RESUMO
Numerous endocytic accessory proteins (EAPs) mediate assembly and maturation of clathrin-coated pits (CCPs) into cargo-containing vesicles. Analysis of EAP function through bulk measurement of cargo uptake has been hampered due to potential redundancy among EAPs and, as we show here, the plasticity and resilience of clathrin-mediated endocytosis (CME). Instead, EAP function is best studied by uncovering the correlation between variations in EAP association to individual CCPs and the resulting variations in maturation. However, most EAPs bind to CCPs in low numbers, making the measurement of EAP association via fused fluorescent reporters highly susceptible to detection errors. Here, we present a framework for unbiased measurement of EAP recruitment to CCPs and their direct effects on CCP dynamics. We identify dynamin and the EAP-binding α-adaptin appendage domain of the AP2 adaptor as switches in a regulated, multistep maturation process and provide direct evidence for a molecular checkpoint in CME.
Assuntos
Complexo 2 de Proteínas Adaptadoras/fisiologia , Subunidades mu do Complexo de Proteínas Adaptadoras/fisiologia , Dinamina II/fisiologia , Endocitose/fisiologia , Proteínas de Ligação a Ácido Graxo/fisiologia , Microscopia de Fluorescência/métodos , Vesículas Transportadoras/fisiologia , Complexo 2 de Proteínas Adaptadoras/química , Subunidades mu do Complexo de Proteínas Adaptadoras/química , Algoritmos , Cadeias Leves de Clatrina/fisiologia , Vesículas Revestidas por Clatrina/fisiologia , Dinamina II/química , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Fluorescência Verde , Humanos , Proteínas Luminescentes , Estrutura Terciária de Proteína/fisiologia , RNA Interferente Pequeno/genética , Epitélio Pigmentado da Retina/citologia , Razão Sinal-Ruído , Proteína Vermelha FluorescenteRESUMO
Clathrin-coated vesicles (CCVs) are major carriers for endocytic cargo and mediate important intracellular trafficking events at the trans-Golgi network (TGN) and endosomes. Whereas clathrin heavy chain provides the structural backbone of the clathrin coat, the role of clathrin light chains (CLCs) is poorly understood. We now demonstrate that CLCs are not required for clathrin-mediated endocytosis but are critical for clathrin-mediated trafficking between the TGN and the endosomal system. Specifically, CLC knockdown (KD) causes the cation-independent mannose-6 phosphate receptor (CI-MPR) to cluster near the TGN leading to a delay in processing of the lysosomal hydrolase cathepsin D. A recently identified binding partner for CLCs is huntingtin-interacting protein 1-related (HIP1R), which is required for productive interactions of CCVs with the actin cytoskeleton. CLC KD causes mislocalization of HIP1R and overassembly of actin, which accumulates in patches around the clustered CI-MPR. A dominant-negative CLC construct that disrupts HIP1R/CLC interactions causes similar alterations in CI-MPR trafficking and actin assembly. Thus, in mammalian cells CLCs function in intracellular membrane trafficking by acting as recruitment proteins for HIP1R, enabling HIP1R to regulate actin assembly on clathrin-coated structures.
Assuntos
Actinas/química , Cadeias Leves de Clatrina/fisiologia , Lectinas Tipo C/metabolismo , Lectinas de Ligação a Manose/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Transporte Biológico , Antígenos CD8/biossíntese , Células COS , Catepsina D/química , Catepsina D/metabolismo , Chlorocebus aethiops , Cadeias Leves de Clatrina/química , Endocitose , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Receptor de Manose , Modelos Biológicos , Proteínas do Tecido Nervoso/química , RNA Interferente Pequeno/metabolismo , Rede trans-GolgiRESUMO
Clathrin-mediated endocytosis is a major pathway for uptake of lipid and protein cargo at the plasma membrane. The lattices of clathrin-coated pits and vesicles are comprised of triskelions, each consisting of three oligomerized heavy chains (HC) bound by a light chain (LC). In addition to binding HC, LC interacts with members of the Hip1/R family of endocytic proteins, including the budding yeast homologue, Sla2p. Here, using in vivo analysis in yeast, we provide novel insight into the role of this interaction. We find that overexpression of LC partially restores endocytosis to cells lacking clathrin HC. This suppression is dependent on the Sla2p binding region of LC. Using live cell imaging techniques to visualize endocytic vesicle formation, we find that the N-terminal Sla2p binding region of LC promotes the progression of arrested Sla2p patches that form in the absence of HC. We propose that LC binding to Sla2p positively regulates Sla2p for efficient endocytic vesicle formation.
Assuntos
Proteínas de Transporte/metabolismo , Cadeias Leves de Clatrina/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Vesículas Transportadoras/fisiologia , Sítios de Ligação , Cadeias Pesadas de Clatrina/genética , Cadeias Leves de Clatrina/genética , Vesículas Revestidas por Clatrina/fisiologia , Proteínas do Citoesqueleto , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
While clathrin heavy chains from different species are highly conserved in amino acid sequence, clathrin light chains are much more divergent. Thus clathrin light chain may have different functions in different organisms. To investigate clathrin light chain function, we cloned the clathrin light chain, clcA, from Dictyostelium and examined clathrin function in clcA-mutants. Phenotypic deficiencies in development, cytokinesis, and osmoregulation showed that light chain was critical for clathrin function in Dictyostelium. In contrast with budding yeast, we found the light chain did not influence steady-state levels of clathrin, triskelion formation, or contribute to clathrin over-assembly on intracellular membranes. Imaging GFP-CHC in clcA- mutants showed that the heavy chain formed dynamic punctate structures that were remarkably similar to those found in wild-type cells. However, clathrin light chain knockouts showed a decreased association of clathrin with intracellular membranes. Unlike wild-type cells, half of the clathrin in clcA- mutants was cytosolic, suggesting that the absence of light chain compromised the assembly of triskelions onto intracellular membranes. Taken together, these results suggest a role for the Dictyostelium clathrin light chain in regulating the self-assembly of triskelions onto intracellular membranes, and demonstrate a crucial contribution of the light chain to clathrin function in vivo.
