Table II

In vitro reconstitution of a Pex5p-containing peroxisomal protein import subcomplex

Microtiter plate wells, coated with 1 μg of human serum albumin cross-linked to the peptide CSYHKHLKPLQSKL (PTS1), were exposed in three subsequent incubations to bacterial lysates containing no recombinant protein (−), (His)6-Pex5p, (His)6-Pex14p, and (His)6-Pex13p/SH3 as indicated schematically. After extensive washings, bound Pex5p, Pex14p, and Pex13p were detected photometrically using peroxin-specific rabbit antisera and anti-rabbit IgGs coupled to alkaline phosphatase and p-nitrophenyl phosphate. To substract the background of the anti-Pex13p antiserum due to a weak cross-reactivity with human serum albumin, data were corrected by performing simultaneously control experiments using one microgram of human serum albumin cross-linked to the peptide CVHESYHKHLKPLQ as immobilized ligand. The experiments were performed in triplicate. The positive interactions are underlined.

PTS1123A405 nm
Pex5pPex14pPex13p/SH3α-Pex5pα-Pex14pα-Pex13p
+++0.000 ± 0.0000.026 ± 0.0150.035 ± 0.030
+++ 0.698 ± 0.032 0.053 ± 0.0050.014 ± 0.012
+++ 0.471 ± 0.008 0.584 ± 0.031 0.010 ± 0.014
++++ 0.440 ± 0.015 0.537 ± 0.015 0.122 ± 0.055