Extended Data Fig. 4. DIESL is an ER membrane glycosylated acyltransferase that physically interacts with TMX1.
a, Localization of 3xHA-DIESL (green) in HeLa cells co-stained with the ER marker CANX (red). Hoechst 33342, blue; scale bar, 10 microns. b, HAP1 cells rescued with 3xHA-DIESL were subcloned, and three independent clones were then transduced with Cas9 and a control sgRNA (sgCTRL) or an sgRNA targeting TMX1 (sgTMX1). DIESL levels were assessed by immunoblot (left) and quantified (right). Bars represent mean ± SEM (Student’s t test, two-tailed) of n = 3 independent cell lines from one representative experiment. c, TMX1-dependent DIESL half-life was analyzed by a 24 hour-long cycloheximide chase in HAP1 cells rescued with 3xHA-DIESL, with or without TMX1 deletion. DIESL protein levels were analyzed by immunoblot (top) and quantified (bottom), normalized to ACTB. The cycloheximide concentration ranged from 200 to 3.125 µg/ml, using successive two-fold dilutions. Bars represent mean ± SEM of n = 3 independent experiments (two-way ANOVA, Bonferroni correction; ns, not significant). d, HeLa cells expressing 3xHA-DIESL were transduced with Cas9 and a control sgRNA (sgCTRL) or an sgRNA targeting TMX1 (sgTMX1). Cells were crosslinked with PFA prior to lysis and immunoblot analysis. x and m indicate the cross-linked TMX1-DIESL complex and corresponding monomer, respectively. e, Quantification of the 3xHA-DIESL coimmunoprecipitation of TMX1 in HAP1 cells shown in Fig. 3b. Fold enrichment over input is shown for TMX1, as well as CANX, PDI an EIF4G for n = 3 independent experiments (two-way ANOVA, Bonferroni correction; ns, not significant). f, TMX1 was co-immunoprecipitated from HAP1 cells rescued with DIESL in a buffer containing the indicated detergent (* indicates an antibody chain). Tw-20, Tween-20; DDM, n-dodecyl-beta-maltoside. g, An N-glycosylation motif (N-X-S/T) is conserved at the DIESL N-terminus. h, Deglycosylation of DIESL N-glycans by PNGaseF. Glycosylation was assessed by immunoblot, where LAMP1 served as a positive control. i, DIESL is glycosylated at N5 at the steady-state. HAP1 ∆DIESL cells reconstituted with the indicated 3xHA-DIESL construct were treated with 1 µg/ml tunicamycin (an inhibitor of N-linked glycosylation) for 16 h. The lower and higher HA bands represent the unglycosylated and N-glycosylated forms of 3xHA-DIESL, respectively. j, HAP1 ∆DIESL cells rescued with 3xHA-DIESL were treated with 5 µg/ml brefeldin A for the indicated period of time, and DIESL glycosylation status was assessed by immunoblot. The N5Q mutant is not glycosylated. k, N5Q is the single N-glycosylation site, as this construct and a mutant lacking arginines (N0, where all arginines have been mutated to glutamine) show an identical band pattern upon brefeldin A treatment. l, TLC analysis of neutral lipids (top) and quantification of TAG (bottom) in HAP1 DGAT DIESL 3KO cells, with or without additional ablation of TMX1, reconstituted with the indicated 3xHA-DIESL construct. Bars represent mean ± SEM of n = 4 independent experiments (one-way ANOVA, Bonferroni correction). m, AlphaFold model of human DIESL (Q96MH6) colored by pLDDT (left), active site histidine (centre) or hydrophobicity (right). n, Schematic of DIESL in the ER membrane with the lumenal domain indicated (top) and depiction of DIESL constructs with or without the 49 amino acid-containing, N-terminal lumenal domain (bottom). o, Immunoblot of HAP1 ∆DIESL cells reconstituted with full-length DIESL (FL) or DIESL lacking the lumenal domain (50-324), each with (WT) or without (H130A) an intact active site. * indicates a non-specific band. p, Depiction of the DIESL catalytic pocket as modeled by Alphafold (left) with H130 in blue, depicting both the backbone (left) and the surface (right), as viewed from the surface of the membrane, as well as the DIESL catalytic dyad (right).