scholarly journals In vitro fusion between Saccharomyces cerevisiae secretory vesicles and cytoplasmic-side-out plasma membrane vesicles

2003 ◽  
Vol 370 (2) ◽  
pp. 641-649 ◽  
Author(s):  
Lorena ARRASTUA ◽  
Eider SAN SEBASTIAN ◽  
Ana F. QUINCOCES ◽  
Claude ANTONY ◽  
Unai UGALDE
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Secretory Pathway ◽  
Membrane Vesicles ◽  
Fusion Process ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Plasma Membrane Vesicles ◽  
Fusion Event ◽  
Cytoplasmic Side

The final step in the secretory pathway, which is the fusion event between secretory vesicles and the plasma membrane, was reconstructed using highly purified secretory vesicles and cytoplasmic-side-out plasma membrane vesicles from the yeast Saccharomyces cerevisiae. Both organelle preparations were obtained from a sec 6-4 temperature-sensitive mutant. Fusion was monitored by means of a fluorescence assay based on the dequenching of the lipophilic fluorescent probe octadecylrhodamine B-chloride (R18). The probe was incorporated into the membrane of secretory vesicles, and it diluted in unlabelled cytoplasmic-side-out plasma membrane vesicles as the fusion process took place. The obtained experimental dequenching curves were found by mathematical analysis to consist of two independent but simultaneous processes. Whereas one of them reflected the fusion process between both vesicle populations as confirmed by its dependence on the assay conditions, the other represented a non-specific transfer of the probe. The fusion process may now be examined in detail using the preparation, validation and analytical methods developed in this study.

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

2012 ◽  
Vol 11 (5) ◽  
pp. 590-600 ◽  
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.

Electroimmunochemical analysis of plasma membrane vesicles from Saccharomyces cerevisiae

1982 ◽  
Vol 60 (6) ◽  
pp. 659-667
Author(s):  
James H. Gerlach ◽  
Ole J. Bjerrum ◽  
Gerald H. Rank
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Concanavalin A ◽  
Periodic Acid ◽  
Membrane Vesicles ◽  
Free Form ◽  
Plasma Membrane Vesicles ◽  
Crossed Immunoelectrophoresis ◽  
Lentil Lectin ◽  
Triton X

Plasma membrane vesicles of Saccharomyces cerevisiae were extracted with 1% (w/v) Triton X-100 and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies against the vesicles. Solubilization was shown to be nonselective and 23 immunoprecipitates were observed reproducibly.Four glycoproteins were identified by interaction with concanavalin A and lentil lectin, either immobilized on agarose beads in an intermediate gel or incorporated in the free form in the first dimension gel. One glycoprotein was stainable by the periodic acid – Schiff procedure. None of the glycoproteins had their origin in the cell wall.Five amphiphilic proteins were identified on the basis of charge-shift and hydrophobic interaction crossed immunoelectrophoresis as well as [14C]Triton X-100 and Sudan black B binding. Three of the amphiphilic proteins were also glycoproteins.Based on the carbohydrate content and amphiphilic properties of the proteins, purification schemes using concanavalin A-Sepharose and phenyl-Sepharose were proposed. Trial separations using 1-mL columns were monitored by fused rocket and crossed immunoelectrophoresis.

Evidence for a selective and electroneutral K+/H+-exchange in Saccharomyces cerevisiae using plasma membrane vesicles

Yeast ◽  
1996 ◽  
Vol 12 (13) ◽  
pp. 1301-1313 ◽  
Author(s):  
Carole Camarasa ◽  
Susana Prieto ◽  
Roc Ros ◽  
Jean-Michel Salmon ◽  
Pierre Barre
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Plasma Membrane Vesicles

scholarly journals Sec6p Anchors the Assembled Exocyst Complex at Sites of Secretion

2009 ◽  
Vol 20 (3) ◽  
pp. 973-982 ◽  
Author(s):  
Jennifer A. Songer ◽  
Mary Munson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Protein Stability ◽  
Protein Complex ◽  
Mutational Analysis ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Conserved Residues ◽  
Temperature Sensitive Mutants ◽  
Exocyst Complex

The exocyst is an essential protein complex required for targeting and fusion of secretory vesicles to sites of exocytosis at the plasma membrane. To study the function of the exocyst complex, we performed a structure-based mutational analysis of the Saccharomyces cerevisiae exocyst subunit Sec6p. Two “patches” of highly conserved residues are present on the surface of Sec6p; mutation of either patch does not compromise protein stability. Nevertheless, replacement of SEC6 with the patch mutants results in severe temperature-sensitive growth and secretion defects. At nonpermissive conditions, although trafficking of secretory vesicles to the plasma membrane is unimpaired, none of the exocyst subunits are polarized. This is consistent with data from other exocyst temperature-sensitive mutants, which disrupt the integrity of the complex. Surprisingly, however, these patch mutations result in mislocalized exocyst complexes that remain intact. Our results indicate that assembly and polarization of the exocyst are functionally separable events, and that Sec6p is required to anchor exocyst complexes at sites of secretion.

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