Localization of the exocyst, Sec4 and actin in the exo70 sec3ΔN double mutant. (A) exo70-45 or exo70-45 sec3ΔN cells expressing the GFP-tagged exocyst components under the control of their endogenous promoters were grown to early log phase at 34°C, and then shifted to 18°C for 6 h. Cells were then fixed for fluorescence microscopy. Members of the exocyst including Sec3 (or sec3ΔN in the exo70-45 sec3ΔN cells), Sec5, Sec8 and the mutant exo70-45 protein remained polarized at the bud tip in the exo70-45 single mutant, but were largely diffused in the mother cell or in the daughter cell cytosol in the exo70-45 sec3ΔN double mutant cells. (B) The localization of the Rab protein Sec4 in exo70-45 and exo70-45 sec3ΔN cells was detected with anti-Sec4 antibody. Sec4 was polarized but diffused in the exo70-45 sec3ΔN double mutant. The cells were also stained with Alex488-phaloidin for F-actin. Actin cables and cortical patches remained polarized in the exo70-45 sec3ΔN double mutant.