Extended Data Fig. 8: In vitro polyubiquitination activity of the RFs.
From: A peroxisomal ubiquitin ligase complex forms a retrotranslocation channel
a, Polyubiquitination reactions were performed with purified GST-fusions of RF2, RF12, or RF10 (E3 enzyme) in the presence of E1 enzyme, the E2 enzyme Ubc4, and Dylight800-labeled ubiquitin. Where indicated, components were omitted. The samples were analyzed by SDS-PAGE and fluorescence scanning. b, As in a, but with different combinations of wild-type (WT) or mutant RF10 and RF12. The results in a and b are representative of three biological repeats. For gel source data, see Supplementary Fig. 1. c, WT Pex10 or Pex12, or the indicated mutants, were expressed from endogenous promoters in S. cerevisiae cells lacking Pex10 and Pex12 (pex10Δ pex12Δ). Peroxisomal protein import was determined by the reduction in the formation of a fluorescent compound (PDV). Fluorescence was measured in cell lysates and the data were normalized, setting the fluorescence of pex10Δ pex12Δ cells as 100% and that of cells expressing WT Pex10 and Pex12 as 0%. Shown are the means and standard errors of three experiments. d, WT or mutant Pex5 was overexpressed (Pex5OE) in Pex2 mutant cells containing mutations in the L1 loop of RF2 (pex2Δ Pex2L1). Vec, vector without Pex5. Cells lacking Pex5 (pex5Δ) served as a control. Protein import was measured as in c. The bar graphs in c and d show the individual data points, the mean and S.E.M. from three biological repeats. Statistical significance between the wild type and mutants was calculated by one-way analysis of variance. ns, not significant; ** P < 0.05; ****P < 0.001. See also Source Data file
