The Peroxisomal Targeting Signal 1 in sterol carrier protein 2 is autonomous and essential for receptor recognition

To investigate the function of this secondary interface we have mutated two key residues from the ancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitative measurement of the binding affinity and kinetics of purified protein components and finally, by in vivo measurements, to determine translocation capability. While a moderate but significant reduction of the interaction was found in binding assays, we were not able to measure any significant defects in vivo.Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of the second binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that other cargo proteins may require this ancillary binding site.Pex5p, the major import receptor for peroxisomal matrix proteins, is known to carry folded proteins across the peroxisomal membrane by a signal assembled shuttling mechanism [1,2]. It has been found to recognise the type 1 Peroxisomal Targeting Signal (PTS1) - a C-terminal tripeptide of consensus sequence -[S/A/C]-[K/H/R]-[L/M]-CO2-, carried by some 40 human proteins destined for the peroxisomal lumen. The PTS1 sequence binds to Pex5p in an extended conformation, which is accommodated in a deep cavity in the tetratricopeptide repeat (TPR) domain of Pex5p [3,4] (Figure 1A). An additional interface, remote from the PTS1 binding site, of about 500 ？2 has been found to form between Pex5p and the model cargo protein sterol carrier protein 2 (SCP2), as demonstrated by the crystal structure and solution studies of SCP2 in complex with the C-terminal region of Pex5p [4]. In the following we will refer to the interface as "ancillary" or "secondary". This interface utilises the first and fourth α-helices of SCP2 and helices 15 and 16 from the Pex5p C-terminal helical bundle (Figure 1A & 1B) and its formation confers an increased binding affinity of around 6-fold when compared to a minimal PTS1