Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

Wen, Wenyu; Chen, Lu; Wu, Hao; Sun, Xin; Zhang, Mingjie; Banfield, David K

doi:10.1091/mbc.e06-02-0128

Cited by 41 publications

(53 citation statements)

References 56 publications

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“…Other SNARE N-domain functions have been explored. The Nyv1p N-terminal longin domain is needed for its trafficking to the vacuole (30), although it may have additional functions as well. Several syntaxins have three-coiled coils as N-domains, but only in certain isoforms do they interact directly with the Cterminal SNARE domains (31).…”

Section: Discussionmentioning

confidence: 99%

N-terminal domain of vacuolar SNARE Vam7p promotes trans -SNARE complex assembly

Wickner

2012

Proc. Natl. Acad. Sci. U.S.A.

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SNARE-dependent membrane fusion in eukaryotic cells requires that the heptad-repeat SNARE domains from R-and Q-SNAREs, anchored to apposed membranes, assemble into four-helix coiled-coil bundles. In addition to their SNARE and transmembrane domains, most SNAREs have N-terminal domains (N-domains), although their functions are unclear. The N-domain of the yeast vacuolar Qc-SNARE Vam7p is a binding partner for the homotypic fusion and vacuole protein sorting complex (a master regulator of vacuole fusion) and has Phox homology, providing a phosphatidylinositol 3-phosphate (PI3P)-specific membrane anchor. We now report that this Vam7p N-domain has yet another role, one that does not depend on its physical connection to the Vam7p SNARE domain. By attaching a transmembrane anchor to the C terminus of Vam7p to create Vam7tm, we bypass the requirement for the N-domain to anchor Vam7tm to reconstituted proteoliposomes. The N-domain of Vam7tm is indispensible for trans-SNARE complex assembly in SNARE-only reactions. Introducing Vam7(1-125)p as a separate recombinant protein suppresses the defect caused by N-domain deletion from Vam7tm, demonstrating that the function of this N-domain is not constrained to covalent attachment to Vam7p. The Vam7p N-domain catalyzes the docking of apposed membranes by promoting transinteractions between R-and Q-SNAREs. This function of the Vam7p Ndomain depends on the presence of PI3P and its affinity for PI3P. Added N-domain can even promote SNARE complex assembly when Vam7 still bears its own N-domain.phosphoinositide | Phox homology domain M embranes fuse by conserved mechanisms during endocytic and exocytic vesicular trafficking (1). The initial step of association between membranes, termed tethering, is mediated by a Rab-family GTPase and its associated tethering proteins (2). Selected proteins and lipids then become enriched in a fusioncompetent microdomain. SNARE proteins anchored to apposed membranes form four-helix bundles, the trans-SNARE complex, in conjunction with SNARE-binding factors such as Sec1-Munc18 proteins and, at neuronal synapses, Munc13, complexin, and synaptotagmin (3, 4). Although SNARE proteins are central to the ensuing fusion, little is known of the roles of their N-domains.We study fusion mechanisms with yeast vacuoles (lysosomes) (5). Vacuoles undergo constant homotypic fusion and fission in the cell in response to the osmolarity of the growth environment. When fusion is genetically impaired, continuing fission causes a fragmented vacuole morphology, the vam phenotype (6), which allowed identification of the genes for proteins that catalyze vacuole fusion. Their roles were confirmed and extended through studies of the fusion of the isolated organelle (7). Vacuole clustering (tethering) is mediated by the Rab GTPase Ypt7p through its direct binding to the hexameric homotypic fusion and vacuole protein sorting (HOPS) complex (8-11). As vacuole membranes are drawn together, the proteins and lipids needed for fusion become enriched in a ring-shaped microdomain whic...

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Section: Discussionmentioning

confidence: 99%

N-terminal domain of vacuolar SNARE Vam7p promotes trans -SNARE complex assembly

Wickner

2012

Proc. Natl. Acad. Sci. U.S.A.

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“…Longin domains are ϳ100 amino acid extension that occur in a small subset of R-SNAREs (three in yeast: Nyv1p, Ykt6p, and Sec22p) as well in several other proteins required for vesicle trafficking (53). The longin domain of Nyv1p has been shown to aid in its membrane targeting and thus facilitate protein sorting to the vacuole (46). Currently there are very few studies that report direct actin-SNARE interactions (54); however several studies report functional co-localization of actin and SNAREs (52,(55)(56)(57).…”

Section: Discussionmentioning

confidence: 99%

“…Because Nyv1p was not identified under native conditions it is likely that actin interacts with SNAREs that are not part of a SNARE complex, and therefore the interaction with Ykt6p may be related its non-SNARE complex functions (44,45). Interestingly, the structures of both Nyv1p and Ykt6p contain N-terminal longin domains extension, which are thought to fold into profilin-like domains (44,46). Profilin is known to bind actin and stimulate cytoskeletal remodeling (47) and because both longin domain SNAREs have the potential to interact with actin, we speculate that this domain may have an important role in directing actin remodeling specifically needed for membrane fusion.…”

Section: Actin-vacuolar Proteinmentioning

confidence: 99%

Stimulation of Actin Polymerization by Vacuoles via Cdc42p-dependent Signaling

Isgandarova

Jones

Forsberg

et al. 2007

Journal of Biological Chemistry

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We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.

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“…In fact, we detected some patterns of coevolution in the different SNARE units, in particular in the Q-SNAREs of group I, but promiscuous interactions between different SNARE subunits may have precluded further diversifications. The phylogenetic trees obtained from SNARE proteins of different eukaryotic species (Figure 3 and Supplementary Material), together with the relative simple domain architecture of the SNAREs (Figure 1), allow for some tentative speculations about the nature of a prototypic SNARE machinery: Because all ancestral R-SNARE types appear to contain an N-terminal domain with a profilin-like fold (Figure 1; Gonzalez et al, 2001;Tochio et al, 2001;Wen et al, 2006), they may have originated from a common ancestor that contained this N-terminal extension. Note that this domain has been lost in the secretory R.IV-SNAREs in fungi and animals.…”

Section: Evolution Of the Snare Apparatus Is Linked To The Developmenmentioning

confidence: 99%

An Elaborate Classification of SNARE Proteins Sheds Light on the Conservation of the Eukaryotic Endomembrane System

Kloepper

Kienle

Fasshauer

2007

MBoC

231

286

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Proteins of the SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) family are essential for the fusion of transport vesicles with an acceptor membrane. Despite considerable sequence divergence, their mechanism of action is conserved: heterologous sets assemble into membrane-bridging SNARE complexes, in effect driving membrane fusion. Within the cell, distinct functional SNARE units are involved in different trafficking steps. These functional units are conserved across species and probably reflect the conservation of the particular transport step. Here, we have systematically analyzed SNARE sequences from 145 different species and have established a highly accurate classification for all SNARE proteins. Principally, all SNAREs split into four basic types, reflecting their position in the four-helix bundle complex. Among these four basic types, we established 20 SNARE subclasses that probably represent the original repertoire of a eukaryotic cenancestor. This repertoire has been modulated independently in different lines of organisms. Our data are in line with the notion that the ur-eukaryotic cell was already equipped with the various compartments found in contemporary cells. Possibly, the development of these compartments is closely intertwined with episodes of duplication and divergence of a prototypic SNARE unit. INTRODUCTIONThe elaborate endomembrane system of eukaryotes is thought to have evolved by invagination of the plasma membrane during perfection of a phagotrophic lifestyle. Subsequently, primitive endomembranes differentiated into the various spatially and functionally separated compartments found in contemporary eukaryotes (Roger, 1999;Cavalier-Smith, 2002). Material exchange is mediated by cargo-loaded vesicles that bud from the donor and eventually fuse with the acceptor compartment. This allows the cells to take up nutrients through the endocytic pathway. Conversely, newly synthesized proteins and lipids are transported within the cell through the exocytic pathway. As each organelle must maintain its identity, vesicular trafficking is tightly regulated (Bonifacino and Glick, 2004). Although some protists possess highly divergent compartments, it is becoming clear that the underlying molecular machineries involved in vesicular trafficking are highly conserved among all eukaryotes. Key players in the concluding step, the fusion of a vesicle with its acceptor membrane, are the so-called SNARE (soluble N-ethylmalemide-sensitive factor attachment protein receptor) proteins (reviewed in Hong, 2005;Jahn and Scheller, 2006). They form a family of small cytoplasmically orientated membrane-associated proteins that comprise a relatively simple domain architecture. Their characteristic is the so-called SNARE motif, an extended segment arranged in heptad repeats. In most SNAREs the motif is C-terminally connected to a single transmembrane domain by a short linker.As general mechanism it is thought that the SNAREs on the transport vesicle form a tight complex with the SNAREs in the ac...

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Identification of the Yeast R-SNARE Nyv1p as a Novel Longin Domain-containing Protein

Cited by 41 publications

References 56 publications

N-terminal domain of vacuolar SNARE Vam7p promotes trans -SNARE complex assembly

N-terminal domain of vacuolar SNARE Vam7p promotes trans -SNARE complex assembly

Stimulation of Actin Polymerization by Vacuoles via Cdc42p-dependent Signaling

An Elaborate Classification of SNARE Proteins Sheds Light on the Conservation of the Eukaryotic Endomembrane System

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