The peroxisomal matrix protein import is facilitated by soluble receptor molecules which cycle between cytosol and the peroxisomal membrane. At the end of the receptor cycle, the import receptors are exported back to the cytosol in an ATP-dependent manner catalyzed by Pex1p and Pex6p, two AAA (ATPases associated with various cellular activities) type ATPases. Pex1p and Pex6p interact and form a heteromeric complex. In order to gain more insight into the stoichiometry and mechanism of assembly of the complex, we heterologously expressed and purified Saccharomyces cerevisiae Pex1p and Pex6p. Size exclusion chromatography studies of the recombinant proteins demonstrate that they form a hexameric complex in a one-to-one ratio of both AAA-proteins. The recombinant AAA-complex exhibits an ATPase activity with a k(m) of 0.17 mM and V(max) of 0.35 nmol min(-1) μg(-1). In the presence of N-ethylmaleimide, ATPase activity of the peroxisomal AAA-complex is drastically decreased and the complex dissociates. Disassembly of the complex into its Pex1p and Pex6p subunits is also observed upon ATP-depletion, indicating that formation of the Pex1p/Pex6p-complex requires the presence of ATP.
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Receptor recognition by the peroxisomal AAA complex depends on the presence of the ubiquitin moiety and is mediated by Pex1p.
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From the Institute of Biochemistry and Pathobiochemistry, Faculty of Medicine, Systems Biochemistry, Ruhr-University Bochum, D-44780 Bochum, Germany
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Abteilung für Systembiochemie, Institut für Biochemie und Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, Universitätsstr. 150, D-44780 Bochum.
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Institut für Biochemie und Pathobiochemie, Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany
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