RESUMO
Clathrin light chains (CLCa and CLCb) are major constituents of clathrin-coated vesicles. Unique functions for these evolutionary conserved paralogs remain elusive, and their role in clathrin-mediated endocytosis in mammalian cells is debated. Here, we find and structurally characterize a direct and selective interaction between CLCa and the long isoform of the actin motor protein myosin VI, which is expressed exclusively in highly polarized tissues. Using genetically-reconstituted Caco-2 cysts as proxy for polarized epithelia, we provide evidence for coordinated action of myosin VI and CLCa at the apical surface where these proteins are essential for fission of clathrin-coated pits. We further find that myosin VI and Huntingtin-interacting protein 1-related protein (Hip1R) are mutually exclusive interactors with CLCa, and suggest a model for the sequential function of myosin VI and Hip1R in actin-mediated clathrin-coated vesicle budding.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cadeias Leves de Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Cadeias Pesadas de Miosina/metabolismo , Actinas/metabolismo , Células CACO-2 , Técnicas de Cultura de Células , Cadeias Leves de Clatrina/ultraestrutura , Cistos , Endocitose , Humanos , Espectroscopia de Ressonância Magnética , Cadeias Pesadas de Miosina/ultraestrutura , Ligação Proteica , Conformação Proteica , Isoformas de ProteínasRESUMO
Clathrin-dependent transport processes require the polymerization of clathrin triskelia into polygonal scaffolds. Together with adapter proteins, clathrin collects cargo and induces membrane bud formation. It is not known to what extent clathrin light chains affect the structural and functional properties of clathrin lattices and the ability of clathrin to deform membranes. To address these issues, we have developed a novel procedure for analyzing clathrin lattice formation on rigid surfaces. We found that lattices can form on adaptor-coated convex-, planar- and even shallow concave surfaces, but the rate of formation and resistance to thermal dissociation of the lattice are greatly enhanced on convex surfaces. Atomic force microscopy on planar clathrin lattices demonstrates that the stiffness of the clathrin lattice is strictly dependent on light chains. The reduced stiffness of the lattice also compromised the ability of clathrin to generate coated buds on the surface of rigid liposomal membranes.
Assuntos
Cadeias Leves de Clatrina/ultraestrutura , Vesículas Revestidas por Clatrina/ultraestrutura , Modelos Biológicos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/ultraestrutura , Animais , Sítios de Ligação , Cadeias Leves de Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Lipossomos/ultraestrutura , Microscopia de Força Atômica , Microscopia Eletrônica de Transmissão , Polivinil/química , Propriedades de SuperfícieRESUMO
Clathrin-coated vesicles are major carriers of vesicular traffic in eukaryotic cells. This endocytic pathway relies on cycles of clathrin coat assembly and Hsc70-mediated disassembly. Here we identify histidine residues as major determinants of lattice assembly and stability. They are located at the invariant interface between the proximal and distal segments of clathrin heavy chains, in triskelions centered on two adjacent vertices of the coated-vesicle lattice. Mutation of these histidine residues to glutamine alters the pH dependence of coat stability. We then describe single-particle fluorescence imaging experiments in which we follow the effect of these histidine mutations on susceptibility to Hsc70-dependent uncoating. Coats destabilized by these mutations require fewer Hsc70 molecules to initiate disassembly, as predicted by a model in which Hsc70 traps conformational distortions during the auxilin- and Hsc70:ATP-mediated uncoating reaction.