Interplay between β-propeller subunits WDR26 and muskelin regulates the CTLH E3 ligase supramolecular complex

The Pro/N-degron recognizing C-terminal to LisH (CTLH) complex is an E3 ligase of emerging interest in the developmental biology field and for targeted protein degradation (TPD) modalities. The human CTLH complex forms distinct supramolecular ring-shaped structures dependent on the multimerization of WDR26 or muskelin β-propeller proteins. Here, we find that, in HeLa cells, CTLH complex E3 ligase activity is dictated by an interplay between WDR26 and muskelin in tandem with muskelin autoregulation. Proteomic experiments revealed that complex-associated muskelin protein turnover is a major ubiquitin-mediated degradation event dependent on the CTLH complex in unstimulated HeLa cells. We observed that muskelin and WDR26 binding to the scaffold of the complex is interchangeable, indicative of the formation of separate WDR26 and muskelin complexes, which correlated with distinct proteomes in WDR26 and muskelin knockout cells. We found that mTOR inhibition-induced degradation of Pro/N-degron containing protein HMGCS1 is distinctly regulated by a muskelin-specific CTLH complex. Finally, we found that mTOR inhibition also activated muskelin degradation, likely as an autoregulatory feedback mechanism to regulate CTLH complex activity. Thus, rather than swapping substrate receptors, the CTLH E3 ligase complex controls substrate selectivity through the differential association of its β-propeller oligomeric subunits WDR26 and muskelin. WDR26 and muskelin mediate the formation of two distinct CTLH E3 ligase complexes that have different targets. Inhibition of mTOR signalling regulates muskelin-specific CTLH complex activity in human cells and leads to muskelin autoregulation.