Two subunits of the exocyst, Sec3p and Exo70p, can function exclusively on the plasma membrane

The exocyst is an octameric complex that tethers secretory vesicles to the plasma membrane in preparation for fusion. We anchored each subunit with a transmembrane (TM) domain at its N- or C-terminus. Only N-terminally anchored TM-Sec3p and C-terminally anchored Exo70p-TM proved functional. These findings orient the complex with respect to the membrane and establish that Sec3p and Exo70p can function exclusively on the membrane. The functions of TM-Sec3p and Exo70p-TM were largely unaffected by blocks in endocytic recycling, suggesting that they act on the plasma membrane rather than on secretory vesicles. Cytosolic pools of the other exocyst subunits were unaffected in TM-sec3 cells, while they were partially depleted in exo70-TM cells. Blocking actin-dependent delivery of secretory vesicles in act1-3 cells results in loss of Sec3p from the purified complex. Our results are consistent with a model in which Sec3p and Exo70p can function exclusively on the plasma membrane while the other subunits are brought to them on secretory vesicles.