Figure 5.

PBD054 is mutated in PEX12. (A) Sequence chromatographs of PEX12 genomic DNA showing the wild-type sequence (top) and PBD054 sequence (bottom) in the region corresponding to nucleotides 950–968 of the PEX12 ORF. Note the C to T transition mutation, S320F, which changes the serine codon TCT to the phenylalanine codon TTT (underlined). (B) The S320F mutation attenuates the interaction between PEX12 and PEX10. Two-hybrid reporter strains expressing the indicated fusion proteins were assayed for β-galactosidase activity (top). To assess the effects of this mutation by blot overlay assay, purified MBP-LacZα, MBP-PEX12C/S320F, and MBP-PEX12C were separated by SDS-PAGE, transferred to membranes, and probed with ³⁵S-labeled PEX10 (upper half of lower panel). These proteins were also separated by SDS-PAGE and stained with Coomassie blue (lower half of lower panel). (C) The S320F mutation attenuates the PEX12-PEX5 interaction in the yeast two-hybrid assay. Two-hybrid reporter strains expressing the listed proteins were assayed for β-galactosidase activity (top). The three strains expressing AD-5S were also lysed and assayed for BD-PEX12 levels by immunoblot with anti–PEX12 antibodies.