Molecular snapshots of the Pex1/6 AAA + complex in action

The peroxisomal proteins Pex1 and Pex6 form a heterohexameric type II AAA+ ATPase complex, which fuels essential protein transport across peroxisomal membranes. Mutations in either ATPase in humans can lead to severe peroxisomal disorders and early death. We present an extensive structural and biochemical analysis of the yeast Pex1/6 complex. The heterohexamer forms a trimer of Pex1/6 dimers with a triangular geometry that is atypical for AAA+ complexes. While the C-terminal nucleotide-binding domains (D2) of Pex6 constitute the main ATPase activity of the complex, both D2 harbour essential sub-strate-binding motifs. ATP hydrolysis results in a pumping motion of the complex, suggesting that Pex1/6 function involves substrate translocation through its central channel. Mutation of the Walker B motif in one D2 domain leads to ATP hydrolysis in the neighbouring domain, giving structural insights into inter-domain communication of these unique heterohexameric AAA + assemblies

Assembly of the heteromeric Pex1p-Pex6p-complex of the peroxisomal matrix protein import machinery

Der peroxisomale Matrixproteinimport basiert auf zyklisierenden Rezeptoren, die zwischen dem Cytosol und der peroxisomalen Membran pendeln. Ein wichtiger Schritt im Rahmen dieses Zyklus ist die ATP-abhängige Freisetzung des Rezeptors, bewerkstelligt durch einen Proteinkomplex bestehend aus Pex1p und Pex6p. Bei diesen beiden Proteinen handelt es sich um Mitglieder der AAA ("ATPases associated with various cellular activities")-Familie, die im Rahmen dieser Arbeit näher charakterisiert wurden. Mithilfe eines speziellen $\textit {Escherichia coli}$-Expressionssystems wurden Pex1p und Pex6p der Bäckerhefe $\textit {Saccharomyces cerevisiae}$ rekombinant gewonnen. Das Reinigungsverfahren konnte derart optimiert werden, dass die Kriterien für funktionelle als auch strukturelle Untersuchungen in Qualität und Quantität erfüllt wurden. Funktionell konnte mit den isolierten Proteinen deren Assemblierung zu einem heterohexameren Komplex gezeigt werden

Nucleotide-dependent assembly of the peroxisomal receptor export complex

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 $\textit {in vivo}$. We reconstituted binding of the Pex1/6p-complex to Pex15p $\textit {in vitro}$ 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 AAAdomain 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