RESUMEN
BACKGROUND AND AIMS: Vacuolar H+-ATP complex (V-ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V-ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X-linked mutations lead to autophagic myopathy. APPROACH AND RESULTS: Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low-density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V-ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element-binding protein-mediated cholesterol synthesis pathways. CONCLUSIONS: Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V-ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.
Asunto(s)
Autofagia/genética , Trastornos Congénitos de Glicosilación/genética , Hepatopatías/genética , ATPasas de Translocación de Protón Vacuolares/genética , Adulto , Biopsia , Células Cultivadas , Trastornos Congénitos de Glicosilación/sangre , Trastornos Congénitos de Glicosilación/diagnóstico , Trastornos Congénitos de Glicosilación/patología , Análisis Mutacional de ADN , Fibroblastos , Humanos , Hígado/citología , Hígado/patología , Hepatopatías/sangre , Hepatopatías/diagnóstico , Hepatopatías/patología , Masculino , Mutación Missense , Linaje , Cultivo Primario de CélulasRESUMEN
ATP6AP2 (also known as the [pro]renin receptor) is a type I transmembrane protein that can be cleaved into two fragments in the Golgi apparatus. While in Drosophila ATP6AP2 functions in the planar cell polarity (PCP) pathway, recent human genetic studies have suggested that ATP6AP2 could participate in the assembly of the V-ATPase in the endoplasmic reticulum (ER). Using a yeast model, we show here that the V-ATPase assembly factor Voa1 can functionally be replaced by Drosophila ATP6AP2. This rescue is even more efficient when coexpressing its binding partner ATP6AP1, indicating that these two proteins together fulfill Voa1 functions in higher organisms. Structure-function analyses in both yeast and Drosophila show that proteolytic cleavage is dispensable, while C-terminus-dependent ER retrieval is required for ATP6AP2 function. Accordingly, we demonstrate that both overexpression and lack of ATP6AP2 causes ER stress in Drosophila wing cells and that the induction of ER stress is sufficient to cause PCP phenotypes. In summary, our results suggest that full-length ATP6AP2 contributes to the assembly of the V-ATPase proton pore and that impairment of this function affects ER homeostasis and PCP signaling.
Asunto(s)
Proteínas de Drosophila/metabolismo , Proteínas de la Membrana/metabolismo , Receptores de Superficie Celular/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Animales , Polaridad Celular/fisiología , Proteínas de Drosophila/genética , Drosophila melanogaster/enzimología , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/genética , Receptores de Superficie Celular/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
The V-ATPase is the main regulator of intra-organellar acidification. Assembly of this complex has extensively been studied in yeast, while limited knowledge exists for man. We identified 11 male patients with hemizygous missense mutations in ATP6AP1, encoding accessory protein Ac45 of the V-ATPase. Homology detection at the level of sequence profiles indicated Ac45 as the long-sought human homologue of yeast V-ATPase assembly factor Voa1. Processed wild-type Ac45, but not its disease mutants, restored V-ATPase-dependent growth in Voa1 mutant yeast. Patients display an immunodeficiency phenotype associated with hypogammaglobulinemia, hepatopathy and a spectrum of neurocognitive abnormalities. Ac45 in human brain is present as the common, processed â¼40-kDa form, while liver shows a 62-kDa intact protein, and B-cells a 50-kDa isoform. Our work unmasks Ac45 as the functional ortholog of yeast V-ATPase assembly factor Voa1 and reveals a novel link of tissue-specific V-ATPase assembly with immunoglobulin production and cognitive function.
Asunto(s)
Disfunción Cognitiva/genética , Síndromes de Inmunodeficiencia/genética , Hepatopatías/genética , Mutación Missense , ATPasas de Translocación de Protón Vacuolares/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Disfunción Cognitiva/metabolismo , Salud de la Familia , Glicosilación , Humanos , Síndromes de Inmunodeficiencia/metabolismo , Lactante , Hepatopatías/metabolismo , Masculino , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/deficiencia , Adulto JovenRESUMEN
Studies of homotypic vacuole-vacuole fusion in the yeast Saccharomyces cerevisiae have been instrumental in determining the cellular machinery required for eukaryotic membrane fusion and have implicated the vacuolar H(+)-ATPase (V-ATPase). The V-ATPase is a multisubunit, rotary proton pump whose precise role in homotypic fusion is controversial. Models formulated from in vitro studies suggest that it is the proteolipid proton-translocating pore of the V-ATPase that functions in fusion, with further studies in worms, flies, zebrafish, and mice appearing to support this model. We present two in vivo assays and use a mutant V-ATPase subunit to establish that it is the H(+)-translocation/vacuole acidification function, rather than the physical presence of the V-ATPase, that promotes homotypic vacuole fusion in yeast. Furthermore, we show that acidification of the yeast vacuole in the absence of the V-ATPase rescues vacuole-fusion defects. Our results clarify the in vivo requirements of acidification for membrane fusion.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Pirofosfatasa Inorgánica/metabolismo , Fusión de Membrana/fisiología , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/metabolismo , Animales , Arabidopsis/metabolismo , Fluorescencia , Concentración de Iones de Hidrógeno , Ratones , Mutación/genética , Bombas de Protones , Saccharomyces cerevisiae/genética , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
Subunit a of the yeast vacuolar-type, proton-translocating ATPase enzyme complex (V-ATPase) is responsible for both proton translocation and subcellular localization of this highly conserved molecular machine. Inclusion of the Vph1p isoform causes the V-ATPase complex to traffic to the vacuolar membrane, whereas incorporation of Stv1p causes continued cycling between the trans-Golgi and endosome. We previously demonstrated that this targeting information is contained within the cytosolic, N-terminal portion of V-ATPase subunit a (Stv1p). To identify residues responsible for sorting of the Golgi isoform of the V-ATPase, a random mutagenesis was performed on the N terminus of Stv1p. Subsequent characterization of mutant alleles led to the identification of a short peptide sequence, W(83)KY, that is necessary for proper Stv1p localization. Based on three-dimensional homology modeling to the Meiothermus ruber subunit I, we propose a structural model of the intact Stv1p-containing V-ATPase demonstrating the accessibility of the W(83)KY sequence to retrograde sorting machinery. Finally, we characterized the sorting signal within the context of a reconstructed Stv1p ancestor (Anc.Stv1). This evolutionary intermediate includes an endogenous W(83)KY sorting motif and is sufficient to compete with sorting of the native yeast Stv1p V-ATPase isoform. These data define a novel sorting signal that is both necessary and sufficient for trafficking of the V-ATPase within the Golgi/endosomal network.
Asunto(s)
Endosomas/enzimología , Señales de Clasificación de Proteína/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Red trans-Golgi/enzimología , Secuencias de Aminoácidos , Endosomas/genética , Evolución Molecular , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Transporte de Proteínas/fisiología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología Estructural de Proteína , ATPasas de Translocación de Protón Vacuolares/genética , Red trans-Golgi/genéticaRESUMEN
Many cellular processes are carried out by molecular 'machines'-assemblies of multiple differentiated proteins that physically interact to execute biological functions. Despite much speculation, strong evidence of the mechanisms by which these assemblies evolved is lacking. Here we use ancestral gene resurrection and manipulative genetic experiments to determine how the complexity of an essential molecular machine--the hexameric transmembrane ring of the eukaryotic V-ATPase proton pump--increased hundreds of millions of years ago. We show that the ring of Fungi, which is composed of three paralogous proteins, evolved from a more ancient two-paralogue complex because of a gene duplication that was followed by loss in each daughter copy of specific interfaces by which it interacts with other ring proteins. These losses were complementary, so both copies became obligate components with restricted spatial roles in the complex. Reintroducing a single historical mutation from each paralogue lineage into the resurrected ancestral proteins is sufficient to recapitulate their asymmetric degeneration and trigger the requirement for the more elaborate three-component ring. Our experiments show that increased complexity in an essential molecular machine evolved because of simple, high-probability evolutionary processes, without the apparent evolution of novel functions. They point to a plausible mechanism for the evolution of complexity in other multi-paralogue protein complexes.
Asunto(s)
Evolución Molecular , Hongos/enzimología , Modelos Biológicos , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/metabolismo , Algoritmos , Biología Computacional , Extinción Biológica , Hongos/clasificación , Hongos/genética , Duplicación de Gen , Mutagénesis , Filogenia , Conformación Proteica , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
The vacuolar-type, proton-translocating ATPase (V-ATPase) is a multisubunit enzyme responsible for organelle acidification in eukaryotic cells. Many organisms have evolved V-ATPase subunit isoforms that allow for increased specialization of this critical enzyme. Differential targeting of the V-ATPase to specific subcellular organelles occurs in eukaryotes from humans to budding yeast. In Saccharomyces cerevisiae, the two subunit a isoforms are the only difference between the two V-ATPase populations. Incorporation of Vph1p or Stv1p into the V-ATPase dictates the localization of the V-ATPase to the vacuole or late Golgi/endosome, respectively. A duplication event within fungi gave rise to two subunit a genes. We used ancestral gene reconstruction to generate the most recent common ancestor of Vph1p and Stv1p (Anc.a) and tested its function in yeast. Anc.a localized to both the Golgi/endosomal network and vacuolar membrane and acidified these compartments as part of a hybrid V-ATPase complex. Trafficking of Anc.a did not require retrograde transport from the late endosome to the Golgi that has evolved for retrieval of the Stv1p isoform. Rather, Anc.a localized to both structures through slowed anterograde transport en route to the vacuole. Our results suggest an evolutionary model that describes the differential localization of the two yeast V-ATPase isoforms.
Asunto(s)
Subunidades de Proteína/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Secuencia de Consenso , Endosomas/metabolismo , Eliminación de Gen , Técnicas de Inactivación de Genes , Aparato de Golgi/metabolismo , Membranas Intracelulares/metabolismo , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Subunidades de Proteína/genética , Transporte de Proteínas , Proteínas Recombinantes/genética , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Alineación de Secuencia , ATPasas de Translocación de Protón Vacuolares/genética , Vacuolas/metabolismoRESUMEN
The function of the vacuolar H(+)-ATPase (V-ATPase) enzyme complex is to acidify organelles; this process is critical for a variety of cellular processes and has implications in human disease. There are five accessory proteins that assist in assembly of the membrane portion of the complex, the V(0) domain. To identify additional elements that affect V-ATPase assembly, trafficking, or enzyme activity, we performed a genome-wide enhancer screen in the budding yeast Saccharomyces cerevisiae with two mutant assembly factor alleles, VMA21 with a dysfunctional ER retrieval motif (vma21QQ) and vma21QQ in combination with voa1Δ, a nonessential assembly factor. These alleles serve as sensitized genetic backgrounds that have reduced V-ATPase enzyme activity. Genes were identified from a variety of cellular pathways including a large number of trafficking-related components; we characterized two redundant gene pairs, HPH1/HPH2 and ORM1/ORM2. Both sets demonstrated synthetic growth defects in combination with the vma21QQ allele. A loss of either the HPH or ORM gene pairs alone did not result in a decrease in vacuolar acidification or defects in V-ATPase assembly. While the Hph proteins are not required for V-ATPase function, Orm1p and Orm2p are required for full V-ATPase enzyme function. Consistent with the documented role of the Orm proteins in sphingolipid regulation, we have found that inhibition of sphingolipid synthesis alleviates Orm-related growth defects.
Asunto(s)
Elementos de Facilitación Genéticos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/enzimología , Esfingolípidos/genética , ATPasas de Translocación de Protón Vacuolares/metabolismo , Genoma Fúngico , Péptidos y Proteínas de Señalización Intracelular/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Esfingolípidos/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Vacuolas/enzimología , Vacuolas/genética , Vacuolas/metabolismoRESUMEN
In 1992, Raymond et al. published a compilation of the 41 yeast vacuolar protein sorting (vps) mutant groups and described a large class of mutants (class E vps mutants) that accumulated an exaggerated prevacuolar endosome-like compartment. Further analysis revealed that this "class E compartment" contained soluble vacuolar hydrolases, vacuolar membrane proteins, and Golgi membrane proteins unable to recycle back to the Golgi complex, yet these class E vps mutants had what seemed to be normal vacuoles. The 13 class E VPS genes were later shown to encode the proteins that make up the complexes required for formation of intralumenal vesicles in late endosomal compartments called multivesicular bodies, and for the sorting of ubiquitinated cargo proteins into these internal vesicles for eventual delivery to the vacuole or lysosome.
Asunto(s)
Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Cuerpos Multivesiculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Endosomas/metabolismo , Aparato de Golgi/metabolismo , Historia del Siglo XX , Cuerpos Multivesiculares/genética , Mutación , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/genética , Vacuolas/metabolismo , Vacuolas/ultraestructura , Proteínas de Transporte Vesicular/genéticaRESUMEN
The yeast Saccharomyces cerevisiae vacuolar ATPase (V-ATPase) is a multisubunit complex divided into two sectors: the V(1) sector catalyzes ATP hydrolysis and the V(0) sector translocates protons, resulting in acidification of its resident organelle. Four protein factors participate in V(0) assembly. We have discovered a fifth V(0) assembly factor, Voa1p (YGR106C); an endoplasmic reticulum (ER)-localized integral membrane glycoprotein. The role of Voa1p in V(0) assembly was revealed in cells expressing an ER retrieval-deficient form of the V-ATPase assembly factor Vma21p (Vma21pQQ). Loss of Voa1p in vma21QQ yeast cells resulted in loss of V-ATPase function; cells were unable to acidify their vacuoles and exhibited growth defects typical of cells lacking V-ATPase. V(0) assembly was severely compromised in voa1 vma21QQ double mutants. Isolation of V(0)-Vma21p complexes indicated that Voa1p associates most strongly with Vma21p and the core proteolipid ring of V(0) subunits c, c', and c''. On assembly of the remaining three V(0) subunits (a, d, and e) into the V(0) complex, Voa1p dissociates from the now fully assembled V(0)-Vma21p complex. Our results suggest Voa1p functions with Vma21p early in V(0) assembly in the ER, but then it dissociates before exit of the V(0)-Vma21p complex from the ER for transport to the Golgi compartment.
Asunto(s)
Retículo Endoplásmico/enzimología , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Aparato de Golgi/metabolismo , Humanos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Complejos Multiproteicos/metabolismo , Estructura Secundaria de Proteína , Subunidades de Proteína/química , Subunidades de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/química , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
How individual protein subunits assemble into the higher order structure of a protein complex is not well understood. Four proteins dedicated to the assembly of the V(0) subcomplex of the V-adenosine triphosphatase (V-ATPase) in the endoplasmic reticulum (ER) have been identified in yeast, but their precise mode of molecular action remains to be identified. In contrast to the highly conserved subunits of the V-ATPase, orthologs of the yeast assembly factors are not easily identified based on sequence similarity. We show in this study that two ER-localized Arabidopsis proteins that share only 25% sequence identity with Vma21p can functionally replace this yeast assembly factor. Loss of AtVMA21a function in RNA interference seedlings caused impaired cell expansion and changes in Golgi morphology characteristic for plants with reduced V-ATPase activity, and we therefore conclude that AtVMA21a is the first V-ATPase assembly factor identified in a multicellular eukaryote. Moreover, VMA21p acts as a dedicated ER escort chaperone, a class of substrate-specific accessory proteins so far not identified in higher plants.
Asunto(s)
Proteínas de Arabidopsis/biosíntesis , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Chaperoninas/biosíntesis , Chaperoninas/genética , Chaperoninas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/ultraestructura , Electroforesis en Gel de Poliacrilamida , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/ultraestructura , Aparato de Golgi/enzimología , Aparato de Golgi/ultraestructura , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Plásmidos , Subunidades de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/biosíntesis , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/biosíntesis , ATPasas de Translocación de Protón Vacuolares/genéticaRESUMEN
The yeast Saccharomyces cerevisiae vacuolar H(+)-ATPase (V-ATPase) is a multisubunit complex responsible for acidifying intracellular organelles and is highly regulated. One of the regulatory subunits, subunit H, is encoded by the VMA13 gene in yeast and is composed of two domains, the N-terminal domain (amino acids (aa) 1-352) and the C-terminal domain (aa 353-478). The N-terminal domain is required for the activation of the complex, whereas the C-terminal domain is required for coupling ATP hydrolysis to proton translocation (Liu, M., Tarsio, M., Charsky, C. M., and Kane, P. M. (2005) J. Biol. Chem. 280, 36978-36985). Experiments with epitope-tagged copies of Vma13p revealed that there is only one copy of Vma13p/subunit H per V-ATPase complex. Analysis of the N-terminal domain shows that the first 179 amino acids are not required for the activation and full function of the V-ATPase complex and that the minimal region of Vma13p/subunit H capable of activating the V-ATPase is aa 180-353 of the N-terminal domain. Subunit H is expressed as two splice variants in mammals, and deletion of 18 amino acids in yeast Vma13p corresponding to the mammalian subunit H beta isoform results in reduced V-ATPase activity and significantly lower coupling of ATPase hydrolysis to proton translocation. Intriguingly, the yeast Vma13p mimicking the mammalian subunit H beta isoform is functionally equivalent to Vma13p lacking the entire C-terminal domain. These results suggest that the mammalian V-ATPase complexes with subunit H splice variant SFD-alpha or SFD-beta are likely to have different activities and may perform distinct cellular functions.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , ATPasas de Translocación de Protón Vacuolares/metabolismo , Secuencia de Aminoácidos , Bioquímica/métodos , Epítopos/química , Concentración de Iones de Hidrógeno , Hidrólisis , Modelos Biológicos , Conformación Molecular , Datos de Secuencia Molecular , Plásmidos/metabolismo , Conformación Proteica , Isoformas de Proteínas , Estructura Terciaria de ProteínaRESUMEN
Endosomal transport is critical for cellular processes ranging from receptor down-regulation and retroviral budding to the immune response. A full understanding of endosome sorting requires a comprehensive picture of the multiprotein complexes that orchestrate vesicle formation and fusion. Here, we use unsupervised, large-scale phenotypic analysis and a novel computational approach for the global identification of endosomal transport factors. This technique effectively identifies components of known and novel protein assemblies. We report the characterization of a previously undescribed endosome sorting complex that contains two well-conserved proteins with four predicted membrane-spanning domains. Vps55p and Vps68p form a complex that acts with or downstream of ESCRT function to regulate endosomal trafficking. Loss of Vps68p disrupts recycling to the TGN as well as onward trafficking to the vacuole without preventing the formation of lumenal vesicles within the MVB. Our results suggest the Vps55/68 complex mediates a novel, conserved step in the endosomal maturation process.
Asunto(s)
Proteínas Portadoras/metabolismo , Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Biológico Activo , Carboxipeptidasas/genética , Carboxipeptidasas/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Fúngicos , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Familia de Multigenes , Complejos Multiproteicos , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Receptores del Factor de Conjugación/genética , Receptores del Factor de Conjugación/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte VesicularRESUMEN
Deletion of the yeast gene PKR1 (YMR123W) results in an inability to grow on iron-limited medium. Pkr1p is localized to the membrane of the endoplasmic reticulum. Cells lacking Pkr1p show reduced levels of the V-ATPase subunit Vph1p due to increased turnover of the protein in mutant cells. Reduced levels of the V-ATPase lead to defective copper loading of Fet3p, a component of the high affinity iron transport system. Levels of Vph1p in cells lacking Pkr1p are similar to cells unable to assemble a functional V-ATPase due to lack of a V0 subunit or an endoplasmic reticulum (ER) assembly factor. However, unlike yeast mutants lacking a V0 subunit or a V-ATPase assembly factor, low levels of Vph1p present in cells lacking Pkr1p are assembled into a V-ATPase complex, which exits the ER and is present on the vacuolar membrane. The V-ATPase assembled in the absence of Pkr1p is fully functional because the mutant cells are able to weakly acidify their vacuoles. Finally, overexpression of the V-ATPase assembly factor Vma21p suppresses the growth and acidification defects of pkr1Delta cells. Our data indicate that Pkr1p functions together with the other V-ATPase assembly factors in the ER to efficiently assemble the V-ATPase membrane sector.
Asunto(s)
Proteínas Fúngicas/química , Proteínas de la Membrana/química , Proteínas de Saccharomyces cerevisiae/química , ATPasas de Translocación de Protón Vacuolares/fisiología , Secuencia de Aminoácidos , Clonación Molecular , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Eliminación de Gen , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/fisiología , Chaperonas Moleculares , Datos de Secuencia Molecular , Mutación , Conformación Proteica , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiología , Homología de Secuencia de Aminoácido , ATPasas de Translocación de Protón Vacuolares/metabolismo , Vacuolas/metabolismoRESUMEN
Previous two-hybrid analysis of the 17 soluble class E Vps yeast proteins revealed that Vps46p/Did2p interacts with Vta1p and the AAA (ATPase associated with a variety of cellular activities) ATPase Vps4p. Here we report that the binding of Vps46p to Vps4p and Vta1p is direct and not mediated by additional proteins, and the binding of Vps46p to Vps4p is ATP independent. Vps46p regulates the membrane association of Vps4p and is required for the interaction of Vta1p with Vps32p/Snf7p of the ESCRT-III complex. Vta1p is a potent activator of Vps4p, stimulating the ATPase activity by 6- to 8-fold. These results reveal functional roles for the Vps46p and Vta1p proteins in regulating the ESCRT complex assembly/disassembly cycle in protein sorting at the yeast late endosome.