PEX14 binding to Arabidopsis PEX5 has differential effects on PTS1 and PTS2 cargo occupancy of the receptor

PEX5 acts as a cycling receptor for import of PTS1 proteins into peroxisomes and as a co-receptor for PEX7, the PTS2 receptor, but the mechanism of cargo unloading has remained obscure. Using recombinant protein domains we show PEX5 binding to the PEX14N-terminal domain (PEX14N) has no effect on the affinity of PEX5 for a PTS1 containing peptide. PEX5 can form a complex containing both recombinant PTS1 cargo and endogenous PEX7-thiolase simultaneously but isolation of the complex via the PEX14 construct resulted in an absence of thiolase, suggesting a possible role for PEX14 in the unloading of PTS2 cargos

A novel Pex14 protein-interacting site of human Pex5 is critical for matrix protein import into peroxisomes.

Protein import into peroxisomes relies on the import receptor Pex5, which recognizes proteins with a peroxisomal targeting signal 1 (PTS1) in the cytosol and directs them to a docking complex at the peroxisomal membrane. Receptor-cargo docking occurs at the membrane-associated protein Pex14. In human cells, this interaction is mediated by seven conserved diaromatic penta-peptide motifs (WXXX(F/Y) motifs) in the N-terminal half of Pex5 and the N-terminal domain of Pex14. A systematic screening of a Pex5 peptide library by ligand blot analysis revealed a novel Pex5-Pex14 interaction site of Pex5. The novel motif composes the sequence LVAEF with the evolutionarily conserved consensus sequence LVXEF. Replacement of the amino acid LVAEF sequence by alanines strongly affects matrix protein import into peroxisomes in vivo. The NMR structure of a complex of Pex5-(57-71) with the Pex14-N-terminal domain showed that the novel motif binds in a similar α-helical orientation as the WXXX(F/Y) motif but that the tryptophan pocket is now occupied by a leucine residue. Surface plasmon resonance analyses revealed 33 times faster dissociation rates for the LVXEF ligand when compared with a WXXX(F/Y) motif. Surprisingly, substitution of the novel motif with the higher affinity WXXX(F/Y) motif impairs protein import into peroxisomes. These data indicate that the distinct kinetic properties of the novel Pex14-binding site in Pex5 are important for processing of the peroxisomal targeting signal 1 receptor at the peroxisomal membrane. The novel Pex14-binding site may represent the initial tethering site of Pex5 from which the cargo-loaded receptor is further processed in a sequential manner

Solution structure of human Pex5.Pex14.PTS1 protein complexes obtained by small angle X-ray scattering

The Pex5p receptor recognizes newly synthesized peroxisomal matrix proteins which have a C-terminal peroxisomal targeting signal to the peroxisome. After docking to protein complexes on the membrane, these proteins are translocated across the membrane. The docking mechanism remains unclear, as no structural data on the multicomponent docking complex are available. As the interaction of the cargo-loaded Pex5p receptor and the peroxisomal membrane protein Pex14p is the essential primary docking step, we have investigated the solution structure of these complexes by small angle x-ray scattering and static light scattering. Titration studies yielded a 1:6 stoichiometry for the Pex5p·Pex14p complex, and low resolution structural models were reconstructed from the x-ray scattering data. The free full-length human Pex5p is monomeric in solution, with an elongated, partially unfolded N-terminal domain. The model of the complex reveals that the N terminus of Pex5p remains extended in the presence of cargo and Pex14p, the latter proteins being significantly intermingled with the Pex5p moiety. These results suggest that the extended structure of Pex5p may play a role in interactions with other substrates such as lipids and membrane proteins during the formation of functional multiprotein complexes

Peroxin 5–peroxin 14 association in the protozoan Leishmania donovani involves a novel protein–protein interaction motif

Import of proteins with a PTS1 (peroxisomal targeting signal 1) into the Leishmania glycosomal organelle involves docking of a PTS1-laden LdPEX5 [Leishmania donovani PEX5 (peroxin 5)] receptor to LdPEX14 on the surface of the glycosomal membrane. In higher eukaryotes, the PEX5–PEX14 interaction is mediated by a conserved diaromatic WXXXY/F motif. Site-directed and deletion mutageneses of the three WXXXY/F repeats in LdPEX5 did not abolish the LdPEX5–LdPEX14 association. Analysis of the equilibrium dissociation constant (K(d)) revealed that ldpex5-W53A (Trp(53)→Ala), ldpex5-W293A, ldpex5-W176,293A and ldpex5-W53,176,293A mutant receptors were capable of binding LdPEX14 with affinities comparable with wild-type LdPEX5. That the diaromatic motifs were not required for the LdPEX5–LdPEX14 interaction was further verified by deletion analysis that showed that ldpex5 deletion mutants or ldpex5 fragments lacking the WXXXY/F motifs retained LdPEX14 binding activity. Mapping studies of LdPEX5 indicated that the necessary elements required for LdPEX14 association were localized to a region between residues 290 and 323. Finally, mutational analysis of LdPEX14 confirmed that residues 23–63, which encompass the conserved signature sequence AX(2)FLX(7)SPX(6)FLKGKGL/V present in all PEX14 proteins, are essential for LdPEX5 binding