RESUMO
Adaptor protein (AP) complexes bind to transmembrane proteins destined for internalization and to membrane lipids, so linking cargo to the accessory internalization machinery. This machinery interacts with the appendage domains of APs, which have platform and beta-sandwich subdomains, forming the binding surfaces for interacting proteins. Proteins that interact with the subdomains do so via short motifs, usually found in regions of low structural complexity of the interacting proteins. So far, up to four motifs have been identified that bind to and partially compete for at least two sites on each of the appendage domains of the AP2 complex. Motifs in individual accessory proteins, their sequential arrangement into motif domains, and partial competition for binding sites on the appendage domains coordinate the formation of endocytic complexes in a temporal and spatial manner. In this work, we examine the dominant interaction sequence in amphiphysin, a synapse-enriched accessory protein, which generates membrane curvature and recruits the scission protein dynamin to the necks of coated pits, for the platform subdomain of the alpha-appendage. The motif domain of amphiphysin1 contains one copy of each of a DX(F/W) and FXDXF motif. We find that the FXDXF motif is the main determinant for the high affinity interaction with the alpha-adaptin appendage. We describe the optimal sequence of the FXDXF motif using thermodynamic and structural data and show how sequence variation controls the affinities of these motifs for the alpha-appendage.
Assuntos
Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Lipídeos de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Subunidades alfa do Complexo de Proteínas Adaptadoras/química , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Subunidades beta do Complexo de Proteínas Adaptadoras/química , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Motivos de Aminoácidos/fisiologia , Animais , Células COS , Chlorocebus aethiops , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/genética , Dinaminas/química , Dinaminas/genética , Dinaminas/metabolismo , Endocitose/fisiologia , Humanos , Lipídeos de Membrana/química , Lipídeos de Membrana/genética , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Estrutura Terciária de Proteína/fisiologia , RatosRESUMO
Clathrin-mediated endocytosis involves the assembly of a network of proteins that select cargo, modify membrane shape and drive invagination, vesicle scission and uncoating. This network is initially assembled around adaptor protein (AP) appendage domains, which are protein interaction hubs. Using crystallography, we show that FxDxF and WVxF peptide motifs from synaptojanin bind to distinct subdomains on alpha-appendages, called 'top' and 'side' sites. Appendages use both these sites to interact with their binding partners in vitro and in vivo. Occupation of both sites simultaneously results in high-affinity reversible interactions with lone appendages (e.g. eps15 and epsin1). Proteins with multiple copies of only one type of motif bind multiple appendages and so will aid adaptor clustering. These clustered alpha(appendage)-hubs have altered properties where they can sample many different binding partners, which in turn can interact with each other and indirectly with clathrin. In the final coated vesicle, most appendage binding partners are absent and thus the functional status of the appendage domain as an interaction hub is temporal and transitory giving directionality to vesicle assembly.
Assuntos
Complexo 2 de Proteínas Adaptadoras/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose , Complexo 2 de Proteínas Adaptadoras/química , Complexo 2 de Proteínas Adaptadoras/genética , Motivos de Aminoácidos , Animais , Sítios de Ligação , Células COS , Chlorocebus aethiops , Cristalografia por Raios X , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Ligantes , Espectrometria de Massas , Modelos Biológicos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/química , Monoéster Fosfórico Hidrolases/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Proteômica , Ratos , Água/químicaRESUMO
EpsinR is a clathrin-coated vesicle (CCV) enriched 70-kD protein that binds to phosphatidylinositol-4-phosphate, clathrin, and the gamma appendage domain of the adaptor protein complex 1 (AP1). In cells, its distribution overlaps with the perinuclear pool of clathrin and AP1 adaptors. Overexpression disrupts the CCV-dependent trafficking of cathepsin D from the trans-Golgi network to lysosomes and the incorporation of mannose-6-phosphate receptors into CCVs. These biochemical and cell biological data point to a role for epsinR in AP1/clathrin budding events in the cell, just as epsin1 is involved in the budding of AP2 CCVs. Furthermore, we show that two gamma appendage domains can simultaneously bind to epsinR with affinities of 0.7 and 45 microM, respectively. Thus, potentially, two AP1 complexes can bind to one epsinR. This high affinity binding allowed us to identify a consensus binding motif of the form DFxDF, which we also find in gamma-synergin and use to predict that an uncharacterized EF-hand-containing protein will be a new gamma binding partner.