Journal: bioRxiv

Article Title: Proteome-wide C-degron activity profiling connects conditional regulation of the CTLH E3 ligase complex to ribosome biogenesis

doi: 10.64898/2026.01.14.698769

Figure Lengend Snippet: (A) Schematic representation of the stability profiling experiment, in which the Ultimate ORFeome GPS expression library was used to identify proteins exhibiting increased stability in MAEA KO and TWA1 KO cells. (B-C) Identification of AAMP, AEN and NATD1 as CTLH substrates. Screen profiles are depicted in (B) , while individual validation experiments by flow cytometry in the presence and absence of Torin-1 (dotted lines) are shown in (C) . See also . (D) Schematic representation of the proteomic experiment, in which TMT mass spectrometry was used to identify proteins more abundant in cells lacking the CTLH subunits TWA1 and Muskelin. Cells expressing GPS-ZMYND19 were used to allow purification of populations of cells in which TWA1 and Muskelin had been successfully ablated by FACS. See also . (E-F) AAMP is more abundant in cells lacking CTLH subunits. The scatterplot in (E) represents the mean fold change of each protein quantified in both TWA1 KO and Muskelin KO cells, with proteins showing a >1.2-fold increase in abundance in both conditions highlighted in red; the performance of AAMP compared to the known substrate HMGCS1 is depicted in (F) . Exogenous expression of ZMYND19 precludes assessment of the true magnitude of the stabilization of the endogenous ZMYND19 protein in this experiment.

Article Snippet: Primary antibodies used were: rabbit α-MAEA (Proteintech, #28363-1-AP), rabbit α-TWA1 (Novus Biologicals, #NBP1-32596), mouse α-Muskelin (Santa Cruz, #sc-398956), mouse α-RanBP9 (Santa Cruz, #sc-271727), rabbit α-RanBP10 (Proteintech, #21107-1-AP), mouse α-ARMC8 (Santa Cruz, #sc-365307), rabbit α-WDR26 (Bethyl, #A302-244A), rabbit α-YPEL5 (Proteintech, #11730-1-AP), rabbit α-AAMP (Abcam, #EPR12369), rabbit α-ISG20L2 (Proteintech, #24639-1-AP), rabbit α-HMGCS1 (Proteintech, #17643-1-AP), mouse α-UBE2H (Santa Cruz, #sc-100620), rabbit α-uL16/RPL10 (Proteintech, #17013-1-AP), rabbit α-uL14/RPL23 (Abcam, #ab264369), mouse α-eS6/RPS6 (Cell Signaling Technology, #2317), mouse α-β-actin (Merck, #A2228), mouse α-vinculin (Merck, #V9131), rabbit α-HA-tag (Cell Signaling Technology, #C29F4), mouse α-FLAG (Merck #F1804), rabbit α-GFP (Abcam, #ab290), mouse α-GFP (Roche, #11814460001) and rabbit α-Phospho-p70 S6 Kinase (Thr389) (Cell Signaling Technology, #9205).

Techniques: Expressing, Biomarker Discovery, Flow Cytometry, Mass Spectrometry, Purification