Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion

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Abstract

Live-cell imaging of the exocyst subunit Sec8 reveals how the protein’s spatiotemporal dynamics correlate with its roles in vesicle fusion.

Abstract

Tethers play ubiquitous roles in membrane trafficking and influence the specificity of vesicle attachment. Unlike soluble N-ethyl-maleimide–sensitive fusion attachment protein receptors (SNAREs), the spatiotemporal dynamics of tethers relative to vesicle fusion are poorly characterized. The most extensively studied tethering complex is the exocyst, which spatially targets vesicles to sites on the plasma membrane. By using a mammalian genetic replacement strategy, we were able to assemble fluorescently tagged Sec8 into the exocyst complex, which was shown to be functional by biochemical, trafficking, and morphological criteria. Ultrasensitive live-cell imaging revealed that Sec8-TagRFP moved to the cell cortex on vesicles, which preferentially originated from the endocytic recycling compartment. Surprisingly, Sec8 remained with vesicles until full dilation of the fusion pore, supporting potential coupling with SNARE fusion machinery. Fluorescence recovery after photobleaching analysis of Sec8 at cell protrusions revealed that a significant fraction was immobile. Additionally, Sec8 dynamically repositioned to the site of membrane expansion, suggesting that it may respond to local cues during early cell polarization.

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Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway.

P. Novick, C. Field, R Schekman (1980)

Cells of a Saccharomyces cerevisiae mutant that is temperature-sensitive for secretion and cell surface growth become dense during incubation at the non-permissive temperature (37 degrees C). This property allows the selection of additional secretory mutants by sedimentation of mutagenized cells on a Ludox density gradient. Colonies derived from dense cells are screened for conditional growth and secretion of invertase and acid phosphatase. The sec mutant strains that accumulate an abnormally large intracellular pool of invertase at 37 degrees C (188 mutant clones) fall into 23 complementation groups, and the distribution of mutant alleles suggests that more complementation groups could be found. Bud emergence and incorporation of a plasma membrane sulfate permease activity stop quickly after a shift to 37 degrees C. Many of the mutants are thermoreversible; upon return to the permissive temperature (25 degrees C) the accumulated invertase is secreted. Electron microscopy of sec mutant cells reveals, with one exception, the temperature-dependent accumulation of membrane-enclosed secretory organelles. We suggest that these structures represent intermediates in a pathway in which secretion and plasma membrane assembly are colinear.

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The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae.

D R TerBush, T Maurice, D. Roth … (1996)

In the yeast Saccharomyces cerevisiae, the products of at least 15 genes are involved specifically in vesicular transport from the Golgi apparatus to the plasma membrane. Previously, we have shown that three of these genes, SEC6, SEC8 and SEC15, encode components of a multisubunit complex which localizes to the tip of the bud, the predominant site of exocytosis in S. cerevisiae. Mutations in three more of these genes, SEC3, SEC5 and SEC10, were found to disrupt the subunit integrity of the Sec6-Sec8-Sec15 complex, indicating that these genes may encode some of the remaining components of this complex. To examine this possibility, we cloned and sequenced the SEC5 and SEC10 genes, disrupted them, and either epitope tagged them (Sec5p) or prepared polyclonal antisera (Sec10p) to them for co-immunoprecipitation studies. Concurrently, we biochemically purified the remaining unidentified polypeptides of the Sec6-Sec8-Sec15 complex for peptide microsequencing. The genes encoding these components were identified by comparison of predicted amino acid sequences with those obtained from peptide microsequencing of the purified complex components. In addition to Sec6p, Sec8p and Sec15p, the complex contains the proteins encoded by SEC3, SEC5, SEC10 and a novel gene, EXO70. Since these seven proteins function together in a complex required for exocytosis, and not other intracellular trafficking steps, we have named it the Exocyst.

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The exocyst complex in polarized exocytosis.

Bing He, Wei. Guo (2009)

The exocyst is an octameric protein complex, which mediates the tethering of post-Golgi secretory vesicles to the plasma membrane before exocytic fusion. The exocyst assembles by side-by-side packing of rod-shaped subunits composed of helical bundles. The targeting of secretory vesicles to the plasma membrane involves direct interactions of the exocyst with PI(4,5)P(2). In addition, a number of small GTP-binding proteins interact with components of the exocyst and regulate the assembly, localization, and function of this complex. Here we review the recent advances in the field, focusing on the function of the exocyst in polarized exocytosis.

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Author and article information

Journal

Journal ID (nlm-ta): J Cell Biol

Journal ID (iso-abbrev): J. Cell Biol

Journal ID (hwp): jcb

Title: The Journal of Cell Biology

Publisher: The Rockefeller University Press

ISSN (Print): 0021-9525

ISSN (Electronic): 1540-8140

Publication date (Print): 27 May 2013

Volume: 201

Issue: 5

Pages: 673-680

Affiliations

Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06510

Author notes

Correspondence to Derek Toomre: derek.toomre@ 123456yale.edu

Article

Publisher ID: 201212103

DOI: 10.1083/jcb.201212103

PMC ID: 3664709

PubMed ID: 23690179

SO-VID: e0ecc6fd-7d6e-4669-b622-e6786e8d1b5f

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This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.rupress.org/terms). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

Live-cell imaging of exocyst links its spatiotemporal dynamics to various stages of vesicle fusion

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Most cited references 42

Identification of 23 complementation groups required for post-translational events in the yeast secretory pathway.

The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae.

The exocyst complex in polarized exocytosis.

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