Revisiting the intraperoxisomal pathway of mammalian PEX7

Abstract

Newly synthesized peroxisomal proteins containing a cleavable type 2 targeting signal (PTS2) are transported to the peroxisome by a cytosolic PEX5-PEX7 complex. There, the trimeric complex becomes inserted into the peroxisomal membrane docking/translocation machinery (DTM), a step that leads to the translocation of the cargo into the organelle matrix. Previous work suggests that PEX5 is retained at the DTM during all the steps occurring at the peroxisome but whether the same applies to PEX7 was unknown. By subjecting different pre-assembled trimeric PEX5-PEX7-PTS2 complexes to in vitro co-import/export assays we found that the export competence of peroxisomal PEX7 is largely determined by the PEX5 molecule that transported it to the peroxisome. This finding suggests that PEX7 is also retained at the DTM during the peroxisomal steps and implies that cargo proteins are released into the organelle matrix by DTM-embedded PEX7. The release step does not depend on PTS2 cleavage. Rather, our data suggest that insertion of the trimeric PEX5-PEX7-PTS2 protein complex into the DTM is probably accompanied by conformational alterations in PEX5 to allow release of the PTS2 protein into the organelle matrix.

Figure 1. The export competence of peroxisomal PEX7 is determined by the PEX5 molecule that transported it to the organelle.

(a) Two sets of three chemically identical co-import reactions of radiolabeled PEX7 and radiolabeled His-tagged PEX7 (His₆PEX7) were performed with the indicated amounts of recombinant ΔC1PEX5L and prePHYH. Recombinant proteins were either added directly to the reaction (lanes 1 and 4) or pre-incubated with one of the radiolabeled PEX7 proteins, as indicated by boxes enclosing the “+” signs, before being added to the reaction containing the other PEX7 protein (lanes 2 and 5 or lanes 3 and 6 for His₆PEX7 or PEX7, respectively). Import assays were made at 23 °C for 5 min to minimize receptor export. Pronase-treated organelles were subjected to SDS-PAGE/western-blot. The Ponceau S-stained membrane (lower panel) and its autoradiography (upper panel) are shown. Lanes I₁ and I₂, 2.5% of the reticulocyte lysates containing ³⁵S-labeled His₆PEX7 and PEX7 used in each reaction. (b) A mixture of radiolabeled His₆PEX7 and PEX7 was pre-incubated with 50 ng of prePHYH in the presence or absence of ΔC1PEX5L (10 ng) and subjected to import assays. Aliquots were removed at 0 and 5 min and processed as above. (c) Reticulocyte lysates containing His₆PEX7 or PEX7 were pre-incubated individually with 25 ng of prePHYH and 5 ng of either ΔC1PEX5L or ΔC1PEX5L(C11A) for 20 min at 23 °C. These mixtures were then added to a rat liver PNS in import buffer containing ATP in three different combinations (#1–#3), as indicated. After 5 min at 23 °C, import was inhibited by adding NDPEX14 (30 μM, final concentration) and the reactions were further incubated at 37 °C. Aliquots from each reaction were taken 0 and 15 min after adding NDPEX14. Pronase-treated organelles were analyzed as above. Lane I, 2.5% of the reticulocyte lysates containing radiolabeled PEX7 and His-tagged PEX7 were mixed and loaded together in the same lane. The bar graph shows averages and standard deviations (n = 4) of the amounts of PEX7 and His-tagged PEX7 exported in 15 min. Numbers to the left indicate the molecular masses of the protein standards in kDa.

Figure 2. Proteolytic cleavage of the PTS2 peptide is not necessary for the cargo-release step.

(a) PrePHYH and an non-cleavable form of it, prePHYH(Δ29-30), accumulate in peroxisomes at similar rates. A rat liver PNS was incubated with either radiolabeled prePHYH or prePHYH(Δ29-30) in import buffer containing ATP, recombinant ΔC1PEX5L and “cold” in vitro synthesized PEX7 at 37 °C. Aliquots were taken at the indicated time points. Pronase-treated organelles were analyzed by SDS-PAGE and blotted onto a nitrocellulose membrane. Lanes I₁ and I₂, 10% of the reticulocyte lysates containing radiolabeled prePHYH or prePHYH(Δ29-30) used in each reaction, respectively. Autoradiographs (upper panels) and corresponding Ponceau S-stained membranes (lower panels) are shown. “pre” and “mat” refer to the precursor and mature forms of PHYH. Numbers to the left indicate the molecular masses of the protein standards in kDa. The graph shows averages and standard deviations (n = 3) of the percentage of imported proteins at different time points. (b) PEX7 proteins that transported prePHYH or prePHYH(Δ29-30) to the peroxisome display similar export kinetics. Radiolabeled PEX7 was incubated with a rat liver PNS in import buffer containing ATP, recombinant ΔC1PEX5L and either recombinant prePHYH or prePHYH(Δ29-30) at 37 °C for 15 min. The organelles were then isolated by centrifugation, resuspended in fresh import buffer and further incubated at 37 °C in the presence of ATP to promote export of receptors. Aliquots were collected at the indicated time points. Pronase-treated organelles were analyzed as above. Lanes I, 5% of the reticulocyte lysate containing radiolabeled PEX7 used in each reaction. An autoradiograph (upper panel) and the corresponding Ponceau S-stained membrane (lower panel) are shown. Numbers to the left indicate the molecular masses of the protein standards in kDa. The graph shows averages and standard deviations (n = 3) of the percentage of exported PEX7 at different time points.

Figure 3. PEX5 dramatically increases the half-life/affinity of the PEX7-PTS2 interaction.

(a) PrePHYH runs in native-PAGE only when mixed with ΔC1PEX5L and PEX7. Radiolabeled prePHYH was pre-incubated for 10 min at 23 °C in the absence or presence of in vitro synthesized “cold” PEX7, recombinant ΔC1PEX5L, or both, as indicated. Samples were analyzed by native-PAGE/autoradiography. (b) The residence time (half-life) of prePHYH in the trimeric ΔC1PEX5L-PEX7-prePHYH complex is very long. Radiolabeled prePHYH and “cold” PEX7 were incubated together for 10 min at 23 °C. Recombinant ΔC1PEX5L was then added and the mixture was incubated for 1 min to allow formation of the trimeric complex. The mixture was divided in two samples (tube A and B, respectively), and aliquots (t = 0′) were withdrawn and immediately flash-frozen in liquid N₂. Recombinant prePHYH and mature PHYH (matPHYH) were then added to tubes A and B, respectively. Both tubes were incubated at 23 °C and aliquots were collected and frozen at the indicated time points. Samples were analyzed by native-PAGE (upper panel) and SDS-PAGE (middle panel). Autoradiographs are shown. The graph shows the logarithm of band intensities as function of time (averages and standard deviations are presented, n = 4; half-life = 3.06 h (95% confidence range of 2.23 h–4.90 h)). (c) The half-life/affinity of the PEX7-PTS2 interaction is very low. Radiolabeled prePHYH was incubated with PEX7 as above. The mixture was divided in two samples (tube A and B, respectively). One aliquot (t = 0′) was withdrawn from tube A, incubated with recombinant ΔC1PEX5L for 1 min and flash-frozen. Recombinant prePHYH was then added to tube A and aliquots were removed at the indicated time points, incubated with recombinant ΔC1PEX5L for 1 min and frozen as above. Tube B received recombinant ΔC1PEX5L and an aliquot (t = 0′) was removed after 1 minute and flash-frozen. Recombinant prePHYH was then added and aliquots were withdrawn and flash-frozen at the indicated time points. Samples were analyzed as above. The open (▷) and solid (▶) arrowheads indicate the wells of the native gel, and the trimeric complex containing ³⁵S-prePHYH, respectively.

Figure 4. Working model for the PTS2-mediated protein import.

PTS2-containing proteins are recognized by the cytosolic receptors PEX5 and PEX7 forming a highly stable trimeric complex. The cargo-receptor complex then interacts with the docking/translocation machinery (DTM) at the peroxisome membrane. The strong protein-protein interactions that occur at this stage result in the insertion of the cargo-receptor complex into the DTM. At this stage PEX5 displays a transmembrane topology, whereas PEX7 exposes a part of its polypeptide chain into the peroxisomal matrix. The insertion step also induces conformational alterations in PEX5, disrupting its strong stabilizing effect on the PEX7-PTS2 interaction, and thus triggering the release of the PTS2 protein into the peroxisomal matrix. Here, the PTS2-containing peptide is cleaved by TYSND1. After cargo release, the receptors are recycled back into the cytosol by an ATP-dependent machinery.