RESUMO
One of the goals of human genetics is to discover the variants that contribute to human diseases [...].
Assuntos
Doenças Neurodegenerativas , Proteínas de Saccharomyces cerevisiae , Humanos , Doenças Neurodegenerativas/genética , Saccharomyces cerevisiaeRESUMO
Mutations in human VPS13A-D genes result in rare neurological diseases, including chorea-acanthocytosis. The pathogenesis of these diseases is poorly understood, and no effective treatment is available. As VPS13 genes are evolutionarily conserved, the effects of the pathogenic mutations could be studied in model organisms, including yeast, where one VPS13 gene is present. In this review, we summarize advancements obtained using yeast. In recent studies, vps13Δ and vps13-I2749 yeast mutants, which are models of chorea-acanthocytosis, were used to screen for multicopy and chemical suppressors. Two of the suppressors, a fragment of the MYO3 and RCN2 genes, act by downregulating calcineurin activity. In addition, vps13Δ suppression was achieved by using calcineurin inhibitors. The other group of multicopy suppressors were genes: FET4, encoding iron transporter, and CTR1, CTR3 and CCC2, encoding copper transporters. Mechanisms of their suppression rely on causing an increase in the intracellular iron content. Moreover, among the identified chemical suppressors were copper ionophores, which require a functional iron uptake system for activity, and flavonoids, which bind iron. These findings point at areas for further investigation in a higher eukaryotic model of VPS13-related diseases and to new therapeutic targets: calcium signalling and copper and iron homeostasis. Furthermore, the identified drugs are interesting candidates for drug repurposing for these diseases.
Assuntos
Neuroacantocitose , Doenças Neurodegenerativas , Proteínas de Saccharomyces cerevisiae , Sinalização do Cálcio , Proteínas de Ligação ao Cálcio/metabolismo , Cobre/metabolismo , Proteínas de Transporte de Cobre , Humanos , Ferro/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Doenças Neurodegenerativas/tratamento farmacológico , Doenças Neurodegenerativas/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Neurodegenerative diseases are a group of age-related diseases and a growing problem in an aging society [...].
Assuntos
Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Modelos Biológicos , Doenças Neurodegenerativas/patologia , Saccharomyces cerevisiae/metabolismo , Envelhecimento , Proteínas Fúngicas/genética , Humanos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
VPS13 proteins are evolutionarily conserved. Mutations in the four human genes (VPS13A-D) encoding VPS13A-D proteins are linked to developmental or neurodegenerative diseases. The relationship between the specific localization of individual VPS13 proteins, their molecular functions, and the pathology of these diseases is unknown. Here we used a yeast model to establish the determinants of Vps13's interaction with the membranes of Golgi apparatus. We analyzed the different phenotypes of the arf1-3 arf2Δ vps13∆ strain, with reduced activity of the Arf1 GTPase, the master regulator of Golgi function and entirely devoid of Vps13. Our analysis led us to propose that Vps13 and Arf1 proteins cooperate at the Golgi apparatus. We showed that Vps13 binds to the Arf1 GTPase through its C-terminal Pleckstrin homology (PH)-like domain. This domain also interacts with phosphoinositol 4,5-bisphosphate as it was bound to liposomes enriched with this lipid. The homologous domain of VPS13A exhibited the same behavior. Furthermore, a fusion of the PH-like domain of Vps13 to green fluorescent protein was localized to Golgi structures in an Arf1-dependent manner. These results suggest that the PH-like domains and Arf1 are determinants of the localization of VPS13 proteins to the Golgi apparatus in yeast and humans.
Assuntos
Fator 1 de Ribosilação do ADP/metabolismo , Complexo de Golgi/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fator 1 de Ribosilação do ADP/genética , Clatrina/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Microscopia Confocal , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Biológicos , Mutação , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Ligação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , TemperaturaRESUMO
Regulation of calcineurin, a Ca2+/calmodulin-regulated phosphatase, is important for the nervous system, and its abnormal activity is associated with various pathologies, including neurodegenerative disorders. In yeast cells lacking the VPS13 gene (vps13Δ), a model of VPS13-linked neurological diseases, we recently demonstrated that calcineurin is activated, and its downregulation reduces the negative effects associated with vps13Δ mutation. Here, we show that overexpression of the RCN2 gene, which encodes a negative regulator of calcineurin, is beneficial for vps13Δ cells. We studied the molecular mechanism underlying this effect through site-directed mutagenesis of RCN2. The interaction of the resulting Rcn2 variants with a MAPK kinase, Slt2, and subunits of calcineurin was tested. We show that Rcn2 binds preferentially to Cmp2, one of two alternative catalytic subunits of calcineurin, and partially inhibits calcineurin. Rcn2 ability to bind to and reduce the activity of calcineurin was important for the suppression. The binding of Rcn2 to Cmp2 requires two motifs in Rcn2: the previously characterized C-terminal motif and a new N-terminal motif that was discovered in this study. Altogether, our findings can help to better understand calcineurin regulation and to develop new therapeutic strategies against neurodegenerative diseases based on modulation of the activity of selected calcineurin isoforms.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Calcineurina/metabolismo , Proteínas de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transdução de Sinal/genética , Sítios de Ligação , Dosagem de Genes , Regulação da Expressão Gênica , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre ProteínasRESUMO
Ion homeostasis is crucial for organism functioning, and its alterations may cause diseases. For example, copper insufficiency and overload are associated with Menkes and Wilson's diseases, respectively, and iron imbalance is observed in Parkinson's and Alzheimer's diseases. To better understand human diseases, Saccharomyces cerevisiae yeast are used as a model organism. In our studies, we used the vps13Δ yeast strain as a model of rare neurological diseases caused by mutations in VPS13A-D genes. In this work, we show that overexpression of genes encoding copper transporters, CTR1, CTR3, and CCC2, or the addition of copper salt to the medium, improved functioning of the vps13Δ mutant. We show that their mechanism of action, at least partially, depends on increasing iron content in the cells by the copper-dependent iron uptake system. Finally, we present that treatment with copper ionophores, disulfiram, elesclomol, and sodium pyrithione, also resulted in alleviation of the defects observed in vps13Δ cells. Our study points at copper and iron homeostasis as a potential therapeutic target for further investigation in higher eukaryotic models of VPS13-related diseases.
Assuntos
Proteínas de Transporte de Cobre/metabolismo , Cobre/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Homeostase , Mutação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Transporte de Cobre/genética , Humanos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Probing the pathogenicity and functional consequences of mitochondrial DNA (mtDNA) mutations from patient's cells and tissues is difficult due to genetic heteroplasmy (co-existence of wild type and mutated mtDNA in cells), occurrence of numerous mtDNA polymorphisms, and absence of methods for genetically transforming human mitochondria. Owing to its good fermenting capacity that enables survival to loss-of-function mtDNA mutations, its amenability to mitochondrial genome manipulation, and lack of heteroplasmy, Saccharomyces cerevisiae is an excellent model for studying and resolving the molecular bases of human diseases linked to mtDNA in a controlled genetic background. Using this model, we previously showed that a pathogenic mutation in mitochondrial ATP6 gene (m.9191T>C), that converts a highly conserved leucine residue into proline in human ATP synthase subunit a (aL222P), severely compromises the assembly of yeast ATP synthase and reduces by 90% the rate of mitochondrial ATP synthesis. Herein, we report the isolation of intragenic suppressors of this mutation. In light of recently described high resolution structures of ATP synthase, the results indicate that the m.9191T>C mutation disrupts a four α-helix bundle in subunit a and that the leucine residue it targets indirectly optimizes proton conduction through the membrane domain of ATP synthase.
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Mitocôndrias/metabolismo , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Trifosfato de Adenosina/metabolismo , Potencial da Membrana Mitocondrial/genética , ATPases Mitocondriais Próton-Translocadoras/química , Modelos Químicos , Mutação , Fosforilação Oxidativa , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
Human Vps13 proteins are associated with several diseases, including the neurodegenerative disorder Chorea-acanthocytosis (ChAc), yet the biology of these proteins is still poorly understood. Studies in Saccharomyces cerevisiae, Dictyostelium discoideum, Tetrahymena thermophila and Drosophila melanogaster point to the involvement of Vps13 in cytoskeleton organization, vesicular trafficking, autophagy, phagocytosis, endocytosis, proteostasis, sporulation and mitochondrial functioning. Recent findings show that yeast Vps13 binds to phosphatidylinositol lipids via 4 different regions and functions at membrane contact sites, enlarging the list of Vps13 functions. This review describes the great potential of simple eukaryotes to decipher disease mechanisms in higher organisms and highlights novel insights into the pathological role of Vps13 towards ChAc.
Assuntos
Neuroacantocitose/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Dictyostelium/metabolismo , Drosophila melanogaster/metabolismo , Humanos , Mutação , Neuroacantocitose/genética , Neuroacantocitose/patologia , Transporte Proteico , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidade da Espécie , Proteínas de Transporte Vesicular/genéticaRESUMO
The rare human disorder chorea-acanthocytosis (ChAc) is caused by mutations in hVPS13A gene. The hVps13A protein interacts with actin and regulates the level of phosphatidylinositol 4-phosphate (PI4P) in the membranes of neuronal cells. Yeast Vps13 is involved in vacuolar protein transport and, like hVps13A, participates in PI4P metabolism. Vps13 proteins are conserved in eukaryotes, but their molecular function remains unknown. One of the mutations found in ChAc patients causes amino acids substitution I2771R which affects the localization of hVps13A in skeletal muscles. To dissect the mechanism of pathogenesis of I2771R, we created and analyzed a yeast strain carrying the equivalent mutation. Here we show that in yeast, substitution I2749R causes dysfunction of Vps13 protein in endocytosis and vacuolar transport, although the level of the protein is not affected, suggesting loss of function. We also show that Vps13, like hVps13A, influences actin cytoskeleton organization and binds actin in immunoprecipitation experiments. Vps13-I2749R binds actin, but does not function in the actin cytoskeleton organization. Moreover, we show that Vps13 binds phospholipids, especially phosphatidylinositol 3-phosphate (PI3P), via its SHR_BD and APT1 domains. Substitution I2749R attenuates this ability. Finally, the localization of Vps13-GFP is altered when cellular levels of PI3P are decreased indicating its trafficking within the endosomal membrane system. These results suggest that PI3P regulates the functioning of Vps13, both in protein trafficking and actin cytoskeleton organization. Attenuation of PI3P-binding ability in the mutant hVps13A protein may be one of the reasons for its mislocalization and disrupted function in cells of patients suffering from ChAc.
Assuntos
Substituição de Aminoácidos/genética , Neuroacantocitose/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Transporte Vesicular/genética , Citoesqueleto de Actina/genética , Transporte Biológico/genética , Endossomos/genética , Humanos , Mutação , Neuroacantocitose/patologia , Fosfatos de Fosfatidilinositol/metabolismo , Saccharomyces cerevisiae/genéticaRESUMO
Chorein is a protein of the Vps13 family, and defects in this protein cause the rare neurodegenerative disorder chorea-acanthocytosis (ChAc). Chorein is involved in the actin cytoskeleton organization, calcium ion flux, neuronal cell excitability, exocytosis and autophagy. The function of this protein is poorly understood, and obtaining this knowledge is a key to finding a cure for ChAc. Chorein, as well as the Vps13 protein from yeast, contains the APT1 domain. Our previous research has shown that the APT1 domain from yeast Vps13 (yAPT1v) binds phosphatidylinositol 3-phosphate (PI3P) in vitro. In this study, we showed that although the APT1 domain from chorein (hAPT1) binds to PI3P it could not functionally replace yAPT1v. The hAPT1 domain binds, in addition to PI3P, to phosphatidylinositol 5-phosphate (PI5P). The binding of hAPT1 to PI3P, unlike the binding of yAPT1v to PI3P, is regulated by the bivalent ions, calcium and magnesium. Regulation of PI3P binding via calcium is also observed for the APT1 domain of yeast autophagy protein Atg2. The substitution I2771R, found in chorein of patient suffering from ChAc, reduces the binding of the hAPT1 domain to PI3P and PI5P. These results suggest that the ability of APT1 domains to bind phosphoinositides is regulated differently in yeast and human protein and that this regulation is important for chorein function.
Assuntos
Neuroacantocitose/genética , Proteínas de Saccharomyces cerevisiae/genética , Tioléster Hidrolases/genética , Proteínas de Transporte Vesicular/genética , Autofagia/genética , Proteínas Relacionadas à Autofagia/genética , Cálcio/química , Humanos , Íons/química , Magnésio/química , Mutação/genética , Neuroacantocitose/metabolismo , Neuroacantocitose/patologia , Neurônios/metabolismo , Neurônios/patologia , Fosfatos de Fosfatidilinositol/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Tioléster Hidrolases/química , Proteínas de Transporte Vesicular/químicaRESUMO
Several rare neurodegenerative diseases, including chorea acanthocytosis, are caused by mutations in the VPS13A-D genes. Only symptomatic treatments for these diseases are available. Saccharomyces cerevisiae contains a unique VPS13 gene and the yeast vps13Δ mutant has been proven as a suitable model for drug tests. A library of drugs and an in-house library of natural compounds and their derivatives were screened for molecules preventing the growth defect of vps13Δ cells on medium with sodium dodecyl sulfate (SDS). Seven polyphenols, including the iron-binding flavone luteolin, were identified. The structure-activity relationship and molecular mechanisms underlying the action of luteolin were characterized. The FET4 gene, which encodes an iron transporter, was found to be a multicopy suppressor of vps13Δ, pointing out the importance of iron in response to SDS stress. The growth defect of vps13Δ in SDS-supplemented medium was also alleviated by the addition of iron salts. Suppression did not involve cell antioxidant responses, as chemical antioxidants were not active. Our findings support that luteolin and iron may target the same cellular process, possibly the synthesis of sphingolipids. Unveiling the mechanisms of action of chemical and genetic suppressors of vps13Δ may help to better understand VPS13A-D-dependent pathogenesis and to develop novel therapeutic strategies.
Assuntos
Produtos Biológicos/farmacologia , Descoberta de Drogas/métodos , Ensaios de Triagem em Larga Escala/métodos , Luteolina/farmacologia , Fármacos Neuroprotetores/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Produtos Biológicos/química , Proliferação de Células/efeitos dos fármacos , Proteínas de Transporte de Cobre/genética , Proteínas de Transporte de Cobre/metabolismo , Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas de Ligação ao Ferro/metabolismo , Luteolina/química , Fármacos Neuroprotetores/química , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Relação Estrutura-Atividade , Supressão GenéticaRESUMO
Rsp5p, a yeast S. cerevisiae ubiquitin ligase, is essential for regulation of unsaturated fatty acid synthesis via activation of the transcriptional activators Spt23p and Mga2p. Here we show that the conditional mutant rsp5-19 produces decreased levels of the end products of mevalonate pathway, such as ergosterol, ubiquinone and of dolichols, especially those with 19-24 isoprene units. The mechanism of Rsp5p involvement in the control of these lipid synthesis pathways was addressed by overproduction of Rsp5p-independent Spt23p or Mga2p. Expression of constitutively active forms of these transactivators resulted in excess production of ergosterol, but did not restore a wild-type level of dolichols. Moreover, synthesis of long chain dolichols was decreased in the wild-type and a rsp5-19 background. Finally, overproduction of active Spt23p or Mga2p was accompanied by the appearance of large lipid particles in the wild-type and rsp5-19 strains as observed by Nile Red staining, due to accumulation of unsaturated triacylglycerol. Thus, we conclude that Rsp5p, Spt23p and Mga2p may participate in the control of the homeostasis of lipids and lipid particles.
Assuntos
Ácido Mevalônico/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Triglicerídeos/biossíntese , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Vias Biossintéticas , Grânulos Citoplasmáticos/metabolismo , Dimetilaliltranstransferase/genética , Dimetilaliltranstransferase/metabolismo , Dolicóis/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte , Ergosterol/biossíntese , Metabolismo dos Lipídeos , Proteínas de Membrana , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Esteróis/biossíntese , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição , Transformação Genética , Ubiquinona/biossíntese , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismoRESUMO
Human Nedd4 ubiquitin ligase is involved in protein trafficking, signal transduction and oncogenesis. Nedd4 with an inactive WW4 domain is toxic to yeast cells. We report here that actin cytoskeleton is abnormal in yeast cells expressing the NEDD4 or NEDD4w4 gene and these cells are more sensitive to Latrunculin A, an actin-depolymerizing drug. These phenotypes are less pronounced when a mutation inactivating the catalytic domain of the ligase has been introduced. In contrast, overexpression of the LAS17 gene, encoding an activator of the Arp2/3 actin nucleating complex, is detrimental to NEDD4w4-expressing cells. The level of Las17p is increased in cells overproducing Nedd4w4 and this depends partially on its catalytic domain. Expression of genes encoding Nedd4 variants, like overexpression of LAS17, suppresses the growth defect of the arp2-1 strain. Our results suggest that human Nedd4 ligase inhibits yeast cell growth by disturbing the actin cytoskeleton, in part by increasing Las17p level, and that Nedd4 ubiquitination targets may include actin cytoskeleton-associated proteins conserved in evolution.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligases/fisiologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Complexos Endossomais de Distribuição Requeridos para Transporte , Imunofluorescência , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/genética , Técnicas de Transferência de Genes , Humanos , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/farmacologia , Temperatura , Tiazolidinas/farmacologia , Ubiquitina-Proteína Ligases/genética , Proteína da Síndrome de Wiskott-Aldrich/biossíntese , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/farmacologiaRESUMO
Chorea-acanthocytosis (ChAc) is a rare neurodegenerative disease associated with mutations in the human VPS13A gene. The mechanism of ChAc pathogenesis is unclear. A simple yeast model was used to investigate the function of the single yeast VSP13 orthologue, Vps13. Vps13, like human VPS13A, is involved in vesicular protein transport, actin cytoskeleton organisation and phospholipid metabolism. A newly identified phenotype of the vps13Δ mutant, sodium dodecyl sulphate (SDS) hypersensitivity, was used to screen a yeast genomic library for multicopy suppressors. A fragment of the MYO3 gene, encoding Myo3-N (the N-terminal part of myosin, a protein involved in the actin cytoskeleton and in endocytosis), was isolated. Myo3-N protein contains a motor head domain and a linker. The linker contains IQ motifs that mediate the binding of calmodulin, a negative regulator of myosin function. Amino acid substitutions that disrupt the interaction of Myo3-N with calmodulin resulted in the loss of vps13Δ suppression. Production of Myo3-N downregulated the activity of calcineurin, a protein phosphatase regulated by calmodulin, and alleviated some defects in early endocytosis events. Importantly, ethylene glycol tetraacetic acid (EGTA), which sequesters calcium and thus downregulates calmodulin and calcineurin, was a potent suppressor of vps13Δ. We propose that Myo3-N acts by sequestering calmodulin, downregulating calcineurin and increasing activity of Myo3, which is involved in endocytosis and, together with Osh2/3 proteins, functions in endoplasmic reticulum-plasma membrane contact sites. These results show that defects associated with vps13Δ could be overcome, and point to a functional connection between Vps13 and calcium signalling as a possible target for chemical intervention in ChAc. Yeast ChAc models may uncover the underlying pathological mechanisms, and may also serve as a platform for drug testing.This article has an associated First Person interview with the first author of the paper.
Assuntos
Sinalização do Cálcio , Cálcio/metabolismo , Calmodulina/metabolismo , Modelos Biológicos , Miosinas/metabolismo , Neuroacantocitose/tratamento farmacológico , Neuroacantocitose/metabolismo , Saccharomyces cerevisiae/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Alelos , Substituição de Aminoácidos , Calcineurina/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Canavanina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Endocitose/efeitos dos fármacos , Genes Supressores , Mutação/genética , Domínios Proteicos , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/metabolismo , Dodecilsulfato de Sódio , Transcrição Gênica/efeitos dos fármacos , Vacúolos/metabolismoRESUMO
Rsp5p of Saccharomyces cerevisiae is a member of the C2-WW-HECT family of ubiquitin ligases and it interacts with targets via its WW domains. Spt23p and Mga2p are Rsp5p substrates and Rsp5p activates the OLE1 inducing functions of these membrane-localized transcription factors by ubiquitination. Although it is known that Rsp5p binds Mga2p and Spt23p via an imperfect WW domain-binding site (LPKY) that is located within the carboxy-terminal domain of the proteins, it remains unclear which WW domains mediate binding. We show that Rsp5p mutants harboring mutations in single WW domains are Spt23p/Mga2p binding and ubiquitination proficient. This is also the case for WW domains 1/2 and WW domains 1/3 mutants. However, disrupting WW domains 2 and 3 abrogates a physical and functional interaction with substrates in vitro and in cells. We also show that abrogation of WW domains 2 and 3 eliminates the activity of an Rsp5p dominant-negative mutant and an rsp5 WW domain 2/3 mutant is unable to rescue the proliferative defects of rsp5 Delta cells. Interestingly, while rsp5 Delta cells are able to grow on oleic acid containing YPD media, they as well as those transformed with the WW domain 2/3 mutant are unable to proliferate on oleic acid containing synthetic drop-out media. We conclude from these studies that WW domains 2 and 3 of Rsp5p play overlapping roles in binding to the LPKY site on Spt23p and Mga2p. Also, we propose that WW domains 2 and 3 perform yet to be defined essential function(s) outside of the OLE1 pathway when cells are grown in nutrient restrictive media.
Assuntos
Ácidos Graxos Dessaturases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , DNA Fúngico , Complexos Endossomais de Distribuição Requeridos para Transporte , Indução Enzimática , Técnicas de Transferência de Genes , Proteínas de Membrana , Mutagênese Sítio-Dirigida , Mutação , Domínios e Motivos de Interação entre Proteínas , Ensaio Radioligante , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Estearoil-CoA Dessaturase , Especificidade por Substrato , Transativadores/genética , Fatores de Transcrição , Ativação Transcricional , Complexos Ubiquitina-Proteína Ligase/genética , Ubiquitina-Proteína Ligases/genética , UbiquitinaçãoRESUMO
Rsp5 is a conserved ubiquitin ligase involved in regulation of numerous cellular processes. A growing number of publications describing new functions of the ligase have appeared in recent years. Rsp5 was shown to be involved in the control of intracellular trafficking of proteins via endocytosis and multivesicular body sorting. Moreover, nuclear functions of Rsp5 in response to various stresses have been discovered. Rsp5 is also involved in the regulation of unsaturated fatty acid and sterol synthesis and phospholipid composition. Here, an overview of Rsp5 functions with emphasis on its involvement in the regulation of lipid biosynthesis will be presented.
Assuntos
Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/fisiologia , Complexos Ubiquitina-Proteína Ligase/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Saccharomyces cerevisiae/citologia , UbiquitinaçãoRESUMO
A number of eukaryotic transcription factors are held in a latent state by being embedded in, or tethered to, cellular membranes. Mga2p of Saccharomyces cerevisiae is an endoplasmic reticulum (ER)-localized transcription factor that plays an overlapping role with homologous Spt23p in upregulating expression of OLE1, a gene required for the synthesis of essential oleic acid. Previous studies have documented that proteasome-dependent processing of ER bound 120 kDa Mga2p and Spt23p proteins generates transcriptionally competent 90 kDa polypeptides. In the case of Spt23p90, it is held at the membrane prior to release via a self-interaction with the unprocessed Spt23p120 anchor. It is currently thought that the highly conserved Rsp5p ubiquitin ligase provides the signal for partial degradation of both proteins. Cells lacking Rsp5p function require oleic acid for growth, and Spt23p processing is suppressed in rsp5 Delta cells and in wild-type RSP5 cells upon expression of Rsp5p dominant-negative mutants. We report here that Rsp5p is dispensable for Mga2p90 generation but not for release of the processed product from the ER. In addition, we demonstrate that polyubiquitinated Mga2p120 accumulates in cells lacking Npl4p or proteasome function and Rsp5p is required for Mga2p120 polyubiquitination. Finally, we provide evidence that Mga2p90 and Mga2p120 dimerize and that Rsp5p binds heterodimeric Mga2p complexes both in vitro and in vivo. In light of these experiments, we propose that Rsp5p facilitates Mga2p90 release from the ER by promoting polyubiquitination and Npl4p-proteasome-mediated degradation of the interacting Mga2p120 ER bound anchor.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Transativadores/metabolismo , Transcrição Gênica/fisiologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Western Blotting , Dimerização , Eletroforese em Gel de Poliacrilamida , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas de Membrana , Modelos Biológicos , Testes de Precipitina , Saccharomyces cerevisiae , Fatores de TranscriçãoRESUMO
Pan1 is an actin patch-associated protein involved in endocytosis. Our studies revealed that in oleate-grown cells Pan1 is located in the nucleus as well as in patches. One of three putative nuclear localization signals (NLS) of Pan1, NLS2, directed beta-galactosidase (beta-gal) to the nucleus. However, GFP-Pan1(886-1219), containing NLS2, was found in the cytoplasm indicating that it may contain a nuclear export signal (NES). A putative Pan1 NES, overlapping with NLS3, re-addressed NLS(H2B)-NES/NLS3-beta-gal from the nucleus to the cytoplasm. Inactivation of the NES allowed NLS3 to be effective. Thus, Pan1 contains functional NLSs and a NES and appears to shuttle in certain circumstances.
Assuntos
Núcleo Celular/metabolismo , Proteínas Fúngicas/metabolismo , Sinais de Exportação Nuclear , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Genes Reporter/genética , Proteínas dos Microfilamentos , Dados de Sequência Molecular , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiaeRESUMO
Rsp5p is an ubiquitin-protein ligase of Saccharomyces cerevisiae that has been implicated in numerous processes including transcription, mitochondrial inheritance, and endocytosis. Rsp5p functions at multiple steps of endocytosis, including ubiquitination of substrates and other undefined steps. We propose that one of the roles of Rsp5p in endocytosis involves maintenance and remodeling of the actin cytoskeleton. We report the following. (i) There are genetic interactions between rsp5 and several mutant genes encoding actin cytoskeletal proteins. rsp5 arp2, rsp5 end3, and rsp5 sla2 double mutants all show synthetic growth defects. Overexpressed wild-type RSP5 or mutant rsp5 genes with lesions of some WW domains suppress growth defects of arp2 and end3 cells. The defects in endocytosis, actin cytoskeleton, and morphology of arp2 are also suppressed. (ii) Rsp5p and Sla2p colocalize in abnormal F-actin-containing clumps in arp2 and pan1 mutants. Immunoprecipitation experiments confirmed that Rsp5p and Act1p colocalize in pan1 mutants. (iii) Rsp5p and Sla2p coimmunoprecipitate and partially colocalize to punctate structures in wild-type cells. These studies provide the first evidence for an interaction of an actin cytoskeleton protein with Rsp5p. (iv) rsp5-w1 mutants are resistant to latrunculin A, a drug that sequesters actin monomers and depolymerizes actin filaments, consistent with the fact that Rsp5p is involved in actin cytoskeleton dynamics.
Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Endocitose/fisiologia , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Complexos Ubiquitina-Proteína Ligase , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteínas de Transporte/genética , Proteínas do Citoesqueleto , Complexos Endossomais de Distribuição Requeridos para Transporte , Proteínas Fúngicas/genética , Vetores Genéticos , Ligases/genética , Mutagênese , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Frações Subcelulares , Tiazóis/farmacologia , Tiazolidinas , Ubiquitina-Proteína LigasesRESUMO
The Rsp5 ubiquitin ligase plays a role in many cellular processes including the biosynthesis of unsaturated fatty acids. The PIS1 (phosphatidylinositol synthase gene) encoding the enzyme Pis1p which catalyses the synthesis of phosphatidylinositol from CDP-diacyglycerol and inositol, was isolated in a screen for multicopy suppressors of the rsp5 temperature sensitivity phenotype. Suppression was allele non-specific. Interestingly, expression of PIS1 was 2-fold higher in the rsp5 mutant than in wild-type yeast, whereas the introduction of PIS1 in a multicopy plasmid increased the level of Pis1p 6-fold in both backgrounds. We demonstrate concomitantly that the expression of INO1 (inositol phosphate synthase gene) was also elevated approx. 2-fold in the rsp5 mutant as compared with the wild-type, and that inositol added to the medium improved growth of rsp5 mutants at a restrictive temperature. These results suggest that enhanced phosphatidylinositol synthesis may account for PIS1 suppression of rsp5 defects. Analysis of lipid extracts revealed the accumulation of saturated fatty acids in the rsp5 mutant, as a consequence of the prevention of unsaturated fatty acid synthesis. Overexpression of PIS1 did not correct the cellular fatty acid content; however, saturated fatty acids (C(16:0)) accumulated preferentially in phosphatidylinositol, and (wild-type)-like fatty acid composition in phosphatidylethanolamine was restored.