ATP-dependent assembly of the heteromeric Pex1p-Pex6p-complex of the peroxisomal matrix protein import machinery.
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.
Abteilung für Systembiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany.
Delia
D
Saffian
Immanuel
I
Grimm
Wolfgang
W
Girzalsky
Ralf
R
Erdmann
D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C511586:PEX1 protein, S cerevisiae; C490760:PEX6 protein, S cerevisiae