Extended Data Fig. 8: Cysteine is a signal regulating LRRC58-mediated CDO1 degradation.

(a) Schema of CDO1 post-translational stability reporter including PGK promoter, CDO1-GFP fusion, internal ribosome entry sequence (IRES) and mCherry for transcriptional normalization. (b) Post-translational stability of CDO1 reporter in Hep G2 cells following transfection with siRNA targeting LRRC58 or scramble control (left) or overexpression of LRRC58 (right). n = 3 cell replicates. (c,e) Post-translational stability of CDO1 reporter in Hep G2 cells cultured in media without cystine and with the following compounds added for 26 h. n = 3 cell replicates except n = 6 for 0 mM cystine. Data are normalized to media without cystine. (d,f) Post-translational stability of CDO1 reporter in Hep G2 cells cultured in media with 0.1 mM cystine (equivalent to 0.2 mM cysteine; except the positive control without cystine) and with the following compounds added for 26 h. n = 6 for 0 and 0.2 mM cystine and n = 3 for other treatments. Data are normalized to 0.1 mM cystine media without further treatment. (g) Post-translational stability of the CDO1-GFP reporter or an empty vector (GFP-only) reporter after exposure to media with 0.1 mM cystine supplementation (equivalent to 0.2 mM cysteine), or media without cystine for 24 h. Data for each reporter are normalized to the 0.1 mM condition. n = 3 cell replicates. (h) Comparison of CDO1 abundance in primary hepatocytes cultured under cystine control (0.1 mM, equivalent to 0.2 mM cysteine), 0 mM cystine, or cystine supplementation (0.2 mM, equivalent to 0.4 mM cysteine). n = 7 (Control), n = 5 (0 mM cystine), and n = 3 (0.2 mM cystine) cell replicates. (i) Transcript abundance of Lrrc58 and Cdo1 in primary hepatocytes cultured under cystine control (0.1 mM, equivalent to 0.2 mM cysteine), 0 mM cystine, or cystine supplementation (0.2 mM, equivalent to 0.4 mM cysteine,). n = 3 (Control), n = 3 (0 mM cystine), and n = 3 (0.2 mM cystine) cell replicates. (j) CDO1-GFP/mCherry ratio of WT and 7Ala-CDO1 GFP fusions in media lacking cystine. n = 4 cell replicates. (k) Alphafold predicted structure of interaction between LRRC58 (blue), ELOB (white), and ELOC (tan). The putative ELOB-binding domain containing residues 256–291 of LRRC58 is teal. (l) Co-IP of FLAG-LRRC58 (WT) and FLAG-LRRC58∆256-291 expressed in Hep G2 cells. Immunoblots of CUL5, ELOB, ELOC, FLAG and vinculin are shown for the IP and input. Results are representative of 3 independent experiments. (m) CDO1-GFP/mCherry ratio of LRRC58WT and LRRC58∆256-291 expressing Hep G2 cells under cysteine restriction. n = 4 cell replicates. Statistics relative to untransduced. (two-tailed Student’s t-test for pairwise comparisons in b-j. b (left), c-h, and j were corrected with Bonferroni Dunn test). Data presented as mean ± s.e.m.