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

摘要

Background: The majority of peroxisomal matrix proteins destined for translocation into the peroxisomal lumenare recognised via a C-terminal Peroxisomal Target Signal type 1 by the cycling receptor Pex5p. The only structureto date of Pex5p in complex with a cargo protein is that of the C-terminal cargo-binding domain of the receptorwith sterol carrier protein 2, a small, model peroxisomal protein. In this study, we have tested the contribution of asecond, ancillary receptor-cargo binding site, which was found in addition to the characterised Peroxisomal TargetSignal type 1.Results: To investigate the function of this secondary interface we have mutated two key residues from theancillary binding site and analyzed the level of binding first by a yeast-two-hybrid assay, followed by quantitativemeasurement of the binding affinity and kinetics of purified protein components and finally, by in vivomeasurements, to determine translocation capability. While a moderate but significant reduction of the interactionwas found in binding assays, we were not able to measure any significant defects in vivo.Conclusions: Our data therefore suggest that at least in the case of sterol carrier protein 2 the contribution of thesecond binding site is not essential for peroxisomal import. At this stage, however, we cannot rule out that othercargo proteins may require this ancillary binding site