RESUMO
The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric assembly proteins, also called adaptor proteins (APs), each of which contains a ß subunit. We identified a single ß subunit, named ß1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding ß1/2 resulted in severe defects in growth, cytokinesis and development. Additionally, cells lacking ß1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of ß1/2 null cells were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of ß1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the ß subunit in the stability of the medium subunits. Dictyostelium ß1/2 could resemble a common ancestor of the more specialized ß1 and ß2 subunits of the vertebrate AP complexes. Our results support the essential contribution of a single ß subunit to the stability and function of AP1 and AP2 in a simple eukaryote.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Clatrina/metabolismo , Dictyostelium/fisiologia , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Sequência de Aminoácidos/fisiologia , Agregação Celular/fisiologia , Membrana Celular/metabolismo , Proliferação de Células , Quimiotaxia/fisiologia , Clatrina/genética , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Clonagem Molecular , Citocinese/fisiologia , Dictyostelium/citologia , Carpóforos/citologia , Carpóforos/fisiologia , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Vacúolos/fisiologia , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
The roles of clathrin, its regulators, and the ESCRT (endosomal sorting complex required for transport) proteins are well defined in endocytosis. These proteins can also participate in intracellular pathways that are independent of endocytosis and even independent of the membrane trafficking function of these proteins. These nonendocytic functions involve unconventional biochemical interactions for some endocytic regulators, but can also exploit known interactions for nonendocytic functions. The molecular basis for the involvement of endocytic regulators in unconventional functions that influence the cytoskeleton, cell cycle, signaling, and gene regulation are described here. Through these additional functions, endocytic regulators participate in pathways that affect infection, glucose metabolism, development, and cellular transformation, expanding their significance in human health and disease.