Fig. 1. Prediction and identification of PEX19-binding sites in PEX26. (A) Binding site prediction. The prediction matrix for PEX19-binding sites (Rottensteiner et al., 2004) was applied to human PEX26 and the hits obtained are presented as dotted vertical lines. Two sites with a high probability score are clearly visible. The positions of the high-scoring peptides within these sites as well as the amino acid sequence of each peak scoring peptide are indicated. (B) Peptide scan for PEX26. 15-mer peptides with two amino acid shifts between neighboring peptides and covering the entire PEX26 protein were synthesized on a nitrocellulose membrane and tested for interaction with GST-HsPEX19. Bound PEX19 was visualized by monoclonal anti-GST antibodies in combination with the ECL^TM reaction system. Arrows mark the peak interacting peptide within each binding site, the sequences of which are indicated. (C) Relative PEX19-binding affinity for the two binding sites in PEX26. Nine peptides per binding site were synthesized and assayed for PEX19 interaction as described in B. The sequences of the strongly interacting peptides are indicated. The right panel shows the relative intensities of the signals obtained for each spot. (D) Graphic representation of PEX26. Denoted are the positions of the PEX19-binding sites as determined in vitro and the postulated TMD (Matsumoto et al., 2003a).