Extended Data Fig. 6: MRR strongly promotes MARCHF6 activity.
From: The MARCHF6 E3 ubiquitin ligase acts as an NADPH sensor for the regulation of ferroptosis
a, SDS-PAGE of purified MARCHF63f (5 µg) and \(\small {{{\mathrm{MARF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\) (5 µg), UBE2G2 (10 µg), and UBE2J2ΔTM (10 µg), followed by Coomassie brilliant blue staining. b, In vitro ubiquitylation assays of MARCHF63f or \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\) (0.1 µM) with UBE2G2 (1 µM), UBE2J2ΔTM (0.2 µM), myc-Ub (20 µM), UBA1 (0.1 µM), and ATP (2 mM) for 20 min at 30 °C. UBE2J2ΔTM alone produced, to some extent, poly-Ub chains, whereas few poly-Ub chains were observed with UBE2G2 alone. Both MARCHF63f−UBE2J2ΔTM and MARCHF63f−UBE2G2 strongly promoted poly-Ub chain formation with higher overall yields and processivity. The combinations of MARCHF63f−UBE2J2ΔTM and MARCHF63f−UBE2G2 formed poly-Ub chains more strongly and greater processivity than MARCHF63f−UBE2J2ΔTM or MARCHF63f−UBE2G2 alone. However, the overall yield and processivity of poly-Ub chain formation were most dramatically reduced by \(\small {{{\mathrm{MARCHF}}}}6_{3{{{\mathrm{f}}}}}^{{{{\mathrm{MRR}}}}}\). c, Diagrams for RINGha only and RINGha-MRR fusion. d, Same as in (b) but with RINGha (0.5 µM) and RINGha-MRR (0.5 µM). The purified linear RINGha-MRR fusion strongly promoted poly-Ub chain formation in the presence of UBE2J2ΔTM and/or UBE2G2, whereas RINGha only produced di- or tri-Ub adducts. e, Same as in (d) but with immunoblotting using the anti-K48 linkage-specific Ub antibody. f, In vitro ubiquitylation assays of RINGha-MRR (0.5 µM) with Ub, Lys-lacking K0-Ub, and the indicated single Lys-containing Ub mutants (each 25 µM), followed by immunoblotting with anti-Ub. g, Scheme for di-Ub formation assay using fluorescent dye-labelled UbR48 with a K48-to-R mutation as a donor and UbAA with C-terminal diglycine (GG) to di-alanine (AA) as an acceptor. h, Di-Ub formation assays of RINGha (0.5 µM) or RINGha-MRR (0.5 µM) upon increasing concentrations of the acceptor UbAA for 15 min at 30 °C. i, Same as in (h) but with linkage-specific tetra-Ubs (each 2 µM)]. j, Same as in (g) but with UbR48 and UbAA-GST (each 10 µM). In (a,b,d,e,f,h,i,j), images represent at least two independent experiments.
