Vesicles carry most exocyst subunits to exocytic sites marked by the remaining two subunits, Sec3p and Exo70p

Exocytosis in the budding yeast Saccharomyces cerevisiae occurs at discrete domains of the plasma membrane. The protein complex that tethers incoming vesicles to sites of secretion is known as the exocyst. We have used photobleaching recovery experiments to characterize the dynamic behavior of the eight subunits that make up the exocyst. One subset (Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, and Exo84p) exhibits mobility similar to that of the vesicle-bound Rab family protein Sec4p, whereas Sec3p and Exo70p exhibit substantially more stability. Disruption of actin assembly abolishes the ability of the first subset of subunits to recover after photobleaching, whereas Sec3p and Exo70p are resistant. Immunogold electron microscopy and epifluorescence video microscopy indicate that all exocyst subunits, except for Sec3p, are associated with secretory vesicles as they arrive at exocytic sites. Assembly of the exocyst occurs when the first subset of subunits, delivered on vesicles, joins Sec3p and Exo70p on the plasma membrane. Exocyst assembly serves to both target and tether vesicles to sites of exocytosis.

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Sterol biosensor reveals LAM-family Ltc1-dependent sterol flow to endosomes upon Arp2/3 inhibition

The Journal of Cell Biology ◽

10.1083/jcb.202001147 ◽

2020 ◽

Vol 219 (6) ◽

Cited By ~ 3

Author(s):

Magdalena Marek ◽

Vincent Vincenzetti ◽

Sophie G. Martin

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Schizosaccharomyces Pombe ◽

Fission Yeast ◽

Biological Membranes ◽

Time Lapse ◽

Vesicular Trafficking ◽

Actin Assembly ◽

Contact Sites ◽

Family Protein

Sterols are crucial components of biological membranes, which are synthetized in the ER and accumulate in the plasma membrane (PM). Here, by applying a genetically encoded sterol biosensor (D4H), we visualize a sterol flow between PM and endosomes in the fission yeast Schizosaccharomyces pombe. Using time-lapse and correlative light-electron microscopy, we found that inhibition of Arp2/3-dependent F-actin assembly promotes the reversible relocalization of D4H from the PM to internal sterol-rich compartments (STRIC) labeled by synaptobrevin Syb1. Retrograde sterol internalization to STRIC is independent of endocytosis or an intact Golgi, but depends on Ltc1, a LAM/StARkin-family protein localized to ER-PM contact sites. The PM in ltc1Δ cells over-accumulates sterols and upon Arp2/3 inhibition forms extended ER-interacting invaginations, indicating that sterol transfer contributes to PM size homeostasis. Anterograde sterol movement from STRIC is independent of canonical vesicular trafficking but requires Arp2/3, suggesting a novel role for this complex. Thus, transfer routes orthogonal to vesicular trafficking govern the flow of sterols in the cell.

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Faculty Opinions recommendation of In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1010821.167908 ◽

2002 ◽

Author(s):

Miguel Penalva

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Vesicles ◽

Secretory Vesicles ◽

Plasma Membrane Vesicles ◽

Cytoplasmic Side

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The a-factor transporter (STE6 gene product) and cell polarity in the yeast Saccharomyces cerevisiae.

The Journal of Cell Biology ◽

10.1083/jcb.120.5.1203 ◽

1993 ◽

Vol 120 (5) ◽

pp. 1203-1215 ◽

Cited By ~ 59

Author(s):

K Kuchler ◽

H G Dohlman ◽

J Thorner

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Gene Product ◽

Cell Polarity ◽

Polyclonal Antibodies ◽

Apparent Molecular Weight ◽

Subcellular Fractionation ◽

Mating Pheromone ◽

Yeast Saccharomyces Cerevisiae ◽

Factor Secretion

STE6 gene product is required for secretion of the lipopeptide mating pheromone a-factor by Saccharomyces cerevisiae MATa cells. Radiolabeling and immunoprecipitation, either with specific polyclonal antibodies raised against a TrpE-Ste6 fusion protein or with mAbs that recognize c-myc epitopes in fully functional epitope-tagged Ste6 derivatives, demonstrated that Ste6 is a 145-kD phosphoprotein. Subcellular fractionation, various extraction procedures, and immunoblotting showed that Ste6 is an intrinsic plasma membrane-associated protein. The apparent molecular weight of Ste6 was unaffected by tunicamycin treatment, and the radiolabeled protein did not bind to concanavalin A, indicating that Ste6 is not glycosylated and that glycosylation is not required either for its membrane delivery or its function. The amino acid sequence of Ste6 predicts two ATP-binding folds; correspondingly, Ste6 was photoaffinity-labeled specifically with 8-azido-[alpha-32P]ATP. Indirect immunofluorescence revealed that in exponentially growing MATa cells, the majority of Ste6 showed a patchy distribution within the plasma membrane, but a significant fraction was found concentrated in a number of vesicle-like bodies subtending the plasma membrane. In contrast, in MATa cells exposed to the mating pheromone alpha-factor, which markedly induced Ste6 production, the majority of Ste6 was incorporated into the plasma membrane within the growing tip of the elongating cells. The highly localized insertion of this transporter may establish pronounced anisotropy in a-factor secretion from the MATa cell, and thereby may contribute to the establishment of the cell polarity which restricts partner selection and cell fusion during mating to one MAT alpha cell.

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Secretory Pathway-Dependent Localization of the Saccharomyces cerevisiae Rho GTPase-Activating Protein Rgd1p at Growth Sites

Eukaryotic Cell ◽

10.1128/ec.00042-12 ◽

2012 ◽

Vol 11 (5) ◽

pp. 590-600 ◽

Cited By ~ 6

Author(s):

Fabien Lefèbvre ◽

Valérie Prouzet-Mauléon ◽

Michel Hugues ◽

Marc Crouzet ◽

Aurélie Vieillemard ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Trafficking ◽

Cell Cycle Progression ◽

Secretory Pathway ◽

Rho Gtpase ◽

Secretory Vesicles ◽

Gtpase Activating Protein ◽

Content Type

ABSTRACT Establishment and maintenance of cell polarity in eukaryotes depends upon the regulation of Rho GTPases. In Saccharomyces cerevisiae , the Rho GTPase activating protein (RhoGAP) Rgd1p stimulates the GTPase activities of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively. Consistent with the distribution of Rho3p and Rho4p, Rgd1p is found mostly in areas of polarized growth during cell cycle progression. Rgd1p was mislocalized in mutants specifically altered for Golgi apparatus-based phosphatidylinositol 4-P [PtdIns(4)P] synthesis and for PtdIns(4,5)P 2 production at the plasma membrane. Analysis of Rgd1p distribution in different membrane-trafficking mutants suggested that Rgd1p was delivered to growth sites via the secretory pathway. Rgd1p may associate with post-Golgi vesicles by binding to PtdIns(4)P and then be transported by secretory vesicles to the plasma membrane. In agreement, we show that Rgd1p coimmunoprecipitated and localized with markers specific to secretory vesicles and cofractionated with a plasma membrane marker. Moreover, in vivo imaging revealed that Rgd1p was transported in an anterograde manner from the mother cell to the daughter cell in a vectoral manner. Our data indicate that secretory vesicles are involved in the delivery of RhoGAP Rgd1p to the bud tip and bud neck.

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Species-specific difference in distribution of voltage-gated L-type Ca2+ channels of cardiac myocytes

AJP Cell Physiology ◽

10.1152/ajpcell.2000.279.6.c1963 ◽

2000 ◽

Vol 279 (6) ◽

pp. C1963-C1969 ◽

Cited By ~ 27

Author(s):

Yoshiko Takagishi ◽

Kenji Yasui ◽

Nicholas J. Severs ◽

Yoshiharu Murata

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Cardiac Myocytes ◽

Immunogold Electron Microscopy ◽

Surface Plasma ◽

Gold Particles ◽

Intracellular Ca ◽

Rat Myocytes ◽

T Tubules ◽

Species Specific

Ca2+influx via sarcolemmal voltage-dependent Ca2+ channels (L-type Ca2+ channels) is the fundamental step in excitation-contraction (E-C) coupling in cardiac myocytes. Physiological and pharmacological studies reveal species-specific differences in E-C coupling resulting from a difference in the contribution of Ca2+ influx and intracellular Ca2+ release to activation of contraction. We investigated the distribution of L-type Ca2+ channels in isolated cardiac myocytes from rabbit and rat ventricle by correlative immunoconfocal and immunogold electron microscopy. Immunofluorescence labeling revealed discrete spots in the surface plasma membrane and transverse (T) tubules in rabbit myocytes. In rat myocytes, labeling appeared more intense in T tubules than in the surface sarcolemma. Immunogold electron microscopy extended these findings, showing that the number of gold particles in the surface plasma membrane was significantly higher in rabbit than rat myocytes. In rabbit myocyte plasma membrane, the gold particles were distributed as clusters in both regions that were associated with junctional sarcoplasmic reticulum and those that were not. The findings are consistent with the idea that influx of Ca2+ via surface sarcolemmal Ca2+ channels contributes to intracellular Ca2+ to a greater degree in rabbit than in rat myocytes.

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The R-SNARE Endobrevin/VAMP-8 Mediates Homotypic Fusion of Early Endosomes and Late Endosomes

Molecular Biology of the Cell ◽

10.1091/mbc.11.10.3289 ◽

2000 ◽

Vol 11 (10) ◽

pp. 3289-3298 ◽

Cited By ~ 112

Author(s):

Wolfram Antonin ◽

Claudia Holroyd ◽

Ritva Tikkanen ◽

Stefan Höning ◽

Reinhard Jahn

Keyword(s):

Electron Microscopy ◽

Plasma Membrane ◽

Subcellular Fractionation ◽

Immunogold Electron Microscopy ◽

Fusion Reactions ◽

Coated Pits ◽

Late Endosomes ◽

Early Endosomes ◽

Golgi Network

Endobrevin/VAMP-8 is an R-SNARE localized to endosomes, but it is unknown in which intracellular fusion step it operates. Using subcellular fractionation and quantitative immunogold electron microscopy, we found that endobrevin/VAMP-8 is present on all membranes known to communicate with early endosomes, including the plasma membrane, clathrin-coated pits, late endosomes, and membranes of thetrans-Golgi network. Affinity-purified antibodies that block the ability of endobrevin/VAMP-8 to form SNARE core complexes potently inhibit homotypic fusion of both early and late endosomes in vitro. Fab fragments were as active as intact immunoglobulin Gs. Recombinant endobrevin/VAMP-8 inhibited both fusion reactions with similar potency. We conclude that endobrevin/VAMP-8 operates as an R-SNARE in the homotypic fusion of early and late endosomes.

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Enzymes of phosphoinositide synthesis in secretory vesicles destined for the plasma membrane in Saccharomyces cerevisiae.

Journal of Bacteriology ◽

10.1128/jb.172.7.4115-4117.1990 ◽

1990 ◽

Vol 172 (7) ◽

pp. 4115-4117 ◽

Cited By ~ 20

Author(s):

A J Kinney ◽

G M Carman

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Secretory Vesicles

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The Saccharomyces cerevisiae MYO2 gene encodes an essential myosin for vectorial transport of vesicles.

The Journal of Cell Biology ◽

10.1083/jcb.113.3.539 ◽

1991 ◽

Vol 113 (3) ◽

pp. 539-551 ◽

Cited By ~ 283

Author(s):

G C Johnston ◽

J A Prendergast ◽

R A Singer

Keyword(s):

Saccharomyces Cerevisiae ◽

Molecular Motor ◽

Actin Binding ◽

Restrictive Temperature ◽

Secretory Vesicles ◽

Biosynthetic Activity ◽

Yeast Saccharomyces Cerevisiae ◽

Terminal Domain ◽

Essential Form ◽

Actin Cables

After the initiation of bud formation, cells of the yeast Saccharomyces cerevisiae direct new growth to the developing bud. We show here that this vectorial growth is facilitated by activity of the MYO2 gene. The wild-type MYO2 gene encodes an essential form of myosin composed of an NH2-terminal domain typical of the globular, actin-binding domain of other myosins. This NH2-terminal domain is linked by what appears to be a short alpha-helical domain to a novel COOH-terminal region. At the restrictive temperature the myo2-66 mutation does not impair DNA, RNA, or protein biosynthetic activity, but produces unbudded, enlarged cells. This phenotype suggests a defect in localization of cell growth. Measurements of cell size demonstrated that the continued development of initiated buds, as well as bud initiation itself, is inhibited. Bulk secretion continues in mutant cells, although secretory vesicles accumulate. The MYO2 myosin thus may function as the molecular motor to transport secretory vesicles along actin cables to the site of bud development.

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Cell surface recycling in yeast: mechanisms and machineries

Biochemical Society Transactions ◽

10.1042/bst20150263 ◽

2016 ◽

Vol 44 (2) ◽

pp. 474-478 ◽

Cited By ~ 9

Author(s):

Chris MacDonald ◽

Robert C. Piper

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Membrane Protein ◽

Cell Surface ◽

Mammalian Cells ◽

Genetic System ◽

Integral Membrane Protein ◽

Protein Activity ◽

Yeast Saccharomyces Cerevisiae ◽

Central Process

Sorting internalized proteins and lipids back to the cell surface controls the supply of molecules throughout the cell and regulates integral membrane protein activity at the surface. One central process in mammalian cells is the transit of cargo from endosomes back to the plasma membrane (PM) directly, along a route that bypasses retrograde movement to the Golgi. Despite recognition of this pathway for decades we are only beginning to understand the machinery controlling this overall process. The budding yeast Saccharomyces cerevisiae, a stalwart genetic system, has been routinely used to identify fundamental proteins and their modes of action in conserved trafficking pathways. However, the study of cell surface recycling from endosomes in yeast is hampered by difficulties that obscure visualization of the pathway. Here we briefly discuss how recycling is likely a more prevalent process in yeast than is widely appreciated and how tools might be built to better study the pathway.

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