Fig. 4: Distinct functions of the ring finger domains of the ligase complex.
From: A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel
a, Predicted interaction of RF10 with E2–Ub. UbcH5a bound to ubiquitin (Protein Data Bank ID: 4AP4) was docked onto Pex10 of the RF10–RF12 complex. RF10 is a S. cerevisiae homology model derived from the T. thermophilus cryo-EM structure. RF12 is a crystal structure (Extended Data Fig. 7a). The magnified view shows amino acid interactions (red dashed lines). b, Polyubiquitylation reactions were performed with WT or mutant RF10 or RF12 in the presence of Uba1, Ubc4, Dylight800-labelled ubiquitin and ATP, as indicated. The results are representative of three biological repeats. For gel source data, see Supplementary Fig. 1. c, WT or mutant Pex10 was expressed in S. cerevisiae cells lacking Pex10 (pex10Δ). 'Mock' indicates cells expressing only an antibiotic resistance gene. Protein import was determined by the reduction in fluorescent pigment (PDV) formation. Fluorescence in cell lysates was normalized, setting the fluorescence of pex10Δ cells as 100% and that of WT cells as 0%. Bar graphs show individual data points, the mean and the s.e.m. from three biological repeats. Statistical significance between WT and mutants were calculated by one-way analysis of variance. NS, not significant; *P < 0.1, **P < 0.05 and ****P < 0.001. See also Source Data file. d, As in c, but for pex12Δ. e, As in c, but for pex2Δ. f, As in c, but for pex2Δ pex10Δ. L1 and L2 indicate Pex2 loop mutations. g, WT or mutant Pex5 was overexpressed (Pex5OE) in WT cells or Pex10(R324A)-mutant cells. Vector, vector without Pex5. h, Receptor recycling pathway involving monoubiquitylation by RF2 (left model), and the degradation pathway involving polyubiquitylation by RF10 and RF12 (right model). RF10 is the active domain.
