RESUMEN
Lipid transfer between cell membrane bilayers at contacts between the endoplasmic reticulum (ER) and other membranes help to maintain membrane lipid homeostasis. We found that two similar ER integral membrane proteins, oxysterol-binding protein (OSBP)-related protein 5 (ORP5) and ORP8, tethered the ER to the plasma membrane (PM) via the interaction of their pleckstrin homology domains with phosphatidylinositol 4-phosphate (PI4P) in this membrane. Their OSBP-related domains (ORDs) harbored either PI4P or phosphatidylserine (PS) and exchanged these lipids between bilayers. Gain- and loss-of-function experiments showed that ORP5 and ORP8 could mediate PI4P/PS countertransport between the ER and the PM, thus delivering PI4P to the ER-localized PI4P phosphatase Sac1 for degradation and PS from the ER to the PM. This exchange helps to control plasma membrane PI4P levels and selectively enrich PS in the PM.
Asunto(s)
Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatidilserinas/metabolismo , Receptores de Esteroides/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Receptores de Esteroides/química , Receptores de Esteroides/genéticaRESUMEN
The recruitment of inositol phosphatases to endocytic membranes mediates dephosphorylation of PI(4,5)P2, a phosphoinositide concentrated in the plasma membrane, and prevents its accumulation on endosomes. The importance of the conversion of PI(4,5)P2 to PtdIns during endocytosis is demonstrated by the presence of both a 5-phosphatase and a 4-phosphatase (Sac domain) module in the synaptojanins, endocytic PI(4,5)P2 phosphatases conserved from yeast to humans and the only PI(4,5)P2 phosphatases in yeast. OCRL, another 5-phosphatase that couples endocytosis to PI(4,5)P2 dephosphorylation, lacks a Sac domain. Here we show that Sac2/INPP5F is a PI4P phosphatase that colocalizes with OCRL on endocytic membranes, including vesicles formed by clathrin-mediated endocytosis, macropinosomes, and Rab5 endosomes. An OCRL-Sac2/INPP5F interaction could be demonstrated by coimmunoprecipitation and was potentiated by Rab5, whose activity is required to recruit Sac2/INPP5F to endosomes. Sac2/INPP5F and OCRL may cooperate in the sequential dephosphorylation of PI(4,5)P2 at the 5 and 4 position of inositol in a partnership that mimics that of the two phosphatase modules of synaptojanin.
Asunto(s)
Endocitosis , Endosomas/enzimología , Monoéster Fosfórico Hidrolasas/fisiología , Animales , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Inositol Polifosfato 5-Fosfatasas , Ratones Noqueados , Monoéster Fosfórico Hidrolasas/metabolismo , Transporte de Proteínas , Proteínas de Unión al GTP rab5/metabolismoRESUMEN
Epsin is an evolutionarily conserved endocytic clathrin adaptor whose most critical function(s) in clathrin coat dynamics remain(s) elusive. To elucidate such function(s), we generated embryonic fibroblasts from conditional epsin triple KO mice. Triple KO cells displayed a dramatic cell division defect. Additionally, a robust impairment in clathrin-mediated endocytosis was observed, with an accumulation of early and U-shaped pits. This defect correlated with a perturbation of the coupling between the clathrin coat and the actin cytoskeleton, which we confirmed in a cell-free assay of endocytosis. Our results indicate that a key evolutionary conserved function of epsin, in addition to other roles that include, as we show here, a low affinity interaction with SNAREs, is to help generate the force that leads to invagination and then fission of clathrin-coated pits.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/genética , Clatrina/metabolismo , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Endocitosis/genética , Citoesqueleto de Actina/genética , Citoesqueleto de Actina/ultraestructura , Actinas/genética , Proteínas Adaptadoras del Transporte Vesicular/deficiencia , Animales , Clatrina/genética , Invaginaciones Cubiertas de la Membrana Celular/genética , Invaginaciones Cubiertas de la Membrana Celular/ultraestructura , Embrión de Mamíferos , Fibroblastos/metabolismo , Fibroblastos/ultraestructura , Expresión Génica , Ratones , Ratones Noqueados , Cultivo Primario de Células , Unión Proteica , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Transducción de SeñalRESUMEN
Mutations in the inositol 5-phosphatase OCRL cause Lowe syndrome and Dent's disease. Although OCRL, a direct clathrin interactor, is recruited to late-stage clathrin-coated pits, clinical manifestations have been primarily attributed to intracellular sorting defects. Here we show that OCRL loss in Lowe syndrome patient fibroblasts impacts clathrin-mediated endocytosis and results in an endocytic defect. These cells exhibit an accumulation of clathrin-coated vesicles and an increase in U-shaped clathrin-coated pits, which may result from sequestration of coat components on uncoated vesicles. Endocytic vesicles that fail to lose their coat nucleate the majority of the numerous actin comets present in patient cells. SNX9, an adaptor that couples late-stage endocytic coated pits to actin polymerization and which we found to bind OCRL directly, remains associated with such vesicles. These results indicate that OCRL acts as an uncoating factor and that defects in clathrin-mediated endocytosis likely contribute to pathology in patients with OCRL mutations.