Vesicles mediate protein transport along the secretory pathway in eukaryotic cells. Transport vesicles bud from a donor organelle and are translocated to an acceptor organelle where they dock, fuse, and thereby deliver their cargo (3). Proteins that mediate different steps in vesicle trafficking are highly conserved from yeast to man. For example, proteins that are crucial for neurosecretion in mammals (nSec1, Vamp1, Vamp2, SNAP-25, NSF, and α-SNAP) are homologous to proteins required for vesicle trafficking to the yeast plasma membrane (Sec1p, Snc1p, Snc2p, Sec9p, Sec18p, and Sec17p, respectively). Another group of proteins involved in this transport step in yeast includes Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p, which form a stable complex called the exocyst (4). A mammalian homolog of this protein complex (Sec6/8 complex) has been described (5, 6), and in both yeast and mammals each subunit is represented once, resulting in protein complexes of 845 kDa (yeast) and 736 kDa (rat).
Accumulating evidence indicates that the Sec6/8 complex is required for post-Golgi vesicle trafficking (7, 8). Subcellular localization of the complex correlates with sites of polarized membrane growth. In yeast, Sec3p is present at plasma membrane sites of active vesicle fusion, and the location of these sites changes during the cell cycle. At the beginning of a new cell cycle, the exocyst localizes in a patch at the prebud site, and as the bud emerges the exocyst is localized to its tip. When the growth pattern switches from apical to isotropic the patch disperses around the membrane of the bud. During cytokinesis, the exocyst subunits reconcentrate in a ring-like structure at the neck separating the mother cell and the bud. Bud tip, isotropic bud, and mother–daughter neck represent sites of directed membrane growth that is coordinated with the cell cycle (1). In mammalian cells the sec6/8 complex is also present on plasma membranes at sites of membrane growth. In cultured hippocampal neurons, the Sec6/8 complex was shown to be present in regions of membrane addition—i.e., at neurite outgrowth and potential active zones during synaptogenesis (9). In differentiated PC12 cells the complex is found in the cell body, in the extending neurite, and at the growth cone, whereas it shows a perinuclear localization in undifferentiated PC12 cells (10). Best characterized however is the localization of the Sec6/8 complex in Madin–Darby canine kidney (MDCK) epithelial cells (8). Here the complex is rapidly recruited from the cytosol to cell–cell contacts on initiation of calcium-dependent cell–cell adhesion. As cell polarity develops, the localization of the complex becomes restricted to the apical junctional complex, which includes adherens junctions and tight junctions. It has been proposed that localization of Sec6/8 complex to cell–cell junctions serves to direct trafficking of transport vesicles containing basal-lateral proteins to the developing lateral membrane domain (11).
Functionally, the Sec6/8 complex probably acts as a tethering complex at the plasma membrane. In line with the localization studies, it has been shown that the Sec6/8 complex is involved in specifying docking and/or tethering of postGolgi transport vesicles to the plasma membrane. In yeast exocyst mutants, there is an accumulation of transport vesicles in the cytoplasm, when the cells are shifted to the restrictive temperature (12). And in streptolysin-O permeabilized MDCK cells, antibodies to Sec8 inhibit delivery of vesicles to the basal-lateral membrane, but not the apical membrane (8).
In addition to a primary localization on the plasma membrane, components of the exocyst complex may be present on other membranes. Overexpressed Sec15p cofractionates in sucrose gradients with Sec4p and Sncp, the rab protein, and v-SNARE associated with secretory vesicles. Because Sec15p also binds to activated Sec4p, the exocyst might be an effector for this Rab-like GTPase that is necessary for the targeting or tethering of secretory vesicles to sites of secretion. Sec10p also exists in a free pool, as has been shown by subcellular fractionations in yeast. Sec15p and Sec10p interact with each other in the two-hybrid system and in vitro synthesized Sec15p coimmunoprecipitates with epitope-tagged Sec10p (13). These findings suggest that Sec10p and Sec15p exist in a subcomplex that might act as a bridge between Sec4p on the vesicle and other subunits bound to the plasma membrane.
The localization of Sec3p in yeast to sites at the plasma membrane is reported to be independent of a functional secretory pathway, the actin cytoskeleton, and the other exocyst subunits (1). This led to the model that Sec3p is a spatial landmark for exocytosis and that it may be the component of the complex most proximal to the target membrane. Purification of mammalian Sec6/8 complex hinted at the existence of a Sec3 protein, but the corresponding gene was not previously cloned (14). Coomassie blue-stained SDS/PAGE of purified Sec6/8 complex reveals eight individual bands, seven of which comprise the components of the exocyst. But peptide sequencing of the remaining protein, p106, did not easily lead to its identification in protein databases (14). Now, as whole genomes from higher organisms are sequenced, a blast2 search with the yeast Sec3p sequence lead to the identification of the SEC3 genes from fly, worm, and man. Here we report the cloning of the human SEC3 gene, its expression pattern in different tissues, and a network of two-hybrid interactions that link Sec3 to other subunits of the Sec6/8 complex. Our localization studies employing green fluorescent protein (GFP) fusions of several Sec6/8 complex subunits revealed that only GFP-Exo70 becomes localized to the plasma membrane in polarized epithelial MDCK cells, whereas all other GFP-tagged subunits are cytosolic. These studies suggest that regulation of Sec6/8 complex assembly and localization at the plasma membrane depends on Exo70 targeting interactions, not Sec3 as in yeast.