The multi-subunit C-terminal to LisH (CTLH) complex is the mammalian homologue of the yeast Gid E3 ubiquitin ligase complex. In this study, we investigated the human CTLH complex and characterized its E3 ligase activity. We confirm that the complex immunoprecipitated from human cells comprises RanBPM, ARMC8 α/β, muskelin, WDR26, GID4 and the RING domain proteins RMND5A and MAEA. We find that loss of expression of individual subunits compromises the stability of other complex members and that MAEA and RMND5A protein levels are interdependent. Using in vitro ubiquitination assays, we demonstrate that the CTLH complex has E3 ligase activity which is dependent on RMND5A and MAEA. We report that the complex can pair with UBE2D1, UBE2D2 and UBE2D3 E2 enzymes and that recombinant RMND5A mediates K48 and K63 poly-ubiquitin chains. Finally, we show a proteasome-dependent increase in the protein levels of CTLH complex member muskelin in RMND5A KO cells. Furthermore, muskelin ubiquitination is dependent on RMND5A, suggesting that it may be a target of the complex. Overall, we further the characterization of the CTLH complex as an E3 ubiquitin ligase complex in human cells and reveal a potential autoregulation mechanism.

RanBPM (Ran-binding protein M, also called RanBP9) is an evolutionarily conserved, ubiquitous protein which localizes to both nucleus and cytoplasm. RanBPM has been implicated in the regulation of a number of signalling pathways to regulate several cellular processes such as apoptosis, cell adhesion, migration as well as transcription, and plays a critical role during development. In addition, RanBPM has been shown to regulate pathways implicated in cancer and Alzheimer's disease, implying that RanBPM has important functions in both normal and pathological development. While its functions in these processes are still poorly understood, RanBPM has been identified as a component of a large complex, termed the CTLH (C-terminal to LisH) complex. The yeast homologue of this complex functions as an E3 ubiquitin ligase that targets enzymes of the gluconeogenesis pathway. While the CTLH complex E3 ubiquitin ligase activity and substrates still remain to be characterized, the high level of conservation between the complexes in yeast and mammals infers that the CTLH complex could also serve to promote the degradation of specific substrates through ubiquitination, therefore suggesting the possibility that RanBPM's various functions may be mediated through the activity of the CTLH complex.

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c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation.

Multi-subunit E3 ligases facilitate ubiquitin transfer by coordinating various substrate receptor subunits with a single catalytic center. Small molecules inducing targeted protein degradation have exploited such complexes, proving successful as therapeutics against previously undruggable targets. The C-terminal to LisH (CTLH) complex, also called the glucose-induced degradation deficient (GID) complex, is a multi-subunit E3 ligase complex highly conserved from Saccharomyces cerevisiae to humans, with roles in fundamental pathways controlling homeostasis and development in several species. However, we are only beginning to understand its mechanistic basis. Here, we review the literature of the CTLH complex from all organisms and place previous findings on individual subunits into context with recent breakthroughs on its structure and function.