RESUMO
Regulation of the epithelial Na(+) channel (ENaC) by ubiquitylation is controlled by the activity of two counteracting enzymes, the E3 ubiquitin-protein ligase Nedd4-2 (mouse ortholog of human Nedd4L) and the ubiquitin-specific protease Usp2-45. Previously, Usp2-45 was shown to decrease ubiquitylation and to increase surface function of ENaC in Xenopus laevis oocytes, whereas the splice variant Usp2-69, which has a different N-terminal domain, was inactive toward ENaC. It is shown here that the catalytic core of Usp2 lacking the N-terminal domain has a reduced ability relative to Usp2-45 to enhance ENaC activity in Xenopus oocytes. In contrast, its catalytic activity toward the artificial substrate ubiquitin-AMC is fully maintained. The interaction of Usp2-45 with ENaC exogenously expressed in HEK293 cells was tested by coimmunoprecipitation. The data indicate that different combinations of ENaC subunits, as well as the α-ENaC cytoplasmic N-terminal but not C-terminal domain, coprecipitate with Usp2-45. This interaction is decreased but not abolished when the cytoplasmic ubiquitylation sites of ENaC are mutated. Importantly, coimmunoprecipitation in HEK293 cells and GST pull-down of purified recombinant proteins show that both the catalytic domain and the N-terminal tail of Usp2-45 physically interact with the HECT domain of Nedd4-2. Taken together, the data support the conclusion that Usp2-45 action on ENaC is promoted by various interactions, including through binding to Nedd4-2 that is suggested to position Usp2-45 favorably for ENaC deubiquitylation.
Assuntos
Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Canais Epiteliais de Sódio/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Western Blotting , Catálise , DNA/genética , Endopeptidases/biossíntese , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/biossíntese , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Escherichia coli/metabolismo , Glutationa/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Cinética , Camundongos , Ubiquitina-Proteína Ligases Nedd4 , Oócitos/metabolismo , Técnicas de Patch-Clamp , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfecção , Ubiquitina Tiolesterase , Ubiquitina-Proteína Ligases/biossíntese , Ubiquitina-Proteína Ligases/genética , Proteínas de Xenopus , Xenopus laevisRESUMO
The occupancy of the central cavity of the membrane-embedded c ring of the ATP synthase of Escherichia coli was investigated with a photo-cross-linking approach. Single cysteine mutants were created at c subunit positions 4, 8, and 11, which are oriented to the inside of the ring. These cysteines were alkylated with reagents carrying a photoactivatable substituent and illuminated. Subunit c and derivatives were then isolated and subjected to mass spectrometric analyses. The most noticeable product, which was found exclusively in irradiated samples, had a mass increase of 719 Da, consistent with a cross-link product between the substituted c subunit and phosphatidylethanolamine. Digestion with phospholipase C converted this product into one with a mass diminished by 126 Da, indicating that the phosphoethanolamine moiety was cleaved off. Hence, the cross-link forms to the diacylglycerol moiety of phosphatidylethanolamine. Control experiments showed that the subunit c-phospholipid adducts were formed in the ATP synthase complex in its natural membrane environment and were not artifacts arising from monomeric c subunits. We conclude therefore that the inner lumen of the c ring is occupied with phospholipids. No evidence was found for an extension of subunit a into this space.