The Sec6/8 complex in mammalian cells: characterization of mammalian Sec3, subunit interactions, and expression of subunits in polarized cells - PubMed (original) (raw)

Comparative Study

. 2001 Aug 14;98(17):9648-53.

doi: 10.1073/pnas.171317898. Epub 2001 Aug 7.

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Comparative Study

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

H T Matern et al. Proc Natl Acad Sci U S A. 2001.

Abstract

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.

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Figures

Figure 1

Figure 1

Sequence alignment of Sec3 proteins. A blast2 search with the ySec3p sequence revealed (in order): C. elegans F52E4.7, accession T16430, high score 105, e value 4.0e-08 with 19% identities;Homo sapiens_BAA91886/FLJ10893, accession NP 060731, high score 83, e value 9.0e-08 with 17% identities; and_Drosophila melanogaster CG3885 protein, accession AAF49347, high score 112, e value 1.4e-06 with 22% identities. The predicted amino acid sequence of human Sec3 was compared with the respective fly and yeast homologues by using the GCG programs PILEUP and PRETTYBOX. Identical residues are in a black box with white letters and similar residues are shaded. Dotted regions represent gaps. Lines above the amino acid sequences indicate the peptides determined by amino-acid sequencing of the p106 subunit purified from rat brain (14). The Sec3 proteins from higher organisms lack an equivalent of the 480-aa N-terminal domain of yeast Sec3p. Therefore, this part of ySec3p is not shown here.

Figure 2

Figure 2

Multiple-tissue Northern blot analysis. Size markers are on the left in Kb. (Upper) hSEC3, one transcript of about 4 Kb is observed in almost all tissues. (Lower) To show an equal loading of mRNA in all lanes, the filter was stripped and reblotted with human β-actin.

Figure 3

Figure 3

Map of two-hybrid interactions. (A) X-Gal filter assays of all pairwise two-hybrid interactions between individual Sec6/8 subunits. CLONTECH vectors pBAD-T-antigene and pGBKT7-p53 combined with Sec6/8 subunits served as negative control, whereas the two vectors together served as positive control. A quantification of the two-hybrid interaction was done by a liquid_o_-nitrophenyl-β-D-galactopyranoside (ONPG) assay. The calculated units are given below the individual dots. Although all interactions were measured, only those 10× higher than background (≥1) are given here. (B) Schematic representation of these two-hybrid interactions. Arrows indicate the direction from bait to prey. (Known two-hybrid interactions in yeast are: Sec3p–Sec5p, Sec5p–Sec10p, Sec10p–Sec15p, Exo84p–Sec5p, and Exo84–Sec10p.)

Figure 4

Figure 4

Expression of GFP-tagged Sec6/8 complex subunits in MDCK cells. Stably transfected MDCK II cells expressing Sec3-GFP (A_–_D), GFP-Sec10 (E_–_H), or Exo70-GFP (I_–_L); all GFP stainings are shown in green. Cells were fixed with 4% paraformaldehyde before extraction with 1% Triton X-100. Cells in C, G, and_J_ were stained with monoclonal antibody 9H5 against endogenous Sec6, and bound antibodies were detected with Texas red donkey anti-mouse IgG. Cells in D and_H_ were stained with polyclonal anti-ZO-1 antibodies, which were detected with Texas red donkey anti-rabbit IgG. Cells in_K_ and L were stained with monoclonal antibody 3G8 against E-cadherin followed by Texas red donkey anti-mouse IgG. (Scale bars, 5 μm.)

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