Figure 5

Nucleotide-dependent association and dissociation of the reconstructed Pex1p/Pex6p/Pex15p-complex.

(a) Hexameric Pex1/6p complexes were isolated in presence of ATP or ADP and incubated with GST-Pex15p1-315His and then subjected to GST pull down assay. Load and 5x concentrates of eluate fractions were analyzed by SDS-PAGE and Coomassie staining (upper panel) and relative binding efficiency (lower panel) was estimated by measuring Pex6p signals from load and eluate fractions and setting Pex1/6p +ATP binding-capacity to 1. (b) GST-Pex15p1-315His pull down assay in presence of ATP using hexameric Pex1/6p wild type, Pex1WB/6p and Pex1/6pWB complexes. Binding efficiency was determined as in (a). In contrast to wild-type complex, Walker B-mutation of Pex6p leads to a significantly higher association of the complex with Pex15p, indicating that ATP-hydrolysis triggers complex-dissociation. (c) 700 kDa Pex1p/Pex6p/Pex15p and Pex1p/Pex6pWB/Pex15p complexes as well as Pex1p/Pex6p/Pex15p wild-type complexes, which had been incubated with 2.5 mM ATPγS during the isolation procedure, were obtained from size exclusion chromatography. Afterwards these complexes were incubated 72 h at 4 °C and subjected again to size exclusion chromatography. Presence of proteins was monitored by SDS-PAGE and immuno-blotting using anti-his-antibodies. Pex1p/Pex6pWB as well as wild-type complexes incubated with ATPγS exhibit a stabilized interaction to Pex15p, proving the direct correlation between ATP hydrolysis and complex-dissociation.