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The gene products (peroxins) of at least 29 PEX genes are known to be necessary for peroxisome biogenesis but for most of them their precise function remains to be established. Here we show that Pex15p, an integral peroxisomal membrane protein, in vivo and in vitro binds the AAA peroxin Pex6p. This interaction functionally interconnects these two hitherto unrelated peroxins. Pex15p provides the mechanistic basis for the reversible targeting of Pex6p to peroxisomal membranes. We could demonstrate that the N-terminal part of Pex6p contains the binding site for Pex15p and that the two AAA cassettes D1 and D2 of Pex6p have opposite effects on this interaction. A point mutation in the Walker A motif of D1 (K489A) decreased the binding of Pex6p to Pex15p indicating that the interaction of Pex6p with Pex15p required binding of ATP. Mutations in Walker A (K778A) and B (D831Q) motifs of D2 abolished growth on oleate and led to a considerable larger fraction of peroxisome bound Pex6p. The nature of these mutations suggested that ATP-hydrolysis is required to disconnect Pex6p from Pex15p. On the basis of these results, we propose that Pex6p exerts at least part of its function by an ATP-dependent cycle of recruitment and release to and from Pex15p.

Phosphopantetheinyl-dependent carrier proteins are part of fatty-acid synthases (primary metabolism), polyketide synthases, and non-ribosomal peptide synthetases (secondary metabolism). For these proteins to become functionally active, they need to be primed with the 4-phosphopantetheine moiety of coenzyme A by a dedicated phosphopantetheine transferase (PPTase). Most organisms that employ more than one phosphopantetheinyl-dependent pathway also have more than one PPTase. Typically, one of these PPTases is optimized for the modification of carrier proteins of primary metabolism and rejects those of secondary metabolism (AcpS-type PPTases), whereas the other, Sfp-type PPTase, efficiently modifies carrier proteins involved in secondary metabolism. We present here a new type of PPTase, the carrier protein synthase of Pseudomonas aeruginosa, an organism that harbors merely one PPTase, namely PcpS. Gene deletion experiments clearly show that PcpS is essential for growth of P. aeruginosa, and biochemical data indicate its association with both fatty acid synthesis and siderophore metabolism. At first sight, PcpS is a PPTase of the monomeric Sfp-type and was consequently expected to have catalytic properties typical for this type of enzyme. However, in vitro characterization of PcpS with natural protein partners and non-cognate substrates revealed that its catalytic properties differ significantly from those of Sfp. Thus, the situation in P. aeruginosa is not simply the result of the loss of an AcpS-type PPTase. PcpS exhibits high catalytic efficiency with the carrier protein of fatty acid synthesis and shows a reduced although significant conversion rate of the carrier proteins of non-ribosomal peptide synthetases from their apo to holo form. This association with enzymes of primary and secondary metabolism indicates that PcpS belongs to a new sub-class of PPTases. 4Ј-Phosphopantetheine (Ppant)1 -dependent carrier proteins (CP) are the central entity in fatty-acid synthases (FAS), polyketide synthases (PKS), and non-ribosomal peptide synthetases (NRPSs) (1, 2). The superfamily of CP includes the acidic acyl and aryl carrier proteins and the neutral peptidyl carrier proteins (PCPs). They can be part of a larger polypeptide chain or exist as distinct proteins but always fulfill the same job; during the multistep assembly of the product, the reaction intermediates of the growing acyl or polypeptide chain remain covalently tethered to the Ppant cofactor moiety of these proteins. This moiety is about 20 Å in length and enables the bound intermediates to move between the reaction centers of multifunctional proteins. The thioester linkage that is used to bind the intermediates and final product is energy-rich, which facilitates cleavage after the final step of the assembly. After ribosomal synthesis, however, the carrier protein exists only in the inactive apo form. The Ppant moiety is post-translationally transferred from coenzyme A to a conserved serine residue of the CP in a Mg 2ϩ -dependent reaction by a dedicated phosph...

Within the extended receptor cycle of peroxisomal matrix import, the function of the import receptor Pex5p comprises cargo recognition and transport. While the C-terminal half (Pex5p-C) is responsible for PTS1 binding, the contribution of the N-terminal half of Pex5p (Pex5p-N) to the receptor cycle has been less clear. Here we demonstrate, using different techniques, that in Saccharomyces cerevisiae Pex5p-N alone facilitates the import of the major matrix protein Fox1p. This finding suggests that Pex5p-N is sufficient for receptor docking and cargo transport into peroxisomes. Moreover, we found that Pex5p-N can be functionally replaced by Pex18p, one of two auxiliary proteins of the PTS2 import pathway. A chimeric protein consisting of Pex18p (without its Pex7p binding site) fused to Pex5p-C is able to partially restore PTS1 protein import in a PEX5 deletion strain. On the basis of these results, we propose that the auxiliary proteins of the PTS2 import pathway fulfill roles similar to those of the N-terminal half of Pex5p in the PTS1 import pathway.