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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 proteins from the endoplasmic reticulum (ER) are transported back into the cytosol for degradation via the ubiquitin-proteasome system. The human cytomegalovirus protein US11 hijacks this ER-associated protein degradation (ERAD) pathway to downregulate human leukocyte antigen (HLA) class I molecules in virus-infected cells, thereby evading elimination by cytotoxic T-lymphocytes. Recently, we identified the E3 ubiquitin ligase transmembrane protein 129 (TMEM129) as a key player in this process, where interference with TMEM129 activity in human cells completely abrogates US11-mediated class I degradation. Here, we set out to further characterize TMEM129. We show that TMEM129 is a non-glycosylated protein containing a non-cleaved signal anchor sequence. By glycosylation scanning mutagenesis, we show that TMEM129 is a tri-spanning ER-membrane protein that adopts an Nexo-Ccyto orientation. This insertion in the ER membrane positions the C-terminal really interesting new gene (RING) domain of TMEM129 in the cytosol, making it available to catalyze ubiquitination reactions that are required for cytosolic degradation of secretory proteins. [ABSTRACT FROM AUTHOR]