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1.
J Cell Sci ; 128(13): 2278-92, 2015 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-25999476

RESUMO

Membrane fusion at the vacuole depends on a conserved machinery that includes SNAREs, the Rab7 homolog Ypt7 and its effector HOPS. Here, we demonstrate that Ypt7 has an unexpected additional function by controlling membrane homeostasis and nutrient-dependent signaling on the vacuole surface. We show that Ivy1, the yeast homolog of mammalian missing-in-metastasis (MIM), is a vacuolar effector of Ypt7-GTP and interacts with the EGO/ragulator complex, an activator of the target of rapamycin kinase complex 1 (TORC1) on vacuoles. Loss of Ivy1 does not affect EGO vacuolar localization and function. In combination with the deletion of individual subunits of the V-ATPase, however, we observed reduced TORC1 activity and massive enlargement of the vacuole surface. Consistent with this, Ivy1 localizes to invaginations at the vacuole surface and on liposomes in a phosphoinositide- and Ypt7-GTP-controlled manner, which suggests a role in microautophagy. Our data, thus, reveal that Ivy1 is a novel regulator of vacuole membrane homeostasis with connections to TORC1 signaling.


Assuntos
Proteínas de Transporte/metabolismo , Homeostase , Membranas Intracelulares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Autofagia , Endocitose , Alvo Mecanístico do Complexo 1 de Rapamicina , Modelos Biológicos , Complexos Multiproteicos , Fosfatidilinositóis/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/ultraestrutura , Transdução de Sinais , Serina-Treonina Quinases TOR , Vacúolos/ultraestrutura
2.
Small GTPases ; 5(3): 1-3, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25483304

RESUMO

Rabs exist in two forms: the inactive GDP- and the active GTP-bound form. GEF proteins mediate the exchange of GDP for GTP and thereby activate Rabs. Although GEFs share a common action, which involves the opening of the Rab nucleotide binding site, they do not contain a conserved catalytic domain. Longin domains have been either found in several GEFs (TRAPP, DENN) or predicted by sequence analyses (Mon1-Ccz1, BLOC-3). At least in TRAPP, they serve as a platform for interaction with a GTPase. We recently generated a model of the predicted longin domains of the Mon1-Ccz1 complex based upon the structure of the respective TRAPP subunits. This allowed us to identify activity-related important regions of the complex. Moreover, we analyzed the GEF activity of Mon1-Ccz1 in the presence of membranes and uncovered that certain acidic phospholipids support the recruitment of the GEF complex. In this commentary, we will discuss our findings in a broader context.


Assuntos
Proteínas de Transporte/metabolismo , Endossomos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte Vesicular/química , Leveduras/metabolismo
3.
J Biol Chem ; 289(48): 33503-12, 2014 Nov 28.
Artigo em Inglês | MEDLINE | ID: mdl-25324549

RESUMO

Membrane fusion at the vacuole, the lysosome equivalent in yeast, requires the HOPS tethering complex, which is recruited by the Rab7 GTPase Ypt7. HOPS provides a template for the assembly of SNAREs and thus likely confers fusion at a distinct position on vacuoles. Five of the six subunits in HOPS have a similar domain prediction with strong similarity to COPII subunits and nuclear porins. Here, we show that Vps18 indeed has a seven-bladed ß-propeller as its N-terminal domain by revealing its structure at 2.14 Å. The Vps18 N-terminal domain can interact with the N-terminal part of Vps11 and also binds to lipids. Although deletion of the Vps18 N-terminal domain does not preclude HOPS assembly, as revealed by negative stain electron microscopy, the complex is instable and cannot support membrane fusion in vitro. We thus conclude that the ß-propeller of Vps18 is required for HOPS stability and function and that it can serve as a starting point for further structural analyses of the HOPS tethering complex.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Complexos Multiproteicos/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/química , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/genética , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
4.
J Cell Sci ; 127(Pt 5): 1043-51, 2014 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-24413168

RESUMO

To function in fusion and signaling, Rab GTPases need to be converted into their active GTP form. We previously identified the conserved Mon1-Ccz1 complex as the guanine nucleotide exchange factor (GEF) of the yeast Rab7 GTPase Ypt7. To address the possible GEF mechanism, we generated a homology model of the predicted longin domains of Mon1 and Ccz1 using the Rab-binding surface of the TRAPP complex as a template. On the basis of this, we identified mutations in both yeast Mon1 and Ccz1 that block Ypt7 activation, without affecting heterodimer formation and intracellular localization of Mon1 and Ccz1 at endosomes. Strikingly, the activity of the isolated Mon1-Ccz1 complex for Ypt7 is highly stimulated on membranes, and is promoted by the same anionic phospholipids such as phosphatidylinositol-3-phosphate (PI3P), which also support membrane association of the GEF complex. Our data imply that the GEF activity of the Mon1-Ccz1 complex towards Rab7/Ypt7 requires the interface formed by their longin domains and profits strongly from its association with the organelle surface.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/química , Membranas Intracelulares/enzimologia , Fosfatos de Fosfatidilinositol/química , Fosfatidilserinas/química , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/química , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Proteínas de Transporte Vesicular/química , Proteínas rab de Ligação ao GTP/química
5.
Proc Natl Acad Sci U S A ; 109(6): 1991-6, 2012 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-22308417

RESUMO

Membrane fusion within the eukaryotic endomembrane system depends on the initial recognition of Rab GTPase on transport vesicles by multisubunit tethering complexes and subsequent coupling to SNARE-mediated fusion. The conserved vacuolar/lysosomal homotypic fusion and vacuole protein sorting (HOPS) tethering complex combines both activities. Here we present the overall structure of the fusion-active HOPS complex. Our data reveal a flexible ≈30-nm elongated seahorse-like structure, which can adopt contracted and elongated shapes. Surprisingly, both ends of the HOPS complex contain a Rab-binding subunit: Vps41 and Vps39. The large head contains in addition to Vps41 the SNARE-interacting Vps33, whereas Vps39 is found in the bulky tip of its tail. Vps11 and Vps18 connect head and tail. Our data suggest that HOPS bridges Ypt7-positive membranes and chaperones SNAREs at fusion sites.


Assuntos
Fusão de Membrana , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Vacúolos/metabolismo , Sítios de Ligação , Proteínas de Fluorescência Verde/metabolismo , Complexos Multiproteicos/isolamento & purificação , Complexos Multiproteicos/ultraestrutura , Ligação Proteica , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Eletricidade Estática , Proteínas rab de Ligação ao GTP/metabolismo
6.
Small GTPases ; 2(3): 182-186, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21776422

RESUMO

Within eukaryotic cells, Rab GTPases control the maturation of early to late endosomes and their subsequent fusion with the vacuole. Within this ExtraView, we will focus on our recent findings regarding the activation of the Rab7 homolog Ypt7 in yeast and its interplay with the two multisubunit tethering complexes CORVET and HOPS.

7.
J Cell Biol ; 191(4): 845-59, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21079247

RESUMO

Tethering factors are organelle-specific multisubunit protein complexes that identify, along with Rab guanosine triphosphatases, transport vesicles and trigger their SNARE-mediated fusion of specific transport vesicles with the target membranes. Little is known about how tethering factors discriminate between different trafficking pathways, which may converge at the same organelle. In this paper, we describe a phosphorylation-based switch mechanism, which allows the homotypic vacuole fusion protein sorting effector subunit Vps41 to operate in two distinct fusion events, namely endosome-vacuole and AP-3 vesicle-vacuole fusion. Vps41 contains an amphipathic lipid-packing sensor (ALPS) motif, which recognizes highly curved membranes. At endosomes, this motif is inserted into the lipid bilayer and masks the binding motif for the δ subunit of the AP-3 complex, Apl5, without affecting the Vps41 function in endosome-vacuole fusion. At the much less curved vacuole, the ALPS motif becomes available for phosphorylation by the resident casein kinase Yck3. As a result, the Apl5-binding site is exposed and allows AP-3 vesicles to bind to Vps41, followed by specific fusion with the vacuolar membrane. This multifunctional tethering factor thus discriminates between trafficking routes by switching from a curvature-sensing to a coat recognition mode upon phosphorylation.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Motivos de Aminoácidos , Animais , Caseína Quinase I/genética , Caseína Quinase I/metabolismo , Membrana Celular/química , Endossomos/metabolismo , Dados de Sequência Molecular , Fosforilação , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo
8.
Curr Biol ; 20(21): R943-52, 2010 Nov 09.
Artigo em Inglês | MEDLINE | ID: mdl-21056839

RESUMO

Protein trafficking within eukaryotic cells depends on vesicular carriers that fuse with organelles to deliver their lipid and protein content. Cells have developed an elaborate system to capture vesicles at organelles that involves the action of Rab GTPases and tethers. Vesicle fusion then takes place with the help of SNARE proteins. In this review we focus on the role of multisubunit tethering complexes of eukaryotic cells. In particular, we discuss the tethering complexes of the secretory pathway and the endolysosomal system and highlight recent evidence for the role of these complexes in interaction with Rabs, coat recognition and cooperation with SNAREs during the fusion cascade.


Assuntos
Fusão de Membrana/fisiologia , Proteínas de Membrana/fisiologia , Proteínas SNARE/fisiologia , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/fisiologia , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dobramento de Proteína , Transporte Proteico , Proteínas SNARE/química , Proteínas SNARE/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo
9.
Curr Biol ; 20(18): 1654-9, 2010 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-20797862

RESUMO

Rab GTPases coordinate membrane fusion reactions [1]. Rab-GDP requires a guanine nucleotide exchange factor (GEF) for its conversion to the active GTP form. It then binds to effectors such as multimeric tethering complexes and supports fusion [2]. GTPase-activating proteins (GAPs) promote GTP hydrolysis to inactivate the Rab. GEFs are thus critical activators of fusion reactions [3, 4]. The Rab GEF family is diverse, ranging from multimeric complexes [5] to monomeric GEFs [6-9]. At the late endosome, Rab7 activation is critical for endosomal maturation. The yeast Rab7 homolog Ypt7 binds to the homotypic fusion and protein sorting (HOPS) complex [10, 11]. Its subunit Vps39/Vam6 has been proposed as a GEF for Ypt7 [12] and the Rag GTPase Gtr1 [13], but other genetic evidence has implicated the endosomal protein Ccz1 as a GEF for Ypt7 [14]. Ccz1 and its binding partner Mon1 have been linked to endosomal transport and maturation [15-20]. We now provide evidence that the dimeric Mon1-Ccz1 complex is the Rab7/Ypt7 GEF. The Mon1-Ccz1 complex, but neither protein alone, counteracts GAP function in vivo, rescues in vitro fusion of vacuoles carrying Ypt7-GDP, and promotes nucleotide exchange on Ypt7 independently of Vps39/HOPS. Our data indicate that the Mon1-Ccz1 complex triggers endosomal maturation by activating Ypt7 on late endosomes.


Assuntos
Endossomos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Complexos Multiproteicos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas rab de Ligação ao GTP/genética , Proteínas Ativadoras de ras GTPase/genética , Proteínas Ativadoras de ras GTPase/metabolismo
10.
Traffic ; 11(10): 1334-46, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20604902

RESUMO

Within the endomembrane system of eukaryotic cells, multisubunit tethering complexes together with their corresponding Rab-GTPases coordinate vesicle tethering and fusion. Here, we present evidence that two homologous hexameric tethering complexes, the endosomal CORVET (Class C core vacuole/endosome transport) and the vacuolar HOPS (homotypic vacuole fusion and protein sorting) complex, have similar subunit topologies. Both complexes contain two Rab-binding proteins at one end, and the Sec1/Munc18-like Vps33 at the opposite side, suggesting a model on membrane bridging via Rab-GTP and SNARE binding. In agreement, HOPS activity can be reconstituted using purified subcomplexes containing the Rab and Vps33 module, but requires all six subunits for activity. At the center of HOPS and CORVET, the class C proteins Vps11 and Vps18 connect the two parts, and Vps11 binds both HOPS Vps39 and CORVET Vps3 via the same binding site. As HOPS Vps39 is also found at endosomes, our data thus suggest that these tethering complexes follow defined but distinct assembly pathways, and may undergo transition by simple subunit interchange.


Assuntos
Subunidades alfa de Proteínas de Ligação ao GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Endossomos/metabolismo , Subunidades alfa de Proteínas de Ligação ao GTP/química , Domínios e Motivos de Interação entre Proteínas , Proteínas de Saccharomyces cerevisiae/química , Vacúolos/metabolismo , Proteínas rab de Ligação ao GTP/química
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