RESUMEN
The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Endocitosis/fisiología , Exocitosis/fisiología , VIH/metabolismo , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Secuencia de Bases , Sitios de Unión , Células Cultivadas , Citoplasma/metabolismo , Citoplasma/fisiología , Complejos de Clasificación Endosomal Requeridos para el Transporte , Receptores ErbB/metabolismo , Técnica del Anticuerpo Fluorescente , Biblioteca de Genes , Productos del Gen gag/metabolismo , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos , Plásmidos/genética , Transporte de Proteínas/fisiología , ARN Interferente Pequeño/genética , Análisis de Secuencia de ADN , Transfección , Técnicas del Sistema de Dos Híbridos , Ubiquitinas/metabolismoRESUMEN
Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.
