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1.
J Biol Chem ; 286(28): 25039-46, 2011 Jul 15.
Article in English | MEDLINE | ID: mdl-21550981

ABSTRACT

Retrograde vesicular transport from the Golgi to the ER requires the Dsl1 tethering complex, which consists of the three subunits Dsl1, Dsl3, and Tip20. It forms a stable complex with the SNAREs Ufe1, Use1, and Sec20 to mediate fusion of COPI vesicles with the endoplasmic reticulum. Here, we analyze molecular interactions between five SNAREs of the ER (Ufe1, Use1, Sec20, Sec22, and Ykt6) and the Dsl1 complex in vitro and in vivo. Of the two R-SNAREs, Sec22 is preferred over Ykt6 in the Dsl-SNARE complex. The NSF homolog Sec18 can displace Ykt6 but not Sec22, suggesting a regulatory function for Ykt6. In addition, our data also reveal that subunits of the Dsl1 complex (Dsl1, Dsl3, and Tip20), as well as the SNAREs Ufe1 and Sec20, are ER-resident proteins that do not seem to move into COPII vesicles. Our data support a model, in which a tethering complex is stabilized at the organelle membrane by binding to SNAREs, recognizes the incoming vesicle via its coat and then promotes its SNARE-mediated fusion.


Subject(s)
COP-Coated Vesicles/metabolism , Endoplasmic Reticulum/metabolism , Membrane Fusion/physiology , Multiprotein Complexes/metabolism , SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/metabolism , COP-Coated Vesicles/genetics , Endoplasmic Reticulum/genetics , Models, Biological , Multiprotein Complexes/genetics , Protein Subunits/genetics , Protein Subunits/metabolism , SNARE Proteins/genetics , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Soluble N-Ethylmaleimide-Sensitive Factor Attachment Proteins/genetics
2.
Traffic ; 9(9): 1510-21, 2008 Sep.
Article in English | MEDLINE | ID: mdl-18541004

ABSTRACT

The dually lipidated SNARE Ykt6 is found on intracellular membranes and in the cytosol. In this study, we show that Ykt6 localizes to the Golgi as well as endosomal and vacuolar membranes in vivo. The ability of Ykt6 to cycle between the cytosol and the membranes depends on the intramolecular interaction of the N-terminal longin and C-terminal SNARE domains and not on either domain alone. A mutant deficient in this interaction accumulates on membranes and--in contrast to the wild-type protein--does not get released from vacuoles. Our data also indicate that Ykt6 is a substrate of the DHHC (Asp-His-His-Cys) acyltransferase network. Overexpression of the vacuolar acyltransferase Pfa3 drives the F42S mutant not only to the vacuole but also into the vacuolar lumen. Thus, depalmitoylation and release of Ykt6 are needed for its recycling and to circumvent its entry into the endosomal multivesicular body pathway.


Subject(s)
Endosomes/metabolism , Intracellular Membranes/metabolism , Lipoylation , R-SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Acyltransferases/metabolism , Cytosol/metabolism , Electrophoresis, Polyacrylamide Gel , Golgi Apparatus/metabolism , Membrane Fusion , Point Mutation , Protein Transport , R-SNARE Proteins/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Vacuoles/metabolism
3.
Autophagy ; 4(1): 5-19, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17932463

ABSTRACT

Vesicular transport in eukaryotic cells is concluded with the consumption of the vesicle at the target membrane. This fusion process relies on Rabs, tethers and SNAREs. Powerful in vitro fusion systems using isolated organelles were crucial to obtain insights into the underlying mechanism of membrane fusion- from the initiation of fusion to lipid bilayer mixing. Among these systems, yeast vacuoles turned out to be particularly useful as they can be manipulated biochemically and genetically. Studies relying on this organelle have revealed insights into the connection of vacuole fusion to endomembrane biogenesis. A number of fusion factors were identified and characterized over the last several years, and placed into the fusion cascade. Within this review, we will present and discuss the current state of our knowledge on vacuole fusion.


Subject(s)
Intracellular Membranes/metabolism , Membrane Fusion/physiology , Saccharomyces cerevisiae/cytology , Vacuoles/metabolism , Endosomes/metabolism , Golgi Apparatus/metabolism , Membrane Lipids/metabolism , SNARE Proteins/metabolism , Saccharomyces cerevisiae Proteins/metabolism , trans-Golgi Network/metabolism
4.
Methods ; 40(2): 171-6, 2006 Oct.
Article in English | MEDLINE | ID: mdl-17012029

ABSTRACT

A protein's function depends on its localization to the right cellular compartment. A number of proteins require lipidation to associate with membranes. Protein palmitoylation is a reversible lipid modification and has been shown to mediate both membrane localization and control protein function. At the yeast vacuole, several palmitoylated proteins have been identified that are required for vacuole biogenesis, including the fusion factor Vac8, the SNARE Ykt6 and the casein kinase Yck3. Moreover, both the DHHC-CRD acyltransferase Pfa3 and Ykt6 are involved in palmitoylation at the vacuole Here, we present and discuss methods to probe for protein palmitoylation at vacuoles.


Subject(s)
Palmitic Acid/metabolism , Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/metabolism , Acylation , Acyltransferases/physiology
5.
J Cell Sci ; 119(Pt 12): 2477-85, 2006 Jun 15.
Article in English | MEDLINE | ID: mdl-16720644

ABSTRACT

Palmitoylation stably anchors specific proteins to membranes, but may also have a direct effect on the function of a protein. The yeast protein Vac8 is required for efficient vacuole fusion, inheritance and cytosol-to-vacuole trafficking. It is anchored to vacuoles by an N-terminal myristoylation site and three palmitoylation sites, also known as the SH4 domain. Here, we address the role of Vac8 palmitoylation and show that the position and number of substrate cysteines within the SH4 domain determine the vacuole localization of Vac8: stable vacuole binding of Vac8 requires two cysteines within the N-terminus, regardless of the combination. Importantly, our data suggest that palmitoylation adds functionality to Vac8 beyond simple localization. A mutant Vac8 protein, in which the palmitoylation sites were replaced by a stretch of basic residues, still localizes to vacuole membranes and functions in cytosol-to-vacuole transport, but can only complement the function of Vac8 in morphology and inheritance if it also contains a single cysteine within the SH4 domain. Our data suggest that palmitoylation is not a mere hydrophobic anchor required solely for localization, but influences the protein function(s).


Subject(s)
Lipoproteins/metabolism , Membrane Proteins/metabolism , Palmitic Acid/metabolism , Protein Processing, Post-Translational/physiology , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Vacuoles/physiology , Cell Membrane/metabolism , Cysteine/metabolism , Mutation , Vesicular Transport Proteins , src Homology Domains/genetics , src Homology Domains/physiology
6.
J Biol Chem ; 280(15): 15348-55, 2005 Apr 15.
Article in English | MEDLINE | ID: mdl-15701652

ABSTRACT

Yeast vacuole fusion requires palmitoylated Vac8. We previously showed that Vac8 acylation occurs early in the fusion reaction, is blocked by antibodies against Sec18 (yeast N-ethylmaleimide-sensitive fusion protein (NSF)), and is mediated by the R-SNARE Ykt6. Here we analyzed the regulation of this reaction on purified vacuoles. We show that Vac8 acylation is restricted to a narrow time window, is independent of ATP hydrolysis by Sec18, and is stimulated by the ion chelator EDTA. Analysis of vacuole protein complexes indicated that Ykt6 is part of a complex distinct from the second R-SNARE, Nyv1. We speculate that during vacuole fusion, Nyv1 is the classical R-SNARE, whereas the Ykt6-containing complex has a novel function in Vac8 palmitoylation.


Subject(s)
Adenosine Triphosphate/metabolism , Lipoproteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Vesicular Transport Proteins/metabolism , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/chemistry , Dose-Response Relationship, Drug , Edetic Acid/chemistry , Electrophoresis, Polyacrylamide Gel , Genotype , Glutathione Transferase/metabolism , Hydrolysis , Immunoprecipitation , Membrane Proteins/chemistry , Membrane Proteins/physiology , Microscopy, Phase-Contrast , Models, Biological , Palmitic Acid/metabolism , Protein Binding , R-SNARE Proteins , Recombinant Proteins/chemistry , SNARE Proteins , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/physiology , Time Factors , Vacuoles/metabolism
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