RESUMO
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
Yeast Rsp5 ubiquitin ligase is involved in several cellular processes, including endocytosis. Actin patches are sites of endocytosis, a process involving actin assembly and disassembly. Here we show Rsp5 localization in cortical patches and demonstrate its involvement in actin cytoskeleton organization and dynamics. We found that the Rsp5-F1-GFP2 N-terminal fragment and full length GFP-Rsp5 were recruited to peripheral patches that temporarily co-localized with Abp1-mCherry, a marker of actin patches. Actin cytoskeleton organization was defective in a strain lacking RSP5 or overexpressing RSP5, and this phenotype was accompanied by morphological abnormalities. Overexpression of RSP5 caused hypersensitivity of cells to Latrunculin A, an actin-depolymerizing drug and was toxic to cells lacking Las17, an activator of actin nucleation. Moreover, Rsp5 was required for efficient actin polymerization in a whole cell extract based in vitro system. Rsp5 interacted with Las17 and Las17-binding proteins, Lsb1 and Lsb2, in a GST-Rsp5-WW2/3 pull down assay. Rsp5 ubiquitinated Lsb1-HA and Lsb2-HA without directing them for degradation. Overexpression of RSP5 increased the cellular level of HA-Las17 in wild type and in lsb1Δ lsb2Δ strains in which the basal level of Las17 was already elevated. This increase was prevented in a strain devoid of Las17-binding protein Sla1 which is also a target of Rsp5 ubiquitination. Thus, Rsp5 together with Lsb1, Lsb2 and Sla1 regulate the level of Las17, an important activator of actin polymerization.
Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Complexos Ubiquitina-Proteína Ligase/metabolismo , Citoesqueleto de Actina/genética , Citoesqueleto/enzimologia , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Regulação Fúngica da Expressão Gênica , Humanos , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Complexos Ubiquitina-Proteína Ligase/genética , Proteína da Síndrome de Wiskott-Aldrich/genética , Proteína da Síndrome de Wiskott-Aldrich/metabolismoRESUMO
The ubiquitin ligase (E3) Rsp5p is the only member of the Nedd (neural-precursor-cell-expressed, developmentally down-regulated) 4 family of E3s present in yeast. Rsp5p has several proteasome-independent functions in membrane protein trafficking, including a role in the ubiquitination of most plasma membrane proteins, leading to their endocytosis. Rsp5p is also required for the ubiquitination of endosomal proteins, leading to their sorting to the internal vesicles of MVBs (multivesicular bodies). Rsp5p catalyses the attachment of non-conventional ubiquitin chains, linked through ubiquitin Lys-63, to some endocytic and MVB cargoes. This modification appears to be required for efficient sorting, possibly because these chains have a greater affinity for the ubiquitin-binding domains present within endocytic or MVB sorting complexes. The mechanisms involved in the recognition of plasma membrane and MVB substrates by Rsp5p remain unclear. A subset of Rsp5/Nedd4 substrates have a 'PY motif' and are recognized directly by the WW (Trp-Trp) domains of Rsp5p. Most Rsp5p substrates do not carry PY motifs, but some may depend on PY-containing proteins for their ubiquitination by Rsp5p, consistent with the latter's acting as specificity factors or adaptors. As in other ubiquitin-conjugating systems, these adaptors are also Rsp5p substrates and undergo ubiquitin-dependent trafficking. In the present review, we discuss recent examples illustrating the role of Rsp5p in membrane protein trafficking and providing new insights into the regulation of this E3 by adaptor proteins.
Assuntos
Transporte Biológico/fisiologia , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexos Ubiquitina-Proteína Ligase/metabolismo , Animais , Endocitose/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Ubiquitinas/metabolismoRESUMO
Rsp5p is an ubiquitin (Ub)-protein ligase of the Nedd4 family that carries WW domains involved in interaction with PPXY-containing proteins. It plays a key role at several stages of intracellular trafficking, such as Ub-mediated internalization of endocytic cargoes and Ub-mediated sorting of membrane proteins to internal vesicles of multivesicular bodies (MVBs), a process that is crucial for their subsequent targeting to the vacuolar lumen. Sna3p is a membrane protein previously described as an Ub-independent MVB cargo, but proteomic studies have since shown it to be an ubiquitylated protein. Sna3p carries a PPXY motif. We observed that this motif mediates its interaction with Rsp5p WW domains. Mutation of either the Sna3p PPXY motif or the Rsp5p WW3 domain or reduction in the amounts of Rsp5 results in the mistargeting of Sna3p to multiple mobile vesicles and prevents its sorting to the endosomal pathway. This sorting defect appears to occur prior to the defect displayed in rsp5 mutants by other MVB cargoes, which are correctly sorted to the endosomal pathway but missorted to the vacuolar membrane instead of the vacuolar lumen. Sna3p is polyubiquitylated on one target lysine, and a mutant Sna3p lacking its target lysine displays defective MVB sorting. Sna3p undergoes Rsp5-dependent polyubiquitylation, with K63-linked Ub chains.