RanBPM, Muskelin, p48EMLP, p44CTLH, and the armadillo-repeat proteins ARMC8alpha and ARMC8beta are components of the CTLH complex

Ran-binding protein in microtubule organising centre (RanBPM) was originally isolated as a protein that binds to the small GTPase Ran. Recently our group and other groups reported that RanBPM was associated with several proteins and composed a large protein complex. Here, we used tandem MS with an antibody against RanBPM to purify this complex from a soluble extract of HEK293 cells: we identified Muskelin, p48EMLP, p44CTLH, and the novel armadillo-repeat proteins ARMC8alpha and ARMC8beta as components. In RanBPM, Muskelin, p48EMLP, and p44CTLH we found LisH/CTLH motifs, which are present in proteins involved in microtubule dynamics, cell migration, nucleokinesis, and chromosome segregation. We renamed the 20S large protein complex the CTLH complex. The N-terminal 364 amino acids of ARMC8alpha and ARMC8beta were completely conserved, suggesting that these proteins are probably alternatively spliced products from the same gene. We confirmed the in vivo association of each component by co-immunoprecipitation assays with Cos-7 cells in which these components were exogenously overexpressed. A pull-down assay with bacterially-expressed Twa1 revealed binding of each in vitro-translated component to Twa1. Finally, we confirmed the cellular localization of these proteins. Taken together, our results reveal that RanBPM, ARMC8alpha, ARMC8beta, Muskelin, p48EMLP, and p44CTLH form complexes in cells.     

GID E3 ligase supramolecular chelate assembly configures multipronged ubiquitin targeting of an oligomeric metabolic enzyme

1. Download : Download high-res image (192KB)
2. Download : Download full-size image

     

Interconversion between Anticipatory and Active GID E3 Ubiquitin Ligase Conformations via Metabolically Driven Substrate Receptor Assembly

1. Download : Download high-res image (210KB)
2. Download : Download full-size image

     

The human GID complex engages two independent modules for substrate recruitment

The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi‐subunit complex remain poorly understood. Using biochemical assays, cross‐linking mass spectrometry, and cryo‐electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N‐end degron. GID4 substrate binding and ligase activity is regulated by ARMC8α, while the shorter ARMC8β isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo‐EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring‐like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non‐overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity.