The transport of peroxisomal membrane proteins (PMPs) requires the soluble PEX19 protein as chaperone and import receptor. Recognition of cargo PMPs by the C-terminal domain (CTD) of PEX19 is required for peroxisome biogenesis in vivo. Farnesylation at a C-terminal CaaX motif in PEX19 enhances the PMP interaction, but the underlying molecular mechanisms are unknown. Here, we report the NMR-derived structure of the farnesylated human PEX19 CTD, which reveals that the farnesyl moiety is buried in an internal hydrophobic cavity. This induces substantial conformational changes that allosterically reshape the PEX19 surface to form two hydrophobic pockets for the recognition of conserved aromatic/aliphatic side chains in PMPs. Mutations of PEX19 residues that either mediate farnesyl contacts or are directly involved in PMP recognition abolish cargo binding and cannot complement a ΔPEX19 phenotype in human Zellweger patient fibroblasts. Our results demonstrate an allosteric mechanism for the modulation of protein function by farnesylation.

Cells can regulate the abundance and composition of peroxisomes to adapt to environmental changes. In the bakers yeast, S. cerevisiae, peroxisomes represent the only site for degradation of fatty acids. Hence, it is not surprising that growth of yeast cells on oleic acid results in a massive proliferation of peroxisomes. New peroxisomes can form either by division of pre-existing peroxisomes or de novo in a Pex25p-dependent process with the involvement of the Endoplasmic Reticulum (ER). In search for further factors involved in de novo formation of peroxisomes, we screened nearly 6,000 yeast mutants that were depleted of peroxisomes by conditional inhibition of PEX19 expression. Screening the mutants for the reappearance of peroxisomes upon expression of PEX19 identified Pex34p, in addition to the well-known component Pex25p, as crucial determinants for de novo biogenesis. Pex34p interacts with Pex19p and with different Peroxisomal Membrane Proteins (PMPs) in a PEX19-dependent manner. Depletion of Pex34p results in reduced numbers of import-competent peroxisomes formed de novo and Pex3p is partly retained and distributed in ER-like structures. We suggest that Pex25p and Pex34p are both required to maintain peroxisome number in a cell and that they perform non-redundant roles in the de novo formation of peroxisomes.