Exocytosis in yeast occurs at plasma membrane subdomains whose locatio
ns vary with the cell cycle, but the primary protein determinants of t
hese sites are unknown. A functional fusion of Sec3 protein with green
fluorescent protein (Sec3-GFP) localizes to the site of polarized exo
cytosis for each cell-cycle stage, where it colocalizes with Sec4p and
Sec8p. Sec3-GFP localization is independent of secretory pathway func
tion, of the actin and septin cytoskeletons, and of the polarity estab
lishment proteins. We propose that Sec3p is a spatial landmark definin
g sites of exocytosis. Polarized secretion would result from the coupl
ing of actin-dependent vesicle targeting with Sec3p-dependent establis
hment of the vesicle fusion site.