Faculty Opinions recommendation of Myosin V transports secretory vesicles via a Rab GTPase cascade and interaction with the exocyst complex.

During membrane trafficking, vesicular carriers are transported and tethered to their cognate acceptor compartments before soluble N-ethylmaleimide–sensitive factor attachment protein (SNARE)-mediated membrane fusion. The exocyst complex was believed to target and tether post-Golgi secretory vesicles to the plasma membrane during exocytosis. However, no definitive experimental evidence is available to support this notion. We developed an ectopic targeting assay in yeast in which each of the eight exocyst subunits was expressed on the surface of mitochondria. We find that most of the exocyst subunits were able to recruit the other members of the complex there, and mistargeting of the exocyst led to secretion defects in cells. On the other hand, only the ectopically located Sec3p subunit is capable of recruiting secretory vesicles to mitochondria. Our assay also suggests that both cytosolic diffusion and cytoskeleton-based transport mediate the recruitment of exocyst subunits and secretory vesicles during exocytosis. In addition, the Rab GTPase Sec4p and its guanine nucleotide exchange factor Sec2p regulate the assembly of the exocyst complex. Our study helps to establish the role of the exocyst subunits in tethering and allows the investigation of the mechanisms that regulate vesicle tethering during exocytosis.

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The Cooh-Terminal Domain of Myo2p, a Yeast Myosin V, Has a Direct Role in Secretory Vesicle Targeting

The Journal of Cell Biology ◽

10.1083/jcb.147.4.791 ◽

1999 ◽

Vol 147 (4) ◽

pp. 791-808 ◽

Cited By ~ 184

Author(s):

Daniel Schott ◽

Jackson Ho ◽

David Pruyne ◽

Anthony Bretscher

Keyword(s):

Secretory Vesicle ◽

Restrictive Temperature ◽

Secretory Vesicles ◽

Tail Domain ◽

Myosin V ◽

Direct Role ◽

Rab Protein ◽

Polarized Delivery ◽

Actin Cables ◽

Type V

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable–dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

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The Neurospora crassa exocyst complex tethers Spitzenkörper vesicles to the apical plasma membrane during polarized growth

Molecular Biology of the Cell ◽

10.1091/mbc.e13-06-0299 ◽

2014 ◽

Vol 25 (8) ◽

pp. 1312-1326 ◽

Cited By ~ 49

Author(s):

Meritxell Riquelme ◽

Erin L. Bredeweg ◽

Olga Callejas-Negrete ◽

Robert W. Roberson ◽

Sarah Ludwig ◽

...

Keyword(s):

Plasma Membrane ◽

Neurospora Crassa ◽

Hyphal Growth ◽

Apical Plasma Membrane ◽

Apical Region ◽

Secretory Vesicles ◽

Polarized Growth ◽

Hyphal Morphogenesis ◽

Exocyst Complex ◽

Dynamic Formation

Fungal hyphae are among the most highly polarized cells. Hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the Spitzenkörper. The exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. We determined that the presence of an octameric exocyst complex is required for the formation of a functional Spitzenkörper and maintenance of regular hyphal growth in Neurospora crassa. Two distinct localization patterns of exocyst subunits at the hyphal tip suggest the dynamic formation of two assemblies. The EXO-70/EXO-84 subunits are found at the peripheral part of the Spitzenkörper, which partially coincides with the outer macrovesicular layer, whereas exocyst components SEC-5, -6, -8, and -15 form a delimited crescent at the apical plasma membrane. Localization of SEC-6 and EXO-70 to the plasma membrane and the Spitzenkörper, respectively, depends on actin and microtubule cytoskeletons. The apical region of exocyst-mediated vesicle fusion, elucidated by the plasma membrane–associated exocyst subunits, indicates the presence of an exocytotic gradient with a tip-high maximum that dissipates gradually toward the subapex, confirming the earlier predictions of the vesicle supply center model for hyphal morphogenesis.

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Secretory vesicle transport velocity in living cells depends on the myosin-V lever arm length

The Journal of Cell Biology ◽

10.1083/jcb.200110086 ◽

2002 ◽

Vol 156 (1) ◽

pp. 35-40 ◽

Cited By ~ 152

Author(s):

Daniel H. Schott ◽

Ruth N. Collins ◽

Anthony Bretscher

Keyword(s):

Molecular Motors ◽

Secretory Vesicle ◽

Genetically Engineered ◽

Secretory Vesicles ◽

Myosin V ◽

Lever Arm ◽

Wide Range ◽

Transport Velocity ◽

Maximal Speed ◽

Muscle Myosin

Myosins are molecular motors that exert force against actin filaments. One widely conserved myosin class, the myosin-Vs, recruits organelles to polarized sites in animal and fungal cells. However, it has been unclear whether myosin-Vs actively transport organelles, and whether the recently challenged lever arm model developed for muscle myosin applies to myosin-Vs. Here we demonstrate in living, intact yeast that secretory vesicles move rapidly toward their site of exocytosis. The maximal speed varies linearly over a wide range of lever arm lengths genetically engineered into the myosin-V heavy chain encoded by the MYO2 gene. Thus, secretory vesicle polarization is achieved through active transport by a myosin-V, and the motor mechanism is consistent with the lever arm model.

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Dominant Negative Alleles ofSEC10Reveal Distinct Domains Involved in Secretion and Morphogenesis in Yeast

Molecular Biology of the Cell ◽

10.1091/mbc.9.7.1725 ◽

1998 ◽

Vol 9 (7) ◽

pp. 1725-1739 ◽

Cited By ~ 28

Author(s):

Dagmar Roth ◽

Wei Guo ◽

Peter Novick

Keyword(s):

Plasma Membrane ◽

Cell Cycle Progression ◽

Secretory Pathway ◽

Dominant Negative ◽

Secretory Vesicles ◽

Cycle Progression ◽

Vesicle Docking ◽

Amino Terminal ◽

Exocyst Complex ◽

Carboxy Terminal

The accurate targeting of secretory vesicles to distinct sites on the plasma membrane is necessary to achieve polarized growth and to establish specialized domains at the surface of eukaryotic cells. Members of a protein complex required for exocytosis, the exocyst, have been localized to regions of active secretion in the budding yeastSaccharomyces cerevisiae where they may function to specify sites on the plasma membrane for vesicle docking and fusion. In this study we have addressed the function of one member of the exocyst complex, Sec10p. We have identified two functional domains of Sec10p that act in a dominant-negative manner to inhibit cell growth upon overexpression. Phenotypic and biochemical analysis of the dominant-negative mutants points to a bifunctional role for Sec10p. One domain, consisting of the amino-terminal two-thirds of Sec10p directly interacts with Sec15p, another exocyst component. Overexpression of this domain displaces the full-length Sec10 from the exocyst complex, resulting in a block in exocytosis and an accumulation of secretory vesicles. The carboxy-terminal domain of Sec10p does not interact with other members of the exocyst complex and expression of this domain does not cause a secretory defect. Rather, this mutant results in the formation of elongated cells, suggesting that the second domain of Sec10p is required for morphogenesis, perhaps regulating the reorientation of the secretory pathway from the tip of the emerging daughter cell toward the mother–daughter connection during cell cycle progression.

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The molecular mechanisms of the mammalian exocyst complex in exocytosis

Biochemical Society Transactions ◽

10.1042/bst0340687 ◽

2006 ◽

Vol 34 (5) ◽

pp. 687-690 ◽

Cited By ~ 31

Author(s):

S. Wang ◽

S.C. Hsu

Keyword(s):

Plasma Membrane ◽

Molecular Mechanisms ◽

Early Stage ◽

Secretory Vesicles ◽

Attachment Protein ◽

Trafficking Pathway ◽

Exocyst Complex ◽

Protein Receptor ◽

Associated Proteins ◽

Protein Attachment

Exocytosis is a highly ordered vesicle trafficking pathway that targets proteins to the plasma membrane for membrane addition or secretion. Research over the years has discovered many proteins that participate at various stages in the mammalian exocytotic pathway. At the early stage of exocytosis, co-atomer proteins and their respective adaptors and GTPases have been shown to play a role in the sorting and incorporation of proteins into secretory vesicles. At the final stage of exocytosis, SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) and SNARE-associated proteins are believed to mediate the fusion of secretory vesicles at the plasma membrane. There are multiple events that may occur between the budding of secretory vesicles from the Golgi and the fusion of these vesicles at the plasma membrane. The most obvious and best-known event is the transport of secretory vesicles from Golgi to the vicinity of the plasma membrane via microtubules and their associated motors. At the vicinity of the plasma membrane, however, it is not clear how vesicles finally dock and fuse with the plasma membrane. Identification of proteins involved in these events should provide important insights into the mechanisms of this little known stage of the exocytotic pathway. Currently, a protein complex, known as the sec6/8 or the exocyst complex, has been implicated to play a role at this late stage of exocytosis.

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Yeast Rgd3 is a phospho-regulated F-BAR-containing RhoGAP involved in the regulation of Rho3 distribution and cell morphology

10.1101/2020.05.04.077206 ◽

2020 ◽

Author(s):

Robert M. Gingras ◽

Kyaw Myo Lwin ◽

Abigail M. Miller ◽

Anthony Bretscher

Keyword(s):

Cell Polarity ◽

Binding Motif ◽

Restrictive Temperature ◽

Secretory Vesicles ◽

Polarized Growth ◽

Related Protein ◽

Myosin V ◽

Over Expression ◽

Dependent Transport

AbstractPolarized growth requires the integration of polarity pathways with the delivery of exocytic vesicles for cell expansion and counterbalancing endocytic uptake. In budding yeast, the myosin-V Myo2 is aided by the kinesin-related protein Smy1 in carrying out the essential Sec4-dependent transport of secretory vesicles to sites of polarized growth. Over-expression suppressors of a conditional myo2 smy1 mutant identified a novel F-BAR-containing RhoGAP, Rgd3, that has activity primarily on Rho3, but also Cdc42. Internally tagged Rho3 is restricted to the plasma membrane in a gradient corresponding to cell polarity that is altered upon Rgd3 over-expression. Rgd3 itself is localized to dynamic polarized vesicles that, while distinct from constitutive secretory vesicles, are dependent on actin and Myo2 function. In vitro Rgd3 associates with liposomes in a PIP2-enhanced manner. Further, the Rgd3 C-terminal region contains several phosphorylatable residues within a reported SH3-binding motif. An unphosphorylated mimetic construct is active and highly polarized, while the phospho-mimetic form is not. Rgd3 is capable of activating Myo2, dependent on its phospho-state and Rgd3 overexpression rescues aberrant Rho3 localization and cell morphologies seen at the restrictive temperature in the myo2 smy1 mutant. We propose a model where Rgd3 functions to modulate and maintain Rho3 polarity during growth.

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Interactions between Rabs, tethers, SNAREs and their regulators in exocytosis

Biochemical Society Transactions ◽

10.1042/bst0340683 ◽

2006 ◽

Vol 34 (5) ◽

pp. 683-686 ◽

Cited By ~ 70

Author(s):

P. Novick ◽

M. Medkova ◽

G. Dong ◽

A. Hutagalung ◽

K. Reinisch ◽

...

Keyword(s):

Amino Acids ◽

Rho Gtpases ◽

Rab Gtpase ◽

Secretory Vesicles ◽

Helical Bundles ◽

Protein Receptor ◽

Lethal Giant Larvae ◽

Target Membrane ◽

A Subunit ◽

Exocytic Pathway

Sec2p is the exchange factor that activates Sec4p, the Rab GTPase controlling the final stage of the yeast exocytic pathway. Sec2p is recruited to secretory vesicles by Ypt32-GTP, a Rab controlling exit from the Golgi. Sec15p, a subunit of the octameric exocyst tethering complex and an effector of Sec4p, binds to Sec2p on secretory vesicles, displacing Ypt32p. Sec2p mutants defective in the region 450–508 amino acids bind to Sec15p more tightly. In these mutants, Sec2p accumulates in the cytosol in a complex with the exocyst and is not recruited to vesicles by Ypt32p. Thus the region 450–508 amino acids negatively regulates the association of Sec2p with the exocyst, allowing it to recycle on to new vesicles. The structures of one nearly full-length exocyst subunit and three partial subunits have been determined and, despite very low sequence identity, all form rod-like structures built of helical bundles stacked end to end. These rods may bind to each other along their sides to form the assembled complex. While Sec15p binds Sec4-GTP on the vesicle, other subunits bind Rho GTPases on the plasma membrane, thus tethering vesicles to exocytic sites. Sec4-GTP also binds Sro7p, a yeast homologue of the Drosophila lgl (lethal giant larvae) tumour suppressor. Sro7 also binds to Sec9p, a SNAP25 (25 kDa synaptosome-associated protein)-like t-SNARE [target-membrane-associated SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor)], and can form a Sec4p–Sro7p–Sec9p ternary complex. Overexpression of Sec4p, Sro7p or Sec1p (another SNARE regulator) can bypass deletions of three different exocyst subunits. Thus promoting SNARE function can compensate for tethering defects.

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