Proteomic analysis of peroxisome-associated PEX5 mutants in Xenopus egg extract.

This project sought to identify the features in PEX5 required for interaction with the peroxisomal PEX2-PEX10-PEX12 ubiquitin ligase complex. Peroxisomes equilibrated with different PEX5 mutants (i.e., C11A, AH1, AH2) were purified from Xenopus egg extract, solubilized in digitonin, and PEX5-associated material was immunoprecipitated via a C-terminal FLAG tag. PEX5 without a FLAG tag was used as a negative control. PEX5-interacting peroxins were identified by LC-MS/MS at the Taplin Mass Spectrometry Facility at Harvard Medical School.