scholarly journals Maturation of the yeast plasma membrane [H+]ATPase involves phosphorylation during intracellular transport.

1991 ◽  
Vol 115 (2) ◽  
pp. 289-295 ◽  
Author(s):  
A Chang ◽  
C W Slayman
Keyword(s):  
Plasma Membrane ◽  
Atpase Activity ◽  
Intracellular Transport ◽  
Secretory Pathway ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Glucose Starvation ◽  
Phosphopeptide Analysis ◽  
Membrane Phosphorylation

In this study we show that the plasma membrane [H+]ATPase of Saccharomyces cerevisiae is phosphorylated on multiple Ser and Thr residues in vivo. Phosphorylation occurs during the movement of newly synthesized ATPase from the ER to the cell surface, as revealed by the analysis of temperature-sensitive sec mutants blocked at successive steps of the secretory pathway. Two-dimensional phosphopeptide analysis of the ATPase indicates that, although most sites are phosphorylated at or before arrival in secretory vesicles, some phosphopeptides are unique to the plasma membrane. Phosphorylation of plasma membrane-specific site(s) is associated with increased ATPase activity during growth on glucose. Upon glucose starvation, dephosphorylation occurs concomitantly with a decrease in enzymatic activity, and both are rapidly reversed (within 2 min) upon readdition of glucose. We suggest that reversible, site-specific phosphorylation serves to adjust ATPase activity in response to nutritional signals.

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.

scholarly journals Promiscuity in Rab–SNARE Interactions

1999 ◽  
Vol 10 (12) ◽  
pp. 4149-4161 ◽  
Author(s):  
Eric Grote ◽  
Peter J. Novick
Keyword(s):  
Plasma Membrane ◽  
Secretory Pathway ◽  
Snare Complex ◽  
Free Form ◽  
Rab Proteins ◽  
Secretory Vesicles ◽  
Rab Protein ◽  
Target Membrane ◽  
Low Efficiency

Fusion of post-Golgi secretory vesicles with the plasma membrane in yeast requires the function of a Rab protein, Sec4p, and a set of v- and t-SNAREs, the Snc, Sso, and Sec9 proteins. We have tested the hypothesis that a selective interaction between Sec4p and the exocytic SNAREs is responsible for ensuring that secretory vesicles fuse with the plasma membrane but not with intracellular organelles. Assembly of Sncp and Ssop into a SNARE complex is defective in asec4-8 mutant strain. However, Snc2p binds in vivo to many other syntaxin-like t-SNAREs, and binding of Sncp to the endosomal/Golgi t-SNARE Tlg2p is also reduced in sec4-8cells. In addition, binding of Sncp to Ssop is reduced by mutations in two other Rab genes and four non-Rab genes that block the secretory pathway before the formation of secretory vesicles. In an alternate approach to look for selective Rab–SNARE interactions, we report that the nucleotide-free form of Sec4p coimmunoprecipitates with Ssop. However, Rab–SNARE binding is nonselective, because the nucleotide-free forms of six Rab proteins bind with similar low efficiency to three SNARE proteins, Ssop, Pep12p, and Sncp. We conclude that Rabs and SNAREs do not cooperate to specify the target membrane.

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 pH-dependent Cargo Sorting from the Golgi

2011 ◽  
Vol 286 (12) ◽  
pp. 10058-10065 ◽  
Author(s):  
Chunjuan Huang ◽  
Amy Chang
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Atpase Activity ◽  
Secretory Pathway ◽  
Ph Dependence ◽  
Glucose Deprivation ◽  
Ph Homeostasis ◽  
Plasma Membrane Proteins ◽  
Cytosolic Ph ◽  
Cargo Sorting

The vacuolar proton-translocating ATPase (V-ATPase) plays a major role in organelle acidification and works together with other ion transporters to maintain pH homeostasis in eukaryotic cells. We analyzed a requirement for V-ATPase activity in protein trafficking in the yeast secretory pathway. Deficiency of V-ATPase activity caused by subunit deletion or glucose deprivation results in missorting of newly synthesized plasma membrane proteins Pma1 and Can1 directly from the Golgi to the vacuole. Vacuolar mislocalization of Pma1 is dependent on Gga adaptors although no Pma1 ubiquitination was detected. Proper cell surface targeting of Pma1 was rescued in V-ATPase-deficient cells by increasing the pH of the medium, suggesting that missorting is the result of aberrant cytosolic pH. In addition to mislocalization of the plasma membrane proteins, Golgi membrane proteins Kex2 and Vrg4 are also missorted to the vacuole upon loss of V-ATPase activity. Because the missorted cargos have distinct trafficking routes, we suggest a pH dependence for multiple cargo sorting events at the Golgi.

scholarly journals Retrograde Transport of Golgi-localized Proteins to the ER

1998 ◽  
Vol 140 (1) ◽  
pp. 1-15 ◽  
Author(s):  
Nelson B. Cole ◽  
Jan Ellenberg ◽  
Jia Song ◽  
Diane DiEuliis ◽  
Jennifer Lippincott-Schwartz
Keyword(s):  
Plasma Membrane ◽  
Golgi Complex ◽  
Fusion Proteins ◽  
Secretory Pathway ◽  
Molecular Mechanisms ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Viral Glycoprotein ◽  
Lumenal Domain

The ER is uniquely enriched in chaperones and folding enzymes that facilitate folding and unfolding reactions and ensure that only correctly folded and assembled proteins leave this compartment. Here we address the extent to which proteins that leave the ER and localize to distal sites in the secretory pathway are able to return to the ER folding environment during their lifetime. Retrieval of proteins back to the ER was studied using an assay based on the capacity of the ER to retain misfolded proteins. The lumenal domain of the temperature-sensitive viral glycoprotein VSVGtsO45 was fused to Golgi or plasma membrane targeting domains. At the nonpermissive temperature, newly synthesized fusion proteins misfolded and were retained in the ER, indicating the VSVGtsO45 ectodomain was sufficient for their retention within the ER. At the permissive temperature, the fusion proteins were correctly delivered to the Golgi complex or plasma membrane, indicating the lumenal epitope of VSVGtsO45 also did not interfere with proper targeting of these molecules. Strikingly, Golgi-localized fusion proteins, but not VSVGtsO45 itself, were found to redistribute back to the ER upon a shift to the nonpermissive temperature, where they misfolded and were retained. This occurred over a time period of 15 min–2 h depending on the chimera, and did not require new protein synthesis. Significantly, recycling did not appear to be induced by misfolding of the chimeras within the Golgi complex. This suggested these proteins normally cycle between the Golgi and ER, and while passing through the ER at 40°C become misfolded and retained. The attachment of the thermosensitive VSVGtsO45 lumenal domain to proteins promises to be a useful tool for studying the molecular mechanisms and specificity of retrograde traffic to the ER.

scholarly journals Yeast Cdc42 functions at a late step in exocytosis, specifically during polarized growth of the emerging bud

2001 ◽  
Vol 155 (4) ◽  
pp. 581-592 ◽  
Author(s):  
Joan E. Adamo ◽  
John J. Moskow ◽  
Amy S. Gladfelter ◽  
Domenic Viterbo ◽  
Daniel J. Lew ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Temperature Sensitive ◽  
Secretory Vesicles ◽  
Polarized Growth ◽  
Vesicle Docking ◽  
Actin Organization ◽  
Late Step ◽  
Rho Family ◽  
Localization Data

The Rho family GTPase Cdc42 is a key regulator of cell polarity and cytoskeletal organization in eukaryotic cells. In yeast, the role of Cdc42 in polarization of cell growth includes polarization of the actin cytoskeleton, which delivers secretory vesicles to growth sites at the plasma membrane. We now describe a novel temperature-sensitive mutant, cdc42-6, that reveals a role for Cdc42 in docking and fusion of secretory vesicles that is independent of its role in actin polarization. cdc42-6 mutants can polarize actin and deliver secretory vesicles to the bud, but fail to fuse those vesicles with the plasma membrane. This defect is manifested only during the early stages of bud formation when growth is most highly polarized, and appears to reflect a requirement for Cdc42 to maintain maximally active exocytic machinery at sites of high vesicle throughput. Extensive genetic interactions between cdc42-6 and mutations in exocytic components support this hypothesis, and indicate a functional overlap with Rho3, which also regulates both actin organization and exocytosis. Localization data suggest that the defect in cdc42-6 cells is not at the level of the localization of the exocytic apparatus. Rather, we suggest that Cdc42 acts as an allosteric regulator of the vesicle docking and fusion apparatus to provide maximal function at sites of polarized growth.

Internalization and intracellular transport of folate-binding protein in rat kidney proximal tubule

1993 ◽  
Vol 264 (2) ◽  
pp. C302-C310 ◽  
Author(s):  
H. Birn ◽  
J. Selhub ◽  
E. I. Christensen
Keyword(s):  
Plasma Membrane ◽  
Proximal Tubule ◽  
Brush Border ◽  
Intracellular Transport ◽  
Brush Border Membrane ◽  
Specific Binding ◽  
Binding Protein ◽  
Rat Kidney ◽  
Folate Binding Protein

Folate-binding protein (FBP) is involved in folate reabsorption in the renal proximal tubule. Immunocytochemical studies have located FBP to the brush-border membrane, endocytic vacuoles, and dense apical tubules. We applied the same polyclonal antibody (anti-FBP) against FBP to investigate the dynamic relationship between FBP in the different compartments by microinjecting the antibody into rat kidney proximal tubules in situ. Specific binding of anti-FBP in vivo to the brush-border membrane was followed by fixation at various times. Protein A-gold labeling shows that anti-FBP is transported from endocytic invaginations into vacuoles followed by transport into dense apical tubules within 15 s. Thus FBP is rapidly internalized, and together with previous studies this study strongly suggests recycling of FBP back to the luminal plasma membrane through dense apical tubules. The results are consistent with reabsorption of folate through endocytosis of the FBP-folate complex followed by dissociation and recycling of FBP. When time is allowed there is a steady accumulation of FBP in dense apical tubules combined with an increase in surface density of the same compartment. A possible explanation involves partial inhibition of the fusion between dense apical tubules and plasma membrane because of the anti-FBP labeling of the receptor.

scholarly journals Glucagon receptors in endothelial and Kupffer cells of mouse liver.

1988 ◽  
Vol 36 (9) ◽  
pp. 1081-1089 ◽  
Author(s):  
J Watanabe ◽  
K Kanai ◽  
S Kanamura
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Kupffer Cells ◽  
Mouse Liver ◽  
Intracellular Transport ◽  
Coated Pits ◽  
Sinusoidal Cells ◽  
Nonparenchymal Cells ◽  
Glucagon Receptors

To determine whether hepatic sinusoidal cells contain glucagon receptors and, if so, to study the significance of the receptors in the cells, binding of [125I]-glucagon to nonparenchymal cells (mainly endothelial cells and Kupffer cells) isolated from mouse liver was examined by quantitative autoradiography and biochemical methods. Furthermore, the pathway of intracellular transport of colloidal gold-labeled glucagon (AuG) was examined in vivo. Autoradiographic and biochemical results demonstrated many glucagon receptors in both endothelial cells and Kupffer cells, and more receptors being present in endothelial cells than in Kupffer cells. In vivo, endothelial cells internalized AuG particles into coated vesicles via coated pits and transported the particles to endosomes, lysosomes, and abluminal plasma membrane. Therefore, receptor-mediated transcytosis of AuG occurs in endothelial cells. The number of particles present on the abluminal plasma membrane was constant if the amount of injected AuG increased. Therefore, the magnitude of receptor-mediated transcytosis of AuG appears to be regulated by endothelial cells. Kupffer cells internalized the ligand into cytoplasmic tubular structures via plasma membrane invaginations and transported the ligand exclusively to endosomes and lysosomes, suggesting that the ligand is degraded by Kupffer cells.

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

1998 ◽  
Vol 9 (7) ◽  
pp. 1725-1739 ◽  
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.

scholarly journals Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport.

1991 ◽  
Vol 114 (4) ◽  
pp. 671-679 ◽  
Author(s):  
T Oka ◽  
S Nishikawa ◽  
A Nakano
Keyword(s):  
Temperature Sensitivity ◽  
Protein Function ◽  
Secretory Pathway ◽  
Protein Transport ◽  
Temperature Sensitive ◽  
Transport Reaction ◽  
Golgi Transport ◽  
Gtp Binding

In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus. In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant. In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products. First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction. Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes. The analysis of Sar1p partially purified by E. coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function.

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