RESUMEN
Results from experiments using an oath to eliminate hypothetical bias in stated preference valuation are presented. An oath has several potential advantages relative to other methods for reducing hypothetical bias. Our empirical results suggest that with an oath, mean hypothetical payments are not different from mean actual payments and that when controlling for experimental participants' characteristics using regression analyses, the oath eliminated hypothetical bias.
Asunto(s)
Ética , Incertidumbre , Humanos , Análisis Multivariante , Análisis de Regresión , Controles Informales de la SociedadRESUMEN
The 100-kDa "a" subunit of the vacuolar proton-translocating ATPase (V-ATPase) is encoded by two genes in yeast, VPH1 and STV1. The Vph1p-containing complex localizes to the vacuole, whereas the Stv1p-containing complex resides in some other intracellular compartment, suggesting that the a subunit contains information necessary for the correct targeting of the V-ATPase. We show that Stv1p localizes to a late Golgi compartment at steady state and cycles continuously via a prevacuolar endosome back to the Golgi. V-ATPase complexes containing Vph1p and Stv1p also differ in their assembly properties, coupling of proton transport to ATP hydrolysis, and dissociation in response to glucose depletion. To identify the regions of the a subunit that specify these different properties, chimeras were constructed containing the cytosolic amino-terminal domain of one isoform and the integral membrane, carboxyl-terminal domain from the other isoform. Like the Stv1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Stv1p localized to the Golgi and the complex did not dissociate in response to glucose depletion. Like the Vph1p-containing complex, the V-ATPase complex containing the chimera with the amino-terminal domain of Vph1p localized to the vacuole and the complex exhibited normal dissociation upon glucose withdrawal. Interestingly, the V-ATPase complex containing the chimera with the carboxyl-terminal domain of Vph1p exhibited a higher coupling of proton transport to ATP hydrolysis than the chimera containing the carboxyl-terminal domain of Stv1p. Our results suggest that whereas targeting and in vivo dissociation are controlled by sequences located in the amino-terminal domains of the subunit a isoforms, coupling efficiency is controlled by the carboxyl-terminal region.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Protones , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , Adenosina Trifosfato/metabolismo , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Western Blotting , Membrana Celular/metabolismo , Eliminación de Gen , Glucosa/metabolismo , Glucosa/farmacología , Aparato de Golgi/metabolismo , Hidrólisis , Microscopía Fluorescente , Datos de Secuencia Molecular , Plásmidos/metabolismo , Pruebas de Precipitina , Isoformas de Proteínas , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/genética , Vacuolas/metabolismo , Levaduras/metabolismoRESUMEN
In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino- and carboxyl-terminal domains also suggests another possible site of interaction with other factors.
Asunto(s)
ATPasas de Translocación de Protón/química , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Caenorhabditis elegans/enzimología , Proteínas de Caenorhabditis elegans , Cristalografía por Rayos X , Drosophila melanogaster/enzimología , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína , Bombas de Protones/química , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
We show that the vacuolar protein sorting gene VPS44 is identical to NHX1, a gene that encodes a sodium/proton exchanger. The Saccharomyces cerevisiae protein Nhx1p shows high homology to mammalian sodium/proton exchangers of the NHE family. Nhx1p is thought to transport sodium ions into the prevacuole compartment in exchange for protons. Pulse-chase experiments show that approximately 35% of the newly synthesized soluble vacuolar protein carboxypeptidase Y is missorted in nhx1 delta cells, and is secreted from the cell. nhx1 delta cells accumulate late Golgi, prevacuole, and lysosome markers in an aberrant structure next to the vacuole, and late Golgi proteins are proteolytically cleaved more rapidly than in wild-type cells. Our results show that efficient transport out of the prevacuolar compartment requires Nhx1p, and that nhx1 delta cells exhibit phenotypes characteristic of the "class E" group of vps mutants. In addition, we show that Nhx1p is required for protein trafficking even in the absence of the vacuolar ATPase. Our analysis of Nhx1p provides the first evidence that a sodium/proton exchange protein is important for correct protein sorting, and that intraorganellar ion balance may be important for endosomal function in yeast.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Transporte de Catión , Canales de Cloruro , Endosomas/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Intercambiadores de Sodio-Hidrógeno , ATPasas de Translocación de Protón Vacuolares , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Proteínas Fúngicas/fisiología , Proteínas de la Membrana/fisiología , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , ATPasas de Translocación de Protón/fisiología , Saccharomyces cerevisiae/genética , Homología de Secuencia , Vacuolas/metabolismoRESUMEN
The Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is composed of at least 13 polypeptides organized into two distinct domains, V(1) and V(0), that are structurally and mechanistically similar to the F(1)-F(0) domains of the F-type ATP synthases. The peripheral V(1) domain is responsible for ATP hydrolysis and is coupled to the mechanism of proton translocation. The integral V(0) domain is responsible for the translocation of protons across the membrane and is composed of five different polypeptides. Unlike the F(0) domain of the F-type ATP synthase, which contains 12 copies of a single 8-kDa proteolipid, the V-ATPase V(0) domain contains three proteolipid species, Vma3p, Vma11p, and Vma16p, with each proteolipid contributing to the mechanism of proton translocation (Hirata, R., Graham, L. A., Takatsuki, A., Stevens, T. H., and Anraku, Y. (1997) J. Biol. Chem. 272, 4795-4803). Experiments with hemagglutinin- and c-Myc epitope-tagged copies of the proteolipids revealed that each V(0) complex contains all three species of proteolipid with only one copy each of Vma11p and Vma16p but multiple copies of Vma3p. Since the proteolipids of the V(0) complex are predicted to possess four membrane-spanning alpha-helices, twice as many as a single F-ATPase proteolipid subunit, only six V-ATPase proteolipids would be required to form a hexameric ring-like structure similar to the F(0) domain. Therefore, each V(0) complex will likely be composed of four copies of the Vma3p proteolipid in addition to Vma11p and Vma16p. Structural differences within the membrane-spanning domains of both V(0) and F(0) may account for the unique properties of the ATP-hydrolyzing V-ATPase compared with the ATP-generating F-type ATP synthase.
Asunto(s)
Proteolípidos/análisis , Bombas de Protones/química , ATPasas de Translocación de Protón/química , Proteínas de Saccharomyces cerevisiae , ATPasas de Translocación de Protón Vacuolares , Vacuolas/enzimología , Escherichia coli/enzimología , Proteínas Fúngicas/análisis , Modelos Moleculares , ATPasas de Translocación de Protón/análisis , Saccharomyces cerevisiae , Especificidad de la EspecieRESUMEN
Mutations in the VPS (vacuolar protein sorting) genes of Saccharomyces cerevisiae have been used to define the trafficking steps that soluble vacuolar hydrolases take en route from the late Golgi to the vacuole. The class D VPS genes include VPS21, PEP12, and VPS45, which appear to encode components of a membrane fusion complex involved in Golgi-to-endosome transport. Vps21p is a member of the Rab family of small Ras-like GTPases and shows strong homology to the mammalian Rab5 protein, which is involved in endocytosis and the homotypic fusion of early endosomes. Although Rab5 and Vps21p appear homologous at the sequence level, it has not been clear if the functions of these two Rabs are similar. We find that Vps21p is an endosomal protein that is involved in the delivery of vacuolar and endocytosed proteins to the vacuole. Vacuolar and endocytosed proteins accumulate in distinct transport intermediates in cells that lack Vps21p function. Therefore, it appears that Vps21p is involved in two trafficking steps into the prevacuolar/late endosomal compartment.
Asunto(s)
Endocitosis , Receptores Acoplados a Proteínas G , Receptores de Feromonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Proteínas de Unión al GTP rab/metabolismo , Fosfatasa Alcalina/metabolismo , Sustitución de Aminoácidos/genética , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/fisiología , Transporte Biológico , Carboxipeptidasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catepsina A , Complejos de Clasificación Endosomal Requeridos para el Transporte , Endosomas/metabolismo , Epistasis Genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos/genética , Genes Fúngicos/fisiología , Membranas Intracelulares/metabolismo , Mutación/genética , Receptores de Superficie Celular/química , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Conjugación , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
The late Golgi of the yeast Saccharomyces cerevisiae receives membrane traffic from the secretory pathway as well as retrograde traffic from post-Golgi compartments, but the machinery that regulates these vesicle-docking and fusion events has not been characterized. We have identified three components of a novel protein complex that is required for protein sorting at the yeast late Golgi compartment. Mutation of VPS52, VPS53, or VPS54 results in the missorting of 70% of the vacuolar hydrolase carboxypeptidase Y as well as the mislocalization of late Golgi membrane proteins to the vacuole, whereas protein traffic through the early part of the Golgi complex is unaffected. A vps52/53/54 triple mutant strain is phenotypically indistinguishable from each of the single mutants, consistent with the model that all three are required for a common step in membrane transport. Native coimmunoprecipitation experiments indicate that Vps52p, Vps53p, and Vps54p are associated in a 1:1:1 complex that sediments as a single peak on sucrose velocity gradients. This complex, which exists both in a soluble pool and as a peripheral component of a membrane fraction, colocalizes with markers of the yeast late Golgi by immunofluorescence microscopy. Together, the phenotypic and biochemical data suggest that VPS52, VPS53, and VPS54 are required for the retrograde transport of Golgi membrane proteins from an endosomal/prevacuolar compartment. The Vps52/53/54 complex joins a growing list of distinct multisubunit complexes that regulate membrane-trafficking events.
Asunto(s)
Proteínas Portadoras , Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular , Fosfatasa Alcalina/metabolismo , Compartimento Celular , Clonación Molecular , Proteínas Fúngicas/genética , Genes Fúngicos , Glicósido Hidrolasas/metabolismo , Glicosilación , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Mutagénesis , Receptores de Superficie Celular/metabolismo , Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , beta-FructofuranosidasaRESUMEN
Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of two distinct SNARE complexes. Pep12p and Vam3p are both t-SNAREs of the syntaxin family that are components of these SNARE complexes. We have used a genetic approach to address the role of Pep12p in vacuolar protein transport. Our screen for temperature-sensitive pep12 mutants yielded six alleles that were rapidly inactivated upon exposure to the non-permissive temperature. Surprisingly, the proteins encoded by these alleles were all truncated immediately prior to the transmembrane domain. Here we demonstrate that Pep12p requires its transmembrane domain for proper localization, but not for its role in vesicle fusion. In addition, we show that although Pep12p can replace Vam3p in the vacuolar SNARE complex, its transmembrane domain is required to function at this step. Therefore, the transmembrane domain of Pep12p performs different roles in the prevacuolar and vacuolar SNARE complexes.
Asunto(s)
Proteínas de la Membrana/metabolismo , Transporte de Proteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/metabolismo , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Fraccionamiento Celular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Immunoblotting , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Microscopía Fluorescente , Datos de Secuencia Molecular , Pruebas de Precipitina , Estructura Terciaria de Proteína , Proteínas Qa-SNARE , Proteínas SNARE , Saccharomyces cerevisiae/genética , Alineación de Secuencia , Temperatura , Transformación GenéticaRESUMEN
Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae requires the function of the endosomal syntaxin, Pep12p. Many vacuolar proteins, such as the soluble vacuolar hydrolase, carboxypeptidase Y (CPY), traverse the prevacuolar compartment (PVC) en route to the vacuole. Here we show that deletion of the carboxy-terminal transmembrane domain of Pep12p results in a temperature-conditional block in transport of CPY to the PVC. The PVC also receives traffic from the early endosome and the vacuole, and mutation in PEP12 also blocks these other trafficking pathways into the PVC. Therefore, Pep12p is a multifunctional syntaxin that is required for all known trafficking pathways into the yeast PVC. Finally, we found that the internalized pheromone receptor, Ste3p, can cycle out of the PVC in a VPS27-independent fashion.
Asunto(s)
Endosomas/metabolismo , Proteínas Fúngicas/fisiología , Proteínas de la Membrana/fisiología , Transporte de Proteínas/fisiología , Receptores Acoplados a Proteínas G , Receptores de Feromonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Fusión de Membrana , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Orgánulos/metabolismo , Proteínas Qa-SNARE , Receptores de Superficie Celular/metabolismo , Receptores del Factor de Conjugación , Proteínas SNARE , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestructura , TemperaturaRESUMEN
The proton-translocating ATPase (H(+)-ATPase) found on the membrane of the yeast vacuole is the best characterized member of the V-type ATPase family. Biochemical and genetic screens have led to the identification of 14 genes, the majority designated VMA (for vacuolar membrane ATPase) encoding subunits of the enzyme complex. At least eight genes encode for proteins comprising the peripherally associated catalytic V(1) subcomplex, and six genes code for proteins forming the proton-translocating membrane V(o) subcomplex. Several additional genes have been identified that encode proteins that are not part of the final V-ATPase complex yet are required for its assembly. These non-subunit Vma proteins function as dedicated V-ATPase assembly factors since their absence appears to inhibit assembly of the V-ATPase only. The assembly factors designated Vma12p, Vma21p and Vma22p have been localized to the membrane of the endoplasmic reticulum and aid the association of newly synthesized V-ATPase subunits translocated into the endoplasmic reticulum membrane. Two of these proteins, Vma12p and Vma22p, function together in an assembly complex that interacts directly with nascent V-ATPase subunits.
Asunto(s)
ATPasas de Translocación de Protón/química , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares , Secuencia de Aminoácidos , Modelos Químicos , Datos de Secuencia Molecular , ATPasas de Translocación de Protón/genética , Saccharomyces cerevisiae/ultraestructura , Vacuolas/enzimologíaRESUMEN
In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.
Asunto(s)
Proteínas Portadoras/química , Proteínas de la Membrana/química , Proteínas de Saccharomyces cerevisiae , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Clonación Molecular , Proteínas Fúngicas/química , Luz , Mamíferos , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Recombinantes , Saccharomyces cerevisiae/metabolismo , Dispersión de RadiaciónRESUMEN
Membrane traffic in eukaryotic cells relies on recognition between v-SNAREs on transport vesicles and t-SNAREs on target membranes. Here we report the identification of AtVTI1a and AtVTI1b, two Arabidopsis homologues of the yeast v-SNARE Vti1p, which is required for multiple transport steps in yeast. AtVTI1a and AtVTI1b share 60% amino acid identity with one another and are 32 and 30% identical to the yeast protein, respectively. By suppressing defects found in specific strains of yeast vti1 temperature-sensitive mutants, we show that AtVTI1a can substitute for Vti1p in Golgi-to-prevacuolar compartment (PVC) transport, whereas AtVTI1b substitutes in two alternative pathways: the vacuolar import of alkaline phosphatase and the so-called cytosol-to-vacuole pathway used by aminopeptidase I. Both AtVTI1a and AtVTI1b are expressed in all major organs of Arabidopsis. Using subcellular fractionation and immunoelectron microscopy, we show that AtVTI1a colocalizes with the putative vacuolar cargo receptor AtELP on the trans-Golgi network and the PVC. AtVTI1a also colocalizes with the t-SNARE AtPEP12p to the PVC. In addition, AtVTI1a and AtPEP12p can be coimmunoprecipitated from plant cell extracts. We propose that AtVTI1a functions as a v-SNARE responsible for targeting AtELP-containing vesicles from the trans-Golgi network to the PVC, and that AtVTI1b is involved in a different membrane transport process.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis/metabolismo , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Vacuolas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Transporte Biológico , Proteínas Portadoras/metabolismo , Compartimento Celular , Epítopos , Regulación de la Expresión Génica de las Plantas , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Pruebas de Precipitina , Proteínas Qb-SNARE , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Sacarosa , Transcripción Genética , Ultracentrifugación , Levaduras/genética , Levaduras/metabolismoRESUMEN
The interaction between v-SNAREs on transport vesicles and t-SNAREs on target membranes is required for membrane traffic in eukaryotic cells. Here we identify Vti1p as the first v-SNARE protein found to be required for biosynthetic traffic into the yeast vacuole, the equivalent of the mammalian lysosome. Certain vti1-ts yeast mutants are defective in alkaline phosphatase transport from the Golgi to the vacuole and in targeting of aminopeptidase I from the cytosol to the vacuole. VTI1 interacts genetically with the vacuolar t-SNARE VAM3, which is required for transport of both alkaline phosphatase and aminopeptidase I to the vacuole. The v-SNARE Nyv1p forms a SNARE complex with Vam3p in homotypic vacuolar fusion; however, we find that Nyv1p is not required for any of the three biosynthetic pathways to the vacuole. v-SNAREs were thought to ensure specificity in membrane traffic. However, Vti1p also functions in two additional membrane traffic pathways: Vti1p interacts with the t-SNAREs Pep12p in traffic from the TGN to the prevacuolar compartment and with Sed5p in retrograde traffic to the cis-Golgi. The ability of Vti1p to mediate multiple fusion steps requires additional proteins to ensure specificity in membrane traffic.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Fosfatasa Alcalina/metabolismo , Aminopeptidasas/metabolismo , Transporte Biológico , Aparato de Golgi/metabolismo , Proteínas Qa-SNARE , Proteínas Qb-SNARERESUMEN
Vacuole SNAREs, including the t-SNAREs Vam3p and Vam7p and the v-SNARE Nyv1p, are found in a multisubunit "cis" complex on isolated organelles. We now identify the v-SNAREs Vti1p and Ykt6p by mass spectrometry as additional components of the immunoisolated vacuolar SNARE complex. Immunodepletion of detergent extracts with anti-Vti1p removes all the Ykt6p that is in a complex with Vam3p, immunodepletion with anti-Ykt6p removes all the Vti1p that is complexed with Vam3p, and immunodepletion with anti-Nyv1p removes all the Ykt6p in complex with other SNAREs, demonstrating that they are all together in the same cis multi-SNARE complex. After priming, which disassembles the cis-SNARE complex, antibodies to any of the five SNARE proteins still inhibit the fusion assay until the docking stage is completed, suggesting that each SNARE plays a role in docking. Furthermore, vti1 temperature-sensitive alleles cause a synthetic fusion-defective phenotype in our reaction. Our data show that vacuole-vacuole fusion requires a cis-SNARE complex of five SNAREs, the t-SNAREs Vam3p and Vam7p and the v-SNAREs Nyv1p, Vti1p, and Ykt6p.
Asunto(s)
Proteínas Portadoras/metabolismo , Fusión de Membrana , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Receptores Citoplasmáticos y Nucleares/genética , Proteínas de Saccharomyces cerevisiae , Vacuolas/química , Proteínas de Transporte Vesicular , Alelos , Anticuerpos/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas Portadoras/aislamiento & purificación , Cromatografía de Afinidad , Proteínas Fúngicas/genética , Proteínas Fúngicas/inmunología , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Membranas Intracelulares/química , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Fusión de Membrana/efectos de los fármacos , Proteínas de la Membrana/análisis , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/aislamiento & purificación , Proteínas Sensibles a N-Etilmaleimida , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/aislamiento & purificación , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/análisis , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Proteínas Nucleares/metabolismo , Fenotipo , Pruebas de Precipitina , Unión Proteica , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas R-SNARE , Receptores Citoplasmáticos y Nucleares/análisis , Receptores Citoplasmáticos y Nucleares/inmunología , Receptores Citoplasmáticos y Nucleares/metabolismo , Proteínas SNARE , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Solubles de Unión al Factor Sensible a la N-Etilmaleimida , Proteína 25 Asociada a Sinaptosomas , Temperatura , Vacuolas/efectos de los fármacos , Vacuolas/metabolismoRESUMEN
The yeast vacuolar proton-translocating ATPase (V-ATPase) is the best characterized member of the V-ATPase family. Biochemical and genetic screens led to the identification of a large number of genes in yeast, designated VMA, encoding proteins required to assemble a functional V-ATPase. A total of thirteen genes encode subunits of the final enzyme complex. In addition to subunit-encoding genes, we have identified three genes that code for proteins that are not part of the final V-ATPase complex yet required for its assembly. We refer to these nonsubunit Vma proteins as assembly factors, since their function is dedicated to assembling the V-ATPase. The assembly factors, Vma12p, Vma21p, and Vma22p are localized to the endoplasmic reticulum (ER) and aid the assembly of newly synthesized V-ATPase subunits that are translocated into the ER membrane. At least two of these proteins, Vma12p and Vma22p, function together in an assembly complex and interact directly with nascent V-ATPase subunits.
Asunto(s)
Bombas de Protones/química , ATPasas de Translocación de Protón/química , Proteínas de Saccharomyces cerevisiae , ATPasas de Translocación de Protón Vacuolares , Levaduras/enzimología , Proteínas Fúngicas/química , Proteínas de la Membrana/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
Newly synthesized proteins that reach the last compartment of the Golgi complex can be sorted into pathways leading either to the cell surface or to the vacuole. It now appears that there are at least two routes from the Golgi to the vacuole: the 'CPY pathway', which involves transit through an endosomal/prevacuolar compartment (PVC), and a recently discovered 'ALP pathway', which bypasses the PVC, but may involve other as yet unidentified intermediate compartments. No cytosolic signal has been identified that directs the entry of membrane proteins into the CPY pathway. In contrast, the transport of ALP through the ALP pathway is saturable and signal mediated. Much recent work has focused on the identification of proteins that regulate trafficking to the vacuole. A number of genes have been identified that are specific for either the CPY or ALP sorting pathways, while other genes affect both types of transport and may therefore act at or after a point of convergence. Progress has also been made in further elucidating the members of the SNARE complexes that act in Golgi-to-PVC transport as well as those that mediate fusion with the vacuole.
Asunto(s)
Proteínas Fúngicas/metabolismo , Aparato de Golgi/metabolismo , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Fosfatasa Alcalina/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Carboxipeptidasas/metabolismo , Catepsina A , Humanos , Fusión de Membrana , Datos de Secuencia MolecularRESUMEN
A large number of trafficking steps occur between the last compartment of the Golgi apparatus (TGN) and the vacuole of the yeast Saccharomyces cerevisiae. To date, two intracellular routes from the TGN to the vacuole have been identified. Carboxypeptidase Y (CPY) travels through a prevacuolar/endosomal compartment (PVC), and subsequently on to the vacuole, while alkaline phosphatase (ALP) bypasses this compartment to reach the same organelle. Proteins resident to the TGN achieve their localization despite a continuous flux of traffic by continually being retrieved from the distal PVC by virtue of an aromatic amino acid-containing sorting motif. In this study we report that a hybrid protein based on ALP and containing this retrieval motif reaches the PVC not by following the CPY sorting pathway, but instead by signal-dependent retrograde transport from the vacuole, an organelle previously thought of as a terminal compartment. In addition, we show that a mutation in VAC7, a gene previously identified as being required for vacuolar inheritance, blocks this trafficking step. Finally we show that Vti1p, a v-SNARE required for the delivery of both CPY and ALP to the vacuole, uses retrograde transport out of the vacuole as part of its normal cellular itinerary.
Asunto(s)
Fosfatasa Alcalina/metabolismo , Endosomas/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Carboxipeptidasas/metabolismo , Proteínas Portadoras/metabolismo , Catepsina A , Proteínas Fúngicas/metabolismo , Aparato de Golgi/enzimología , Aparato de Golgi/metabolismo , Proteínas de la Membrana/metabolismo , Mutación , Proteínas Qb-SNARE , Saccharomyces cerevisiae/enzimología , Saccharomyces cerevisiae/genéticaRESUMEN
Three previously identified genes from Saccharomyces cerevisiae, VMA12, VMA21, and VMA22, encode proteins localized to the endoplasmic reticulum (ER). These three proteins are required for the biogenesis of a functional vacuolar ATPase (V-ATPase), but are not part of the final enzyme complex. Subcellular fractionation and chemical cross-linking studies have revealed that Vma12p and Vma22p form a stable membrane associated complex. Cross-linking analysis also revealed a direct physical interaction between the Vma12p/Vma22p assembly complex and Vph1p, the 100-kD integral membrane subunit of the V-ATPase. The interaction of the Vma12p/Vma22p complex with Vph1p was transient (half-life of approximately 5 min), reflecting trafficking of this V-ATPase subunit through the ER en route to the vacuolar membrane. Analysis of these protein-protein interactions in ER-blocked sec12 mutant cells indicated that the Vph1p-Vma12p/Vma22p interactions are quite stable when transport of the V-ATPase out of the ER is blocked. Fractionation of solubilized membrane proteins on a density gradient revealed comigration of Vma22p and Vma12p, indicating that they form a complex even in the absence of cross-linker. Vma12p and Vma22p migrated to fractions separate from Vma21p. Loss of Vph1p caused the Vma12p/Vma22p complex to sediment to less dense fractions, consistent with association of Vma12p/ Vma22p with nascent Vph1p in ER membranes. This is the first evidence for a dedicated assembly complex in the ER required for the assembly of an integral membrane protein complex (V-ATPase) as it is transported through the secretory pathway.
Asunto(s)
Retículo Endoplásmico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de la Membrana/metabolismo , ATPasas de Translocación de Protón/biosíntesis , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares , Membranas Intracelulares/metabolismo , Chaperonas MolecularesRESUMEN
The vps (vacuolar protein sorting) mutants have been used to dissect and characterize the vacuolar biogenesis pathway in the yeast Saccharomyces cerevisiae. The vps mutants were isolated through their loss of ability to correctly sort the vacuolar hydrolase CPY, which travels from Golgi membranes to the vacuole through a prevacuolar compartment. Over 50 VPS genes have been divided into 6 classes according to vacuolar morphology. Mutations in any one of the class E VPS genes, such as VPS27, lead to an exaggerated form of the prevacuolar compartment. This class E compartment contains endocytosed proteins as well as proteins en route to the vacuole, and is thus taken to represent an intersection point between the endocytic and biosynthetic pathways. Mutations in the class D gene VPS45 can be used to define a second transport intermediate along the vacuolar biogenesis pathway, Golgi-derived transport vesicles carrying vacuolar membrane proteins on their way to the vacuole. Here we demonstrate that the Sec1p-like protein Vps45p is required for the fusion of Golgi-derived vesicles with the prevacuolar compartment indicating that VPS45 functions before VPS27 in the vacuolar biogenesis pathway. In addition, we show that VPS45 function is not required for the delivery of endocytosed proteins to the prevacuolar compartment from the plasma membrane suggesting that the function of Vps45p is restricted to a single vesicular pathway.
Asunto(s)
Endosomas/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Unión al GTP , Proteínas de la Membrana/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Feromonas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Vacuolas/metabolismo , Proteínas de Transporte Vesicular , Transporte Biológico , Carboxipeptidasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Catepsina A , Compartimento Celular , Endocitosis , Epistasis Genética , Proteínas Fúngicas/genética , Proteínas de la Membrana/genética , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores del Factor de ConjugaciónRESUMEN
Delivery of proteins to the vacuole of the yeast Saccharomyces cerevisiae provides an excellent model system in which to study vacuole and lysosome biogenesis and membrane traffic. This organelle receives proteins from a number of different routes, including proteins sorted away from the secretory pathway at the Golgi apparatus and endocytic traffic arising from the plasma membrane. Genetic analysis has revealed at least 60 genes involved in vacuolar protein sorting, numerous components of a novel cytoplasm-to-vacuole transport pathway, and a large number of proteins required for autophagy. Cell biological and biochemical studies have provided important molecular insights into the various protein delivery pathways to the yeast vacuole. This review describes the various pathways to the vacuole and illustrates how they are related to one another in the vacuolar network of S. cerevisiae.