( D) Stably expressed HSS-Armc8 was isolated from HEK-293 cells, and after HA-peptide elution the presence of additional GID proteins in the immunoprecipitate was analyzed using the indicated antibodies. cerevisiae as bona fide GID proteins are colored in yellow. Conserved GID subunits currently not assigned in S. cerevisiae GID complex are labeled in green. GID subunits with no sequence/functional homologue within the S. ( C) Network of Rmnd5a high-confidence interacting proteins (HCIPs) in HEK-239 cells analyzed by SAINT (confidence score ≥0.9, FC ≥2, n = 2). Data are shown as mean of quadruplicates and % change in signal relative to control gRNA-treated cells ± SD, n = 3, ∗∗∗p≤0.0003. ( B) Cell proliferation of RPE control and WDR26-KO cells was quantified by MTT assays between days 6–9 after lentiviral transfection. ( A) Representative clonogenic survival assay and corresponding quantification of RPE cells (technical replicates, n ≥ 2) treated with lentiviruses expressing either control gRNAs (WT) or two gRNAs deleting the GID subunit WDR26 (WDR26-KO). cerevisiae biochemistry cell biology cell proliferation chemical biology human metabolism mouse transcription factors. 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.Į3 ubiquitin ligase N-end rule S. Indeed, Hbp1 accumulates in cells lacking GID/CTLH activity, and Hbp1 physically interacts and is ubiquitinated in vitro by reconstituted GID/CTLH complexes. We identify the negative regulator of pro-proliferative genes Hbp1 as a bonafide GID/CTLH proteolytic substrate. 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. Biochemical reconstitution demonstrates that the mammalian complex possesses inherent E3 ubiquitin ligase activity, using Ube2H as its cognate E2. Here, we identified all subunits of the mammalian GID/CTLH complex and provide a comprehensive map of its hierarchical organization and step-wise assembly. In yeast, the glucose-induced degradation-deficient (GID) E3 ligase selectively degrades superfluous gluconeogenic enzymes.
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