RESUMO
ERdj5 is a member of the protein disulfide isomerase family of proteins localized to the endoplasmic reticulum (ER) of mammalian cells. To date, only a limited number of substrates for ERdj5 are known. Here we identify a number of endogenous substrates that form mixed disulfides with ERdj5, greatly expanding its client repertoire. ERdj5 previously had been thought to exclusively reduce disulfides in proteins destined for dislocation to the cytosol for degradation. However, we demonstrate here that for one of the identified substrates, the low-density lipoprotein receptor (LDLR), ERdj5 is required not for degradation, but rather for efficient folding. Our results demonstrate that the crucial role of ERdj5 is to reduce non-native disulfides formed during productive folding and that this requirement is dependent on its interaction with BiP. Hence, ERdj5 acts as the ER reductase, both preparing misfolded proteins for degradation and catalyzing the folding of proteins that form obligatory non-native disulfides.
Assuntos
Cistina/metabolismo , Retículo Endoplasmático/enzimologia , Proteínas de Choque Térmico HSP40/fisiologia , Chaperonas Moleculares/fisiologia , Processamento de Proteína Pós-Traducional , Receptores de LDL/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP40/química , Humanos , Chaperonas Moleculares/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Proteólise , RNA Interferente Pequeno/genética , Receptores de LDL/químicaRESUMO
Understanding the function of the thousands of cellular proteins is a central question in molecular cell biology. As proteins are typically part of multiple dynamic and often overlapping macromolecular complexes exerting distinct functions, the identification of protein-protein interactions (PPI) and their assignment to specific complexes is a crucial but challenging task. We present a protein fragments complementation assay integrated with the proximity-dependent biotinylation technique BioID. Activated on the interaction of two proteins, split-BioID is a conditional proteomics approach that allows in a single and simple assay to both experimentally validate binary PPI and to unbiasedly identify additional interacting factors. Applying our method to the miRNA-mediated silencing pathway, we can probe the proteomes of two distinct functional complexes containing the Ago2 protein and uncover the protein GIGYF2 as a regulator of miRNA-mediated translation repression. Hence, we provide a novel tool to study dynamic spatiotemporally defined protein complexes in their native cellular environment.