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Cleavage of transmembrane segments on target proteins by the aspartyl intramembrane protease signal peptide peptidase (SPP, encoded by HM13) has been linked to immunity, viral infection and protein quality control. How SPP recognizes its various substrates and specifies their fate remains elusive. Here, we identify the lanosterol demethylase CYP51A1 as an SPP substrate and show that SPPcatalysed cleavage triggers CYP51A1 clearance by endoplasmic reticulum-associated degradation (ERAD). We observe that SPP targets only a fraction of CYP51A1 molecules, and we identify an amphipathic helix in the CYP51A1 N terminus as a key determinant for SPP recognition. SPP recognition is remarkably specific to CYP51A1 molecules with the amphipathic helix aberrantly inserted in the membrane with a type II orientation. Thus, our data are consistent with a role for SPP in topology surveillance, triggering the clearance of certain potentially non-functional conformers. [ABSTRACT FROM AUTHOR]

Misfolded endoplasmic reticulum (ER) proteins are dislocated towards the cytosol and degraded by the ubiquitin--proteasome system in a process called ER-associated protein degradation (ERAD). During infection with human cytomegalovirus (HCMV), the viral US2 protein targets HLA class I molecules (HLA-I) for degradation via ERAD to avoid elimination by the immune system. US2-mediated degradation of HLA-I serves as a paradigm of ERAD and has facilitated the identification of TRC8 (also known as RNF139) as an E3 ubiquitin ligase. No specific E2 enzymes had previously been described for cooperation with TRC8. In this study, we used a lentiviral CRISPR/Cas9 library targeting all known human E2 enzymes to assess their involvement in US2-mediated HLA-I downregulation. We identified multiple E2 enzymes involved in this process, of which UBE2G2 was crucial for the degradation of various immunoreceptors. UBE2J2, on the other hand, counteracted US2- induced ERAD by downregulating TRC8 expression. These findings indicate the complexity of cellular quality control mechanisms, which are elegantly exploited by HCMV to elude the immune system. [ABSTRACT FROM AUTHOR]

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. • The RNF185 ubiquitin ligase, Membralin, and TMUB1/2 assemble into an ERAD complex • RNF185/Membralin complex targets membrane proteins, including CYP51A1 and TMUB2 • RNF185/Membralin and TEB4 ERAD complexes recognize distinct substrate features • TEB4 ERAD complex recognizes substrates through their transmembrane domain Membrane proteins are diverse, and the components involved in their quality control in each case are unclear. By comparing the degradation of two short-lived ER proteins, van de Weijer et al. identified an ERAD complex composed of RNF185, TMUBs, and Membralin, a membrane protein essential to neuronal function. [ABSTRACT FROM AUTHOR]