t-SNARE) and anti-Adh1 antibodies were used to detect the plasma membrane and cytosol fractions. The level of Exo70-45, Sec3 and Sec8 associated with the P2 fraction was greatly reduced in the exo70-45 sec3ΔN double mutant. Note that the full-length Sec3 is mostly degraded to a major band of approximately 110 kDa as previously described (TerBush and Novick, 1995), which has molecular weight similar to that of the Sec3N protein.