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 specific docking sites on the plasma membrane. It is composed of eight proteins (Sec3, -5, -6, -8, -10, and -15, and Exo70 and -84), with molecular weights ranging from 70 to 144 kDa. Mammalian orthologues for seven of these proteins have been described and here we report the cloning and initial characterization of the remaining subunit, Sec3. Human Sec3 (hSec3) shares 17% sequence identity with yeast Sec3p, interacts in the two-hybrid system with other subunits of the complex (Sec5 and Sec8), and is expressed in almost all tissues tested. In yeast, Sec3p has been proposed to be a spatial landmark for polarized secretion (1), and its localization depends on its interaction with Rho1p (2). We demonstrate here that hSec3 lacks the potential Rho1-binding site and GFP-fusions of hSec3 are cytosolic. Green fluorescent protein (GFP)-fusions of nearly every subunit of the mammalian Sec6/8 complex were expressed in Madin-Darby canine kidney (MDCK) cells, but they failed to assemble into a complex with endogenous proteins and localized in the cytosol. Of the subunits tested, only GFP-Exo70 localized to lateral membrane sites of cell-cell contact when expressed in MDCK cells. Cells overexpressing GFP-Exo70 fail to form a tight monolayer, suggesting the Exo70 targeting interaction is critical for normal development of polarized epithelial cells.
Matern
H T
HT
Genentech, Inc., Department of Richard Scheller, South San Francisco, CA 94080-4990, USA.
Yeaman
C
C
Nelson
W J
WJ
Scheller
R H
RH
D000595:Amino Acid Sequence; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line; D016764:Cell Polarity; D003001:Cloning, Molecular; D004285:Dogs; D005089:Exocytosis; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D020869:Gene Expression Profiling; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D021381:Protein Transport; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
D002352:Carrier Proteins; C490685:EXOC3 protein, human; C489607:EXOC4 protein, human; D005656:Fungal Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; C490684:SEC6 protein, S cerevisiae; C489603:SEC8 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins