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Accueil > Agenda > Les séminaires Jean Roche > Control of vacuole biogenesis by protein modifications.

Control of vacuole biogenesis by protein modifications.

Lundi 10 octobre 2006,11h, salle Lissitzky.

Bibliographie

1 : J Cell Sci. 2005 Sep 1 ;118(Pt 17):3819-28.

Functions of SNAREs in intracellular membrane fusion and lipid bilayer mixing. Ungermann C, Langosch D.

Biochemie Zentrum der Universitat Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.

Intracellular membrane fusion occurs with exquisite coordination and specificity. Each fusion event requires three basic components : Rab-GTPases organize the fusion site ; SNARE proteins act during fusion ; and N-ethylmaleimide-sensitive factor (NSF) plus its cofactor alpha-SNAP are required for recycling or activation of the fusion machinery. Whereas Rab-GTPases seem to mediate the initial membrane contact, SNAREs appear to lie at the center of the fusion process. It is known that formation of complexes between SNAREs from apposed membranes is a prerequisite for lipid bilayer mixing ; however, the biophysics and many details of SNARE function are still vague. Nevertheless, recent observations are shedding light on the role of SNAREs in membrane fusion. Structural studies are revealing the mechanisms by which SNARES form complexes and interact with other proteins. Furthermore, it is now apparent that the SNARE transmembrane segment not only anchors the protein but engages in SNARE-SNARE interactions and plays an active role in fusion. Recent work indicates that the fusion process itself may comprise two stages and proceed via a hemifusion intermediate. http://jcs.biologists.org/cgi/content/full/118/17/3819

2 : EMBO Rep. 2004 Nov ;5(11):1053-7.

On the mechanism of protein palmitoylation.

Dietrich LE, Ungermann C.

Biochemie-Zentrum der Universitat Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.

Protein palmitoylation or, more specifically, S-acylation is a reversible post-translational lipid modification. Despite the identification of several proteins that are altered in this way, our understanding of the enzymology of this process has been hampered by the lack of well-characterized acyltransferases. We now know of three proteins in Saccharomyces cerevisiae that promote palmitoylation : effector of Ras function (Erf2), ankyrin-repeat-containing protein (Akr1) and the SNARE protein Ykt6. Erf2 and Akr1 are integral membrane proteins that contain a cysteine-rich domain and an Asp-His-His-Cys motif, both of which catalyse acylation at the carboxyl terminus of their target proteins. Recently, we discovered that Ykt6 mediates the amino-terminal acylation of the fusion protein Vac8. Even though these three proteins differ in sequence, topology, size and substrate specificity, they might function in a similar manner. In this review, we discuss these observations in the context of a potential general mechanism of acylation. http://www.nature.com/embor/journal/v5/n11/full/7400277.html

3 : Trends Biochem Sci. 2004 Dec ;29(12):682-8.

Longins and their longin domains : regulated SNAREs and multifunctional SNARE regulators.

Rossi V, Banfield DK, Vacca M, Dietrich LE, Ungermann C, D’Esposito M, Galli T, Filippini F.

Molecular Biology and Bioinformatics Unit (MOLBINFO), Department of Biology, University of Padua, 35131 Padua, Italy.

Longins are the only R-SNAREs that are common to all eukaryotes and are characterized by a conserved N-terminal domain with a profilin-like fold called a longin domain (LD). These domains seem to be essential for regulating membrane trafficking and they mediate unexpected biochemical functions via a range of protein-protein and intramolecular binding specificities. In addition to the longins, proteins involved in the regulation of intracellular trafficking, such as subunits of the adaptor and transport protein particle complexes, also have LD-like folds. The functions and cellular localization of longins are regulated at several levels and the longin prototypes TI-VAMP, Sec22 and Ykt6 show different distributions among eukaryotes, reflecting their modular and functional diversity. In mammals, TI-VAMP and Ykt6 are crucial for neuronal function, and defects in longin structure or function might underlie some human neurological pathologies. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6TCV-4DSPN6S-5&_coverDate=12%2F01%2F2004&_alid=319141096&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=5180&_sort=d&view=c&_acct=C000009002&_version=1&_urlVersion=0&_userid=113324&md5=a9f789ef00d2e9a726ef3297312e937e

4 : EMBO J. 2004 Jan 14 ;23(1):45-53. Epub 2003 Dec 11.

The SNARE Ykt6 mediates protein palmitoylation during an early stage of homotypic vacuole fusion.

Dietrich LE, Gurezka R, Veit M, Ungermann C.

Biochemie-Zentrum Heidelberg (BZH), University of Heidelberg, Heidelberg, Germany.

The NSF homolog Sec18 initiates fusion of yeast vacuoles by disassembling cis-SNARE complexes during priming. Sec18 is also required for palmitoylation of the fusion factor Vac8, although the acylation machinery has not been identified. Here we show that the SNARE Ykt6 mediates Vac8 palmitoylation and acts during a novel subreaction of vacuole fusion. This subreaction is controlled by a Sec17-independent function of Sec18. Our data indicate that Ykt6 presents Pal-CoA via its N-terminal longin domain to Vac8, while transfer to Vac8’s SH4 domain occurs spontaneously and not enzymatically. The conservation of Ykt6 and its localization to several organelles suggest that its acyltransferase activity may also be required in other intracellular fusion events. http://www.nature.com/emboj/journal/v23/n1/full/7600015a.html

5 : Nat Cell Biol. 2005 Feb ;7(2):103-4.

Expanding dynamin : from fission to fusion.

Peplowska K, Ungermann C.

6 : Biochim Biophys Acta. 2003 Aug 18 ;1641(2-3):111-9.

Control of eukaryotic membrane fusion by N-terminal domains of SNARE proteins.

Dietrich LE, Boeddinghaus C, LaGrassa TJ, Ungermann C.

Biochemie Zentrum Heidelberg (BZH), University of Heidelberg, Im Neuenheimer Feld 328, 69120, Heidelberg, Germany. cu2 ix.urz.uni-heidelberg.de

SNARE proteins function at the center of membrane fusion reactions by forming complexes with each other via their coiled-coil domains. Several SNAREs have N-terminal domains (NTDs) that precede the coiled-coil domain and have critical functions in regulating the fusion cascade. This review will highlight recent findings on NTDs of syntaxins, the longin domain of VAMP proteins and SNAP-23/25 homologues in yeast. Biochemical and genetic experiments as well as the resolution of several NMR and crystal structures of SNARE NTDs shed light on their diverse function. http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T20-491R9WH-1&_coverDate=08%2F18%2F2003&_alid=319143485&_rdoc=1&_fmt=&_orig=search&_qd=1&_cdi=4904&_sort=d&view=c&_acct=C000009002&_version=1&_urlVersion=0&_userid=113324&md5=ae5f34fd2b8f605fcf3e61f3551241fe

7 : J Biol Chem. 2003 Jan 17 ;278(3):1656-62. Epub 2002 Nov 8

The transmembrane domain of Vam3 affects the composition of cis- and trans-SNARE complexes to promote homotypic vacuole fusion.

Rohde J, Dietrich L, Langosch D, Ungermann C.

Interdisziplinares Zentrum fur Neurowissenschaften, University of Heidelberg, Im Neuenheimer Feld 307, 69120 Heidelberg, Germany.

It is presently not clear how the function of SNARE proteins is affected by their transmembrane domains. Here, we analyzed the role of the transmembrane domain of the vacuolar SNARE Vam3 by replacing it by a lipid anchor. Vacuoles with mutant Vam3 fuse poorly and have increased amounts of cis-SNARE complexes, indicating that they are more stable. As a consequence efficient cis-SNARE complex disassembly that occurs at priming as a prerequisite of fusion requires addition of exogenous Sec18. trans-SNARE complexes in this mutant accumulate up to 4-fold over wild type, suggesting that the transmembrane domain of Vam3 is required to transit through this step. Finally, palmitoylation of Vac8, a reaction that also occurs early during priming is reduced by almost one-half. Since palmitoylated Vac8 is required beyond trans-SNARE complex formation, this may partially explain the fusion deficiency. http://www.jbc.org/cgi/content/full/278/3/1656

8 : J Biol Chem. 2005 Apr 15 ;280(15):15348-55. Epub 2005 Feb 8 . ATP-independent control of Vac8 palmitoylation by a SNARE subcomplex on yeast vacuoles.

Dietrich LE, LaGrassa TJ, Rohde J, Cristodero M, Meiringer CT, Ungermann C.

Biochemie-Zentrum der Universitat Heidelberg, Im Neuenheime r Feld 328, 69120 Heidelberg, Germany.

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. http://www.jbc.org/cgi/content/full/280/15/15348

9 : EMBO Rep. 2005 Mar ;6(3):245-50.

The SNARE Ykt6 is released from yeast vacuoles during an early stage of fusion.

Dietrich LE, Peplowska K, LaGrassa TJ, Hou H, Rohde J, Ungermann C.

Biochemie-Zentrum der Universitat Heidelberg (BZH), Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.

The farnesylated SNARE (N-ethylmaleimide-s ensitive factor attachment protein receptor) Ykt6 mediates protein palmitoylation at the yeast vacuole by means of its amino-terminal longin domain. Ykt6 is localized equally to membranes and the cytosol, although it is unclear how this distribution is mediated. We now show that Ykt6 is released efficiently from vacuoles during an early stage of yeast vacuole fusion. This release is dependent on the disassembly of vacuolar SNAREs (priming). In recent literature, it had been demonstrated for mammalian Ykt6 that the membrane-bound form is both palmitoylated and farnesylated at its carboxy-terminal CAAX box, whereas soluble Ykt6 is only farnesylated. In agreement with this, we find that yeast Ykt6 becomes palmitoylated in vitro at its C-terminal CAAX motif. Mutagenesis of the potential palmitoylation site in yeast Ykt6 prevents stable membrane association and is lethal. On the basis of these and other findings, we speculate that Ykt6 is released from membranes by depalmitoylation. Such a mechanism could enable recycling of this lipid-anchored SNARE from the vacuole independent of retrograde transport.

10 : J Cell Biol. 2002 Apr 1 ;157(1):79-89. Epub 2002 Mar 26.

A cycle of Vam7p release from and PtdIns 3-P-dependent rebinding to the yeast vacuole is required for homotypic vacuole fusion.

Boeddi nghaus C, Merz AJ, Laage R, Ungermann C.

Biochemie-Zentrum Heidelberg, University of Heidelberg, 69120 Heidelberg, Germany.

Vacuole fusion requires a coordinated cascade of priming, docking, and fusion. SNARE proteins have been implicated in the fusion itself, although their precise role in the cascade remains unclear. We now report that the vacuolar SNAP-23 homologue Vam7p is a mobile element of the SNARE complex, which moves from an initial association with the cis-SNARE complex via a soluble intermediate to the docking site. Soluble Vam7p is specifically recruited to vacuoles and can rescue a fusion reaction poisoned with antibodies to Vam7p. Both the recombinant Vam7p PX domain and a FYVE domain construct of human Hrs block the recruitment of Vam7p and vacuole fusion, demonstrating that phosphatidylinositol 3-phosphate is a primary receptor of Vam7p on vacuoles. We propose that the Vam7p cycle is linked to the availability of a lipid domain on yeast vacuoles, which is essential for coordinating the fusion reaction prior to and beyond docking. http://www.jcb.org/cgi/content/full/157/1/79

11 : Mol Biol Cell. 2001 Nov ;12(11):3375-85

The N-terminal domain of the t-SNARE Vam3p coordinates priming and docking in yeast vacuole fusion.

Laage R, Ungermann C.

University of Heidelberg, Biochemie Zentrum Heidelberg, 69120 Heidelberg, Germany.

Homotypic fusion of yeast vacuoles require s a regulated sequence of events. During priming, Sec18p disassembles cis-SNARE complexes. The HOPS complex, which is initially associated with the cis-SNARE complex, then mediates tethering. Finally, SNAREs assemble into trans-complexes before the membranes fuse. The t-SNARE of the vacuole, Vam3p, plays a central role in the coordination of these processes. We deleted the N-terminal region of Vam3p to analyze the role of this domain in membrane fusion. The truncated protein (Vam3 Delta N) is sorted normally to the vacuole and is functional, because the vacuolar morphology is unaltered in this strain. However, in vitro vacuole fusion is strongly reduced due to the following reasons : Assembly, as well as disassembly of the cis-SNARE complex is more efficient on Vam3 Delta N vacuoles ; however, the HOPS complex is not associated well with the Vam3 Delta N cis-complex. Thus, primed SNAREs from Vam3 Delta N vacuoles cannot participate efficiently in the reaction because trans-SNARE pairing is substantially reduced. We conclude that the N-terminus of Vam3p is required for coordination of priming and docking during homotypic vacuole fusion. http://www.pubmedcentral.gov/articlerender.fcgi?tool=pubmed&pubmedid=11694574

12 : EMBO J. 2001 Jun15 ;20(12):3145-55

Vac8p release from the SNARE complex and its palmitoylation are coupled and essential for vacuole fusion.

Veit M, Laage R, Dietrich L, Wang L, Ungermann C.

Department of Immunology and Molecular Biology, Vet.-Med. Faculty of the Free University Berlin, Philippstrasse 13, D-10115 Berlin.

Activated fatty acids stimulate budding and fusion in several cell-free assays for vesicular transport. This stimulation is thought to be due to protein palmitoylation, but relevant substrates have not yet been identified. We now report that Vac8p, a protein known to be required for vacuole inheritance, becomes palmitoylated when isolated yeast vacuoles are incubated under conditions that allow membrane fusion. Similar requirements for Vac8p palmitoylation and vacuole fusion, the inhibition of vacuole fusion by antibodies to Vac8p and the strongly reduced fusion of vacuoles lacking Vac8p suggest that palmitoylated Vac8p is essential for homotypic vacuole fusion. Strikingly, palmitoylation of Vac8p is blocked by the addition of antibodies to Sec18p (yeast NSF) only. Consistent with this, a portion of Vac8p is associated with the SNARE complex on vacuoles, which is lost during Sec18p- and ATP-dependent priming. During or after SNARE complex disassembly, palmitoylation occurs and anchors Vac8p to the vacuolar membrane. We propose that palmitoylation of Vac8p is regulated by the same machinery that controls membrane fusion. http://www.nature.com/emboj/journal/v20/n12/abs/7593812a.html

13 : J Biol Chem. 2001 Mar 2 ;276(9):6200-6. Epub 2000 Nov 15

Inhibition of the Ca(2+)-ATPase Pmc1p by the v-SNARE protein Nyv1p.

Takita Y, Engstrom L, Ungermann C, Cunningham KW.

Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.

Pmc1p, the Ca(2+)-ATPase of budding yeast related to plasma membrane Ca(2+)-ATPases of animals, is transcriptionally up-regulated in response to signaling by the calmodulin-calcineurin-Tcn1p/Crz1p signaling pathway. Little is known about post-translational regulation of Pmc1p. In a genetic screen for potential negative regulators of Pmc1p, a vacuolar v-SNARE protein, Nyv1p, was recovered. Cells overproducing Nyv1p show decreased Ca(2+) tolerance and decreased accumulation of Ca(2+) in the vacuole, similar to pmc1 null mutants. Overexpression of Nyv1p had no such effects on pmc1 mutants, suggesting that Nyv1p may inhibit Pmc1p function. Overexpression of Nyv1p did not decrease Pmc1p levels but decreased the specific ATP-dependent Ca(2+) transport activity of Pmc1p in purified vacuoles by at least 2-fold. The effect of Nyv1p on Pmc1p function is likely to be direct because native immunoprecipitation experiments showed that Pmc1p coprecipitated with Nyv1p. Complexes between Nyv1p and its t-SNARE partner Vam3p were also isolated, but these complexes lacked Pmc1p. We conclude that Nyv1p can interact physically with Pmc1p and inhibit its Ca(2+) transport activity in the vacuole membrane. This is the first example of a Ca(2+)-ATPase regulation by a v-SNARE protein involved in membrane fusion reactions. http://www.jbc.org/cgi/content/full/276/9/6200

14 : J Cell Biol. 2000 Mar 20 ;148(6):1231-8.

The docking stage of yeast vacuole fusion requires the transfer of proteins from a cis-SNARE complex to a Rab/Ypt protein.

Price A, Seals D, Wickner W, Ungermann C.

Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03755-3844, USA.

The homotypic fusion of yeast vacuoles requires Sec18p (NSF)-driven priming to allow vacuole docking, but the mechanism that links priming and docking is unknown. We find that a large multisubunit protein called the Vam2/6p complex is bound to cis-paired SNAP receptors (SNAREs) on isolated vacuoles. This association of the Vam2/6p complex with the cis-SNARE complex is disrupted during priming. The Vam2/6p complex then binds to Ypt7p, a guanosine triphosphate binding protein of the Rab family, to initiate productive contact between vacuoles. Thus, cis-SNARE complexes can contain Rab/Ypt effectors, and these effectors can be mobilized by NSF/Sec18p-driven priming, allowing their direct association with a Rab/Ypt protein to activate docking. http://www.jcb.org/cgi/content/full/148/6/1231

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