A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion

Seals, Darren F.; Eitzen, Gary; Margolis, Nathan; Wickner, William; Price, Albert

doi:10.1073/pnas.97.17.9402

Cited by 426 publications

(502 citation statements)

References 69 publications

(84 reference statements)

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“…Among them, yeast Vps11p, Vps18p, and Vps41p are core components of the 38S complex, called the HOPS complex (Wickner, 2002). It has been reported that the yeast counterpart of Rab7, Ypt7p, interacts with the HOPS complex Seals et al, 2000;Ungermann et al, 2000). The HOPS complex is also called the class C Vps complex and consists of Vps11p, Vps16p, Vps18p, Vps33p, Vps39p, and Vps41p.…”

Section: Discussionmentioning

confidence: 99%

Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

Mizuno

Kitamura

Sasaki

2003

MBoC

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Rab7, a member of the Rab family small G proteins, has been shown to regulate intracellular vesicle traffic to late endosome/lysosome and lysosome biogenesis, but the exact roles of Rab7 are still undetermined. Accumulating evidence suggests that each Rab protein has multiple target proteins that function in the exocytic/endocytic pathway. We have isolated a new Rab7 target protein, Rabring7 (Rab7-interacting RING finger protein), using a CytoTrap system. It contains an H2 type RING finger motif at the C termini. Rabring7 shows no homology with RILP, which has been reported as another Rab7 target protein. GST pull-down and coimmunoprecipitation assays demonstrate that Rabring7 specifically binds the GTP-bound form of Rab7 at the N-terminal portion. Rabring7 is found mainly in the cytosol and is recruited efficiently to late endosomes/lysosomes by the GTP-bound form of Rab7 in BHK cells. Overexpression of Rabring7 not only affects epidermal growth factor degradation but also causes the perinuclear aggregation of lysosomes, in which the accumulation of the acidotropic probe LysoTracker is remarkably enhanced. These results suggest that Rabring7 plays crucial roles as a Rab7 target protein in vesicle traffic to late endosome/lysosome and lysosome biogenesis

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Section: Discussionmentioning

confidence: 99%

Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

Mizuno

Kitamura

Sasaki

2003

MBoC

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“…In the initial "priming" step, Sec18p/Sec17p disassemble cis-SNARE complexes (17). The following tethering stage of "docking" requires the Rab-family GTPase Ypt7p (18) and its effector complex, termed HOPS (homotypic fusion and vacuole protein sorting) (19), which has Vps11p, Vps16p, Vps18p, Vps33p, Vps39p, and Vps41p as subunits (20,21). Tethered vacuoles are drawn against each other at closely apposed discshaped microdomains, termed "boundary membranes" to distinguish them from the "outside membranes," which are not in contact (22).…”

mentioning

confidence: 99%

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Fratti¹,

Collins²,

Hickey

et al. 2007

Journal of Biological Chemistry

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SNARE proteins form bundles of four ␣-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q⅐1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7 Q283R , yet Vam7 Q283R is preferred by vacuoles with Nyv1 R191Q . Rotation of the position of the arginine in the 0-layer increases the K m for Vam7p but does not affect the maximal rate of fusion. Vam7 Q283R forms stable 2Q⅐2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion. SNARE 4 proteins (1) are vital for membrane fusion. Initially discovered in neuronal tissues, SNAREs are found on all organelles of the exocytic and endocytic pathways, from yeast to humans. Their characteristic feature is a heptad-repeat "SNARE domain," flanked by varied N-domains and by C-terminal membrane anchors, either a single membrane-spanning apolar polypeptide or an acyl anchor. SNAREs form 4-helical complexes through their SNARE domains (2). Although the residues in each SNARE that face the others in a 4-helical complex are generally apolar, 3 glutamine (Q) and 1 arginine (R) near the center of the 4-complexed SNARE domains form a conserved and polar "0-layer" (3). Recombinant neuronal SNAREs spontaneously assemble into stable bundles, yet SNARE complex assembly in vivo requires SNARE-binding proteins of the Sec1-Munc18 "SM" family. SNARE complexes are disassembled by two chaperones: Sec17p/␣-SNAP, which binds directly to SNARE complexes, and Sec18p/NSF, which binds to Sec17p/␣-SNAP and couples the energy of ATP binding and hydrolysis to SNARE complex disassembly (4). SNARE complexes form in cis, with each SNARE anchored to the same membrane, or in trans, with SNAREs anchored to apposed, "tethered" membranes.SNARE function has been studied in vivo, on isolated organelles, and through the reconstitution of recombinant SNAREs into proteoliposomes. When several SNAREs, which are found in vivo on a "target" membrane, are co-reconstituted into one population of liposomes, they form a t-SNARE complex. These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7,8). This reconstituted reaction shows impress...

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“…Examples include those encoding the ␤3A and ␦ subunits of the AP-3 complex, which recognizes sorting signals of integral membrane proteins for transport to lysosomes and related organelles (17), and the gene encoding Vps33a, an ortholog of the yeast Vps33 protein implicated in membrane fusion events within the endosomal-lysosomal system (18,19). On the other hand, most genes known to be associated with HPS encode polypeptides that display no homology to any characterized protein.…”

mentioning

confidence: 99%

Identification of Snapin and Three Novel Proteins (BLOS1, BLOS2, and BLOS3/Reduced Pigmentation) as Subunits of Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)

Starcevic¹,

Dell’Angelica

2004

Journal of Biological Chemistry

227

229

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Biogenesis of lysosome-related organelles complex-1 (BLOC-1) is a ubiquitously expressed multisubunit protein complex required for the normal biogenesis of specialized organelles of the endosomal-lysosomal system, such as melanosomes and platelet dense granules. The complex is known to contain the coiled-coil-forming proteins, Pallidin, Muted, Cappuccino, and Dysbindin. The genes encoding these proteins are defective in inbred mouse strains that serve as models of Hermansky-Pudlak syndrome (HPS), a genetic disorder characterized by hypopigmentation and platelet storage pool deficiency. In addition, mutation of human Dysbindin causes HPS type 7. Here, we report the identification of another four subunits of the complex. One is Snapin, a coiled-coilforming protein previously characterized as a binding partner of synaptosomal-associated proteins 25 and 23 and implicated in the regulation of membrane fusion events. The other three are previously uncharacterized proteins, which we named BLOC subunits 1, 2, and 3 (BLOS1, -2, and -3). Using specific antibodies to detect endogenous proteins from human and mouse cells, we found that Snapin, BLOS1, BLOS2, and BLOS3 co-immunoprecipitate, and co-fractionate upon size exclusion chromatography, with previously known BLOC-1 subunits. Furthermore, steady-state levels of the four proteins are significantly reduced in cells from pallid mice, which carry a mutation in Pallidin and display secondary loss of other BLOC-1 subunits. Yeast two-hybrid analyses suggest a network of binary interactions involving all of the previously known and newly identified subunits. Interestingly, the HPS mouse model strain, reduced pigmentation, carries a nonsense mutation in the gene encoding BLOS3. As judged from size exclusion chromatographic analyses, the reduced pigmentation mutation affects BLOC-1 assembly less severely than the pallid mutation. Mutations in the human genes encoding Snapin and the BLOS proteins could underlie novel forms of HPS.

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A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion

Cited by 426 publications

References 69 publications

Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

Rabring7, a Novel Rab7 Target Protein with a RING Finger Motif

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Identification of Snapin and Three Novel Proteins (BLOS1, BLOS2, and BLOS3/Reduced Pigmentation) as Subunits of Biogenesis of Lysosome-related Organelles Complex-1 (BLOC-1)

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