irefweb

For Pex5p, we mapped the site required for PTS1 and Pex12p interaction to the tetratricopeptide repeat domains labeled 2–8 and 2–9, respectively (Fig. 4).

These results, combined with the observation that Pex12p(275–359) interacts with Pex5p in the y-2HS (see Refs. 29 and 34, and see Fig. 4), suggest that Pex5p has distinct binding sites for the C3HC4 RING finger domain of Pex12p and PTS1 proteins and that Pex5p and Pex5p-Pex12p(275–359) have similar PTS1 binding properties

Several lines of evidence suggest that this complex is in fact a homodimer of Pex14p. (i) Pex14p can homodimerize when it is expressed in E. coli (Fig. 1); (ii) the cross-linked Pex14p-containing complex of CHO cells migrates during SDS-PAGE to a similar distance as the dimerized fraction of bacterially expressed (His)6-Pex14p (data not shown); and (iii) the cross-linked Pex14p-containing complex is not recognized by antisera raised against the Pex14p-interacting peroxins Pex5p, Pex13p, and Pex19p (data not shown). Interestingly, even in the absence of cross-linker a small but significant fraction of Pex14p migrated as a dimer on SDS-PAGE under non-reducing conditions (Fig. 3). However, deletion analysis studies revealed that the dimeric Pex14p structure is not maintained by disulfide bonding between the subunits but by its coiled-coil structure (see below).

Interestingly, this domain of Pex13p was also found to interact weakly with itself, at least in the y-2HS (Fig. 1).

However, in vitro reconstitution experiments of a putative peroxisomal protein import complex consisting of PTS1 ligand, Pex5p, Pex14p, and the SH3 domain of Pex13p (amino acids 236 to 403) demonstrated that Pex14p, but not the SH3 domain of Pex13p, interacts with Pex5p loaded with PTS1 cargo (Table II). In addition, these experiments show that Pex14p can bind simultaneously to Pex5p and Pex13p/SH3 (Table II). Summarized, these results suggest that in mammals (i) Pex14p, and not Pex13p, functions as the docking protein for cargo-loaded Pex5p, and (ii) the Pex5p-Pex13p interaction is bridged by Pex14p (Table II).

This validation study yielded 9 different interacting peroxin pairs; four pairs (Pex14p-Pex5p, Pex19p-Pex3p, Pex19p-Pex16p, and Pex19p-Pex11pß) were found to interact in both two-hybrid systems, three pairs (Pex19p-Pex10p, Pex19p-Pex12p, and Pex19p-Pex13p) were only detected in the y-2HS, and two pairs (Pex14p-Pex14p and Pex14p-Pex19p) interacted solely in the b-2HS.