Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex

Michael L Van De Weijer,Logesvaran Krshnan,Sabrina Liberatori,Elena Navarro Guerrero,Jacob Robson-Tull,Lilli Hahn,Robert Jan Lebbink,Emmanuel J.H.J Wiertz,Roman Fischer,Daniel Ebner,Pedro Carvalho

doi:10.1016/j.molcel.2020.07.009

Michael L Van De Weijer, Logesvaran Krshnan + Show 9 more

Open Access

https://doi.org/10.1016/j.molcel.2020.07.009

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Abstract

SummaryMisfolded 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.

Highlights

Misfolded proteins in the lumen and membrane of the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD) (Christianson and Ye, 2014; Mehrtash and Hochstrasser, 2019; Wu and Rapoport, 2018)
To gain insight on the quality control of membrane proteins in mammalian cells, we tested whether Erg11TM would be an ERAD substrate when expressed in human HEK293 cells
Like Erg11TM (Monk et al, 2014), CYP51A1TM is predicted to have an N-terminal amphipathic helix (AH) in the ER lumen followed by a single transmembrane alpha helix (TMD)

Summary

Introduction

Misfolded proteins in the lumen and membrane of the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD) (Christianson and Ye, 2014; Mehrtash and Hochstrasser, 2019; Wu and Rapoport, 2018). In a signal-dependent manner, ERAD degrades certain folded proteins, such as rate-limiting enzymes for sterol biosynthesis, thereby controlling the metabolic flux through this vital pathway (Ruggiano et al, 2014). Studies in yeast and mammalian cells showed that, upon recognition, both luminal and membrane substrates are retrotranslocated across the ER bilayer to the cytosol and ubiquitinated (Christianson and Ye, 2014; Ruggiano et al, 2014) These sequential steps are coordinated by ER membrane ubiquitin ligase complexes, each defining an ERAD branch with specificity to certain classes of substrates. The diverse ERAD branches converge on the cytosolic p97 ATPase complex (Cdc in yeast), which extracts ubiquitinated substrates from the ER membrane and hands them to the proteasome for degradation (Wu and Rapoport, 2018)

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