Recombinant human peroxisomal targeting signal receptor PEX5 - Structural basis for interaction of PEX5 with PEX14

Import of matrix proteins into peroxisomes requires two targeting signal-sp ecific import receptors, Pex5p and Pex7p, and their binding partners at the peroxisomal membrane, Pex13p and Pex14p. Several constructs of human PEX5 have been overexpressed and purified by affinity chromatography in order to determine functionally important interactions and provide initial structur al information. Sizing chromatography and electron microscopy suggest that the two isoforms of the human PTS1 receptor, PEX5L and PEX5S, form homotetr amers. Surface plasmon resonance analysis indicates that PEX5 binds to the N-terminal fragment of PEX14-(1-78) with a very high affinity in the low na nomolar range. Stable complexes between recombinant PEX14-(1-78) and both t he full-length and truncated versions of PEX5 were formed in vitro. Analysi s of these complexes revealed that PEX5 possesses multiple binding sites fo r PEX14, which appear to be distributed throughout its N-terminal half. Coi ncidentally, this part of the molecule is also responsible for oligomerizat ion, whereas the C-terminal half with its seven tetratricopeptide repeats h as been reported to bind PTS1-proteins. A pentapeptide motif that is reiter ated seven times in PEX5 is proposed as a determinant for the interaction w ith PEX14.