ABSTRACT
The type-2 peroxisomal targeting signal (PTS2) is one of two peptide motifs destining soluble proteins for peroxisomes. This signal acts as amphiphilic α-helix exposing the side chains of all conserved residues to the same side. PTS2 motifs are recognized by a bipartite protein complex consisting of the receptor PEX7 and a co-receptor. Cargo-loaded receptor complexes are translocated across the peroxisomal membrane by a transient pore and inside peroxisomes, cargo proteins are released and processed in many, but not all species. The components of the bipartite receptor are re-exported into the cytosol by a ubiquitin-mediated and ATP-driven export mechanism. Structurally, PTS2 motifs resemble other N-terminal targeting signals, whereas the functional relation to the second peroxisomal targeting signal (PTS1) is unclear. Although only a few PTS2-carrying proteins are known in humans, subjects lacking a functional import mechanism for these proteins suffer from the severe inherited disease rhizomelic chondrodysplasia punctata.
Subject(s)
Peroxisomal Targeting Signal 2 Receptor/metabolism , Amino Acid Motifs , Chondrodysplasia Punctata, Rhizomelic/metabolism , Chondrodysplasia Punctata, Rhizomelic/pathology , Humans , Membrane Proteins/metabolism , Peroxisomal Targeting Signal 2 Receptor/chemistry , Peroxisomal Targeting Signal 2 Receptor/genetics , Peroxisomes/metabolism , Protein Domains , Protein Structure, Quaternary , Protein TransportABSTRACT
Peroxisomal matrix proteins contain either a peroxisomal targeting sequence 1 (PTS1) or a PTS2 that are recognized by the import receptors PEX5 and PEX7, respectively. PEX5 transports the PTS1 proteins and the PEX7/PTS2 complex to the docking translocation module (DTM) at the peroxisomal membrane. After cargo release PEX5 is monoubiquitinated and extracted from the peroxisomal membrane by the receptor export machinery (REM) comprising PEX26 and the AAA ATPases PEX1 and PEX6. Here, we investigated the protein interactions of monoubiquitinated PEX5 with the docking proteins PEX13, PEX14 and the REM. "Click" chemistry was used to synthesise monoubiquitinated recombinant PEX5. We found that monoubiquitinated PEX5 binds the PEX7/PTS2 complex and restores PTS2 protein import in vivo in ΔPEX5 fibroblasts. In vitro pull-down assays revealed an interaction of recombinant PEX5 and monoubiquitinated PEX5 with PEX13, PEX14 and with the REM components PEX1, PEX6 and PEX26. The interactions with the docking proteins were independent of the PEX5 ubiquitination status whereas the interactions with the REM components were increased when PEX5 is ubiquitinated.
Subject(s)
Peroxisome-Targeting Signal 1 Receptor/chemistry , Peroxisomes/chemistry , Protein Interaction Maps/genetics , Protein Transport/genetics , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/genetics , Amino Acid Sequence/genetics , Animals , Click Chemistry , Cytosol/chemistry , Cytosol/metabolism , Fibroblasts/chemistry , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Mice , Molecular Docking Simulation , Mutation , Peroxisomal Targeting Signal 2 Receptor/chemistry , Peroxisomal Targeting Signal 2 Receptor/genetics , Peroxisomal Targeting Signals/genetics , Peroxisome-Targeting Signal 1 Receptor/genetics , Peroxisomes/genetics , Ubiquitin/chemistry , Ubiquitin/metabolism , Ubiquitination/geneticsABSTRACT
A newly isolated binding protein of peroxisomal targeting signal type 2 (PTS2) receptor Pex7, termed P7BP2, is transported into peroxisomes by binding to the longer isoform of Pex5p, Pex5pL, via Pex7p. The binding to Pex7p and peroxisomal localization of P7BP2 depends on the cleavable PTS2 in the N-terminal region, suggesting that P7BP2 is a new PTS2 protein. By search on human database, three AAA+ domains are found in the N-terminal half of P7BP2. Protein sequence alignment and motif search reveal that in the C-terminal region P7BP2 contains additional structural domains featuring weak but sufficient homology to AAA+ domain. P7BP2 behaves as a monomer in gel-filtration chromatography and the single molecule observed under atomic force microscope shapes a disc-like ring. Collectively, these results suggest that P7BP2 is a novel dynein-type AAA+ family protein, of which domains are arranged into a pseudo-hexameric ring structure.
