RESUMO
The enzymatic A1 chain of cholera toxin retrotranslocates across the endoplasmic reticulum membrane into the cytosol, where it induces toxicity. Almost all other retrotranslocation substrates are modified by the attachment of polyubiquitin chains and moved into the cytosol by the ubiquitin-interacting p97 ATPase complex. The cholera toxin A1 chain, however, can induce toxicity in the absence of ubiquitination, and the motive force that drives retrotranslocation is not known. Here, we use adenovirus expressing dominant-negative mutants of p97 to test whether p97 is required for toxin action. We find that cholera toxin still functions with only a small decrease in potency in cells that cannot retrotranslocate other substrates at all. These results suggest that p97 does not provide the primary driving force for extracting the A1 chain from the endoplasmic reticulum, a finding that is consistent with a requirement for polyubiquitination in p97 function.
Assuntos
Adenosina Trifosfatases/fisiologia , Toxina da Cólera/química , Proteínas Nucleares/fisiologia , Transporte Proteico , Adenosina Trifosfatases/química , Adenoviridae/genética , Adenoviridae/metabolismo , Animais , Astrocitoma/metabolismo , Células COS , Linhagem Celular Tumoral , Toxina da Cólera/metabolismo , AMP Cíclico/metabolismo , Citosol/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Retículo Endoplasmático/metabolismo , Genes Dominantes , Genes MHC Classe I/genética , Humanos , Imunoprecipitação , Mutação , Proteínas Nucleares/química , Ligação Proteica , Dobramento de Proteína , Fatores de Tempo , Ubiquitina/metabolismoRESUMO
The human MHC class I-related neonatal Fc receptor, hFcRn, mediates bidirectional transport of IgG across mucosal barriers. Here, we find that at steady state hFcRn distributes predominantly to an apical intracellular compartment and almost exclusively to the basolateral cell surface of polarized epithelial cells. It moves only transiently to the apical membrane. Ligand binding does not redistribute the steady state location of the receptor. Removal of the cytoplasmic tail that contains di-leucine and tryptophan-based endocytosis motifs or incubation at low temperature (18 degrees C) redistributes the receptor apically. The rates of endocytosis of the full-length hFcRn from the apical or basolateral membrane domains, however, are equal. Thus, the strong cell surface polarity displayed by hFcRn results from dominant basolateral sorting by motifs in the cytoplasmic tail that nonetheless allows for a cycle of bidirectional transcytosis.