title: Nucleotide-dependent assembly of the peroxisomal receptor export complex.
abstract: Pex1p and Pex6p are two AAA-ATPases required for biogenesis of peroxisomes. Both proteins form a hetero-hexameric complex in an ATP-dependent manner, which has a dual localization in the cytosol and at the peroxisomal membrane. At the peroxisomal membrane, the complex is responsible for the release of the import receptor Pex5p at the end of the matrix protein import cycle. In this study, we analyzed the recruitment of the AAA-complex to its anchor protein Pex15p at the peroxisomal membrane. We show that the AAA-complex is properly assembled even under ADP-conditions and is able to bind efficiently to Pex15p in vivo. We reconstituted binding of the Pex1/6p-complex to Pex15p in vitro and show that Pex6p mediates binding to the cytosolic part of Pex15p via a direct interaction. Analysis of the isolated complex revealed a stoichiometry of Pex1p/Pex6p/Pex15p of 3:3:3, indicating that each Pex6p molecule of the AAA-complex binds Pex15p. Binding of the AAA-complex to Pex15p in particular and to the import machinery in general is stabilized when ATP is bound to the second AAA-domain of Pex6p and its hydrolysis is prevented. The data indicate that receptor release in peroxisomal protein import is associated with a nucleotide-depending Pex1/6p-cycle of Pex15p-binding and release.
authors: Abteilung für Systembiochemie, Institut für Biochemie und Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany.
authors: Immanuel
authors: I
authors: Grimm
authors: Abteilung für Systembiochemie, Institut für Biochemie und Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany.
authors: Delia
authors: D
authors: Saffian
authors: Abteilung für Systembiochemie, Institut für Biochemie und Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany.
authors: Wolfgang
authors: W
authors: Girzalsky
authors: Abteilung für Systembiochemie, Institut für Biochemie und Pathobiochemie, Medizinische Fakultät der Ruhr-Universität Bochum, D-44780 Bochum, Germany.
authors: Ralf
authors: R
authors: Erdmann
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D008565:Membrane Proteins; D009711:Nucleotides; C110177:PEX15 protein, S cerevisiae; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D000074183:ATPases Associated with Diverse Cellular Activities; C511586:PEX1 protein, S cerevisiae; C490760:PEX6 protein, S cerevisiae