RESUMO
Endoplasmic reticulum (ER) proteins are degraded by proteasomes in the cytosol through ER-associated degradation (ERAD). This process involves the retrotranslocation of substrates across the ER membrane, their ubiquitination, and membrane extraction by the Cdc48/Npl4/Ufd1 ATPase complex prior to delivery to proteasomes for degradation. How the presence of a folded luminal domain affects substrate retrotranslocation and this event is coordinated with subsequent ERAD steps remains unknown. Here, using a model substrate with a folded luminal domain, we showed that Cdc48 ATPase activity is sufficient to drive substrate retrotranslocation independently of ERAD membrane components. However, the complete degradation of the folded luminal domain required substrate-tight coupling of retrotranslocation and proteasomal degradation, which was ensured by the derlin Dfm1. Mutations in Dfm1 intramembrane rhomboid-like or cytosolic Cdc48-binding regions resulted in partial degradation of the substrate with accumulation of its folded domain. Our study revealed Dfm1 as a critical regulator of Cdc48-driven retrotranslocation and highlights the importance of coordinating substrate retrotranslocation and degradation during ERAD.
Assuntos
Retículo Endoplasmático , Proteínas de Membrana , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatases/genética , Citosol , Retículo Endoplasmático/metabolismo , Degradação Associada com o Retículo Endoplasmático , Complexo de Endopeptidases do Proteassoma/metabolismo , Domínios Proteicos , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Tail-anchored (TA) proteins are anchored to their corresponding membrane via a single transmembrane segment (TMS) at their C-terminus. In yeast, the targeting of TA proteins to the endoplasmic reticulum (ER) can be mediated by the guided entry of TA proteins (GET) pathway, whereas it is not yet clear how mitochondrial TA proteins are targeted to their destination. It has been widely observed that some mitochondrial outer membrane (MOM) proteins are mistargeted to the ER when overexpressed or when their targeting signal is masked. However, the mechanism of this erroneous sorting is currently unknown. In this study, we demonstrate the involvement of the GET machinery in the mistargeting of suboptimal MOM proteins to the ER. These findings suggest that the GET machinery can, in principle, recognize and guide mitochondrial and non-canonical TA proteins. Hence, under normal conditions, an active mitochondrial targeting pathway must exist that dominates the kinetic competition against other pathways.