The multi-subunit GID/CTLH E3 ubiquitin ligase promotes cell proliferation and targets the transcription factor Hbp1 for degradation

Abstract

In yeast, the glucose-induced degradation-deficient (GID) E3 ligase selectively degrades superfluous gluconeogenic enzymes. Here, we identified all subunits of the mammalian GID/CTLH complex and provide a comprehensive map of its hierarchical organization and step-wise assembly. Biochemical reconstitution demonstrates that the mammalian complex possesses inherent E3 ubiquitin ligase activity, using Ube2H as its cognate E2. Deletions of multiple GID subunits compromise cell proliferation, and this defect is accompanied by deregulation of critical cell cycle markers such as the retinoblastoma (Rb) tumor suppressor, phospho-Histone H3 and Cyclin A. We identify the negative regulator of pro-proliferative genes Hbp1 as a bonafide GID/CTLH proteolytic substrate. Indeed, Hbp1 accumulates in cells lacking GID/CTLH activity, and Hbp1 physically interacts and is ubiquitinated in vitro by reconstituted GID/CTLH complexes. Our biochemical and cellular analysis thus demonstrates that the GID/CTLH complex prevents cell cycle exit in G1, at least in part by degrading Hbp1.

Egg activation triggers clearance of maternally deposited RNA binding proteins

“…Nonetheless, so far the CTLH complex appears simpler than the human and yeast complexes. We were unable to identify orthologs of GID4 and GID5, which are seen in the human and yeast versions (Lampert et al, 2018;Maitland et al, 2019;Santt et al, 2008). Similarly, although WDR26 is important in the human complex and we identified a Drosophila ortholog (CG7611), we found no evidence of its association with ME31B or requirement for ME31B degradation.…”

“…But, given the incomplete stabilization of ME31B, we hypothesized that there might be another, as-yet unidentified factor important for ME31B degradation, and so we continued the RNAi screen, this time looking at E2 conjugating enzymes. Strikingly, knockdown of UBC-E2H, an E2 ligase conserved from yeast to humans (Kaiser et al, 1995;1994;Lampert et al, 2018), completely blocked degradation of ME31B, nearly phenocopying the dynamics seen in png 50 mutants, as determined by fluorescence microscopy ( Figure 3A).…”

“…Through a small-scale RNAi screen, we identified that the E2 conjugating enzyme Kondo is required for the ubiquitination and clearance of ME31B, TRAL, and Cup (Figure 3). Kondo is conserved from yeast to humans and, as in those systems (Kaiser et al, 1994;Lampert et al, 2018), appears to work with the CTLH complex, which acts as the E3 ligase. Components of the CTLH complex physically interact with ME31B, and the CTLH complex is also required for the degradation of ME31B, TRAL, and Cup during early embryogenesis (Figure 4).…”

“…Thus, we conclude that it is either a nonessential component or not part of the Drosophila CTLH complex. Interestingly, the role of the CTLH complex during the MZT appears very different from its role in glucose metabolism (in yeast) and DNA damage (in humans) (Lampert et al, 2018;Santt et al, 2008). In the future, it will be important to determine other proteins targeted by the Drosophila CTLH complex and the extent to which ME31B, a ubiquitously expressed protein, is targeted outside of the MZT.…”

“…Kondo is conserved from yeast to humans and is known to work through the CTLH E3 ligase, a multicomponent complex (Lampert et al, 2018;Santt et al, 2008). Using BLAST, we were easily able to identify putative homologs of the CTLH components in Drosophila: RanBPM (homologous to Hs RanBP9), Muskelin, CG3295 (homologous to Hs RMND5A/GID2), CG7611 (homologous to Hs WCR26), CG6617 (homologous to Hs TWA1/GID8), and CG31357 (homologous to Hs MAEA) ( Figure 4A).…”