Recognition of peroxisomal targeting signal type 1 by the import receptor Pex5p

We have studied how Pex5p recognizes peroxisomal targeting signal type I (P TS1)-containing proteins. A randomly mutagenized pex5 library was screened in a two-hybrid setup for mutations that disrupted the interaction with the PTS1 protein Mdh3p or for suppressor mutations that could restore the inte raction with Mdh3p containing a mutation in its PTS1. All mutations localiz ed in the tetratricopeptide repeat (TPR) domain of Pex5p. The Pex5p TPR dom ain was modeled based on the crystal structure of a related TPR protein. Ma pping of the mutations on this structural model revealed that some of the l oss-of-interaction mutations consisted of substitutions in alpha -helices o f TPRs with bulky amino acids, probably resulting in local misfolding and t hereby indirectly preventing binding of PTS1 proteins. The other loss-of-in teraction mutations and most suppressor mutations localized in short, expos ed, intra-repeat loops of TPR2, TPR3, and TPR6, which are predicted to medi ate direct interaction with PTS1 amino acids. Additional site-directed muta nts at conserved positions in intra-repeat loops underscored the importance of the loops of TPR2 and TPR3 for PTS1 interaction. Based on the mutationa l analysis and the structural model, we put forward a model as to how PTS1 proteins are selected by Pex5p.