The Sec6/8 complex in mammalian cells: Characterization of mammalian Sec3,subunit interactions, and expression of subunits in polarized cells

The yeast exocyst complex (also called Sec6/8 complex in higher eukaryotes) is a multiprotein complex essential for targeting exocytic vesicles to spe cific docking sites on the plasma membrane. It is composed of eight protein s (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weigh ts ranging from 70 to 144 kDa. Mammalian orthologues for seven of these pro teins have been described and here we report the cloning and initial charac terization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% se quence identity with yeast Sec3p, interacts in the two-hybrid system with o ther subunits of the complex (Sec5 and Sec8), and is expressed in almost al l tissues tested. In yeast, Sec3p has been proposed to be a spatial landmar k for polarized secretion (1), and its localization depends on its interact ion with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1 -binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent pro tein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed t o assemble into a complex with endogenous proteins and localized in the cyt osol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressi ng GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cel ls.