Journal: bioRxiv

Article Title: SLC33A1 exports oxidized glutathione to maintain endoplasmic reticulum redox homeostasis

doi: 10.64898/2026.02.01.703113

Figure Lengend Snippet: a. Schematic depicting the chemical proteomic workflow for profiling oxidized cysteines. Cells are treated with N-ethylmaleimide (NEM) before lysis and Iodoacetamide (IAM) after lysis to alkylate the reduced cysteines and GSH. Oxidized cysteines are reduced by Tris(2-carboxyethyl) phosphine (TCEP) before being labeled with iodoacetamide-desthiobiotin (IA-DTB) and enrichment via streptavidin agarose beads for further tandem mass tag (TMT) labeling and LC/LC-MS/MS/MS analysis. b. Bar graph representing the percent of proteins within indicated organelles showing higher than 1.5-fold difference (log 2 ) in reactivity of oxidized cysteines from Slc33a1 knockout KPK cells expressing a vector control compared to those expressing SLC33A1 cDNA. c. Fold change (log 2 ) in oxidized cysteine reactivity of indicated proteins from Slc33a1 knockout KPK cells expressing a vector control compared to those expressing SLC33A1 cDNA. Dark red and light red mark cysteine residues with ≥2-fold and ≥1.5-fold reactivity changes compared to protein expression, respectively. Grey marks unchanged cysteines. Green marks protein expression. Data are median values for n = 2 (or more) independent cysteine reactivity profiling experiments and n = 1 independent unenriched proteomics experiments. Each biological independent experiment had 2-3 technical replicates for each condition. d. Immunoblot analysis of 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS) modified proteins from Slc33a1 knockout KPK cells expressing a vector control or SLC33A1 cDNA. Cells were reduced with 10 mM DTT or oxidized with 5 mM diamide for 5 minutes prior to treatment with 25 mM NEM for 10 minutes before lysis. The bands of oxidized and reduced proteins are labeled. e. Immunoblot analysis of indicated proteins from Slc33a1 knockout KPK cells expressing a vector control (EV), WT or 2YA mutant SLC33A1 cDNAs. f. Immunoblot analysis of indicated proteins from SLC33A1 knockout HEK293T cells expressing an inducible cyto-GshF (icyto-GshF) complemented with a vector control WT or 2YA mutant SLC33A1 cDNA treated with 1 µg/ml doxycycline for 48 hours. g. Heatmap of fold change (log 2 ) in unfolded protein response (UPR) target mRNAs from Slc33a1 knockout KPK cells expressing a vector control or SLC33A1 cDNA comparing cells treated with tunicamycin to DMSO. Cells were treated with DMSO or 10 ng/ml tunicamycin (TM) for 8 hours before RNA extraction. h. Immunoblot analysis of indicated proteins from Slc33a1 knockout KPK cells expressing a vector control or SLC33A1 cDNA. Cells were treated with 20 ng/ml tunicamycin for indicated time. i. Left, percent of cell number from Slc33a1 knockout KPK cells expressing a vector control or SLC33A1 cDNA treated with DMSO or tunicamycin for 4 days under indicated concentrations. Right, percent of cell number from SLC33A1 knockout HEK293T cells expressing a vector control or SLC33A1 cDNA treated with DMSO or tunicamycin for 4 days under indicated concentrations. Numbers under tunicamycin treatment were normalized to DMSO untreated. j. Relative metabolite abundance of indicated whole cell or ER metabolites from Slc33a1 knockout KPK cells expressing a sgRNA targeting intergenic region (sgControl) or two different sgRNAs targeting Ero1la . k. Immunoblot analysis of indicated proteins from Slc33a1 knockout KPK cells complemented with a vector control or SLC33A1 cDNA expressing a sgRNA targeting intergenic region (sgControl) or two different sgRNAs targeting Ero1la. l. Left, CRISPR gene scores in SLC33A1 knockout HEK293T cells expressing a vector control or SLC33A1 cDNA cultured for 14 doublings. Top scoring hits color-coded. Pearson correlation coefficient, two-sided. Right, top-scoring genes that sensitize cells to SLC33A1 loss. m. Percent of cell number from SLC33A1 knockout HEK293T cells complemented with a vector control or SLC33A1 cDNA expressing a sgRNA targeting intergenic region (sgControl) or two different sgRNAs targeting SYVN1 after 4-day proliferation. Numbers of sg SYVN1 were normalized to sgCtr. n. Schematic of GSSG export by SLC33A1 to maintain ER redox homeostasis. GSH imported into ER is oxidized to GSSG upon reducing PDI family proteins that catalyze disulfide reduction and isomerization. SLC33A1 exports GSSG into cytosol, where it is reduced back to GSH by glutathione reductase (GR).

Article Snippet: Iodoacetamide-desthiobiotin (IA-DTB, HY-150230) from MedChemExpress; antimycin A (ALX-380-075) from Enzo Life Sciences; tunicamycin (3516) from Tocris Bioscience; X-tremeGENETM HP DNA Transfection Reagent (6366244001) and Dithiothreitol (10197777001) from Roche.

Techniques: Lysis, Labeling, Liquid Chromatography with Mass Spectroscopy, Tandem Mass Spectroscopy, Knock-Out, Expressing, Plasmid Preparation, Control, Western Blot, Modification, Mutagenesis, RNA Extraction, CRISPR, Cell Culture