RESUMO
Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos , Dobramento de Proteína , Proteólise , Proteínas de Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismoRESUMO
The nuclear envelope (NE) is the defining feature of eukaryotic cells, separating the nucleus from the cytoplasm. It has a complex architecture consisting of two lipid bilayers that, despite being continuous between them and with the endoplasmic reticulum, have different protein compositions consistent with their distinct functions. In particular, the unique composition of the inner nuclear membrane (INM), facing the nucleoplasm and its underlying nuclear lamina, is critical for the organisation and function of nuclear processes, from cell fate to gene regulation and DNA repair. Mutations in INM proteins affecting this organisation are associated with muscular dystrophies and premature ageing syndromes highlighting the role of INM architecture in cell homeostasis. Here, we discuss recent progress in understanding how specific proteins concentrate at the INM, as well as the quality control mechanisms involved in remodelling and maintaining INM protein homeostasis.