A cargo-centered perspective on the PEX5 receptor-mediated peroxisomal protein import pathway

Abstract

Peroxisomal matrix proteins are synthesized on cytosolic ribosomes and post-translationally targeted to the organelle by PEX5, the peroxisomal shuttling receptor. The pathway followed by PEX5 during this process is known with reasonable detail. After recognizing cargo proteins in the cytosol, the receptor interacts with the peroxisomal docking/translocation machinery, where it gets inserted; PEX5 is then monoubiquitinated, extracted back to the cytosol and, finally, deubiquitinated. However, despite this information, the exact step of this pathway where cargo proteins are translocated across the organelle membrane is still ill-defined. In this work, we used an in vitro import system to characterize the translocation mechanism of a matrix protein possessing a type 1 targeting signal. Our results suggest that translocation of proteins across the organelle membrane occurs downstream of a reversible docking step and upstream of the first cytosolic ATP-dependent step (i.e. before ubiquitination of PEX5), concomitantly with the insertion of the receptor into the docking/translocation machinery.

Keywords: In Vitro Import Assays; PEX5; PTS1 Protein; Peroxisomes; Protein Sorting; Protein Translocation; Subcellular Organelles; Ubiquitination.

FIGURE 1.

[³⁵S]SCPx is specifically imported into peroxisomes in vitro. A, preincubation of [³⁵S]SCPx with recombinant PEX5 improves its in vitro import efficiency. Two chemically identical import reactions were assembled, differing solely in the step of the protocol where recombinant PEX5 was added. In one reaction (lane 3), [³⁵S]SCPx was preincubated in the absence of PEX5 and added to a PNS in ATP-containing import buffer. After the addition of recombinant PEX5, the reaction was incubated for 15 min at 37 °C. In the other reaction (lane 4), [³⁵S]SCPx was preincubated with recombinant PEX5, added to a PNS in the same buffer, and incubated under the same conditions. Proteinase K-treated organelles were then subjected to SDS-PAGE/Western blotting/autoradiography. Lanes 1 and 2 contain 5% of the preincubated [³⁵S]SCPx proteins used in the assays shown in lanes 3 and 4, respectively. The autoradiograph (upper panel) and the Ponceau S-stained membrane (lower panel) are shown. B, in vitro synthesized [³⁵S]SCPx interacts with PEX5. [³⁵S]SCPx was preincubated for 30 min at room temperature in the absence or presence of 1 μm recombinant PEX5, as indicated. After adding a mixture of protein standards, the samples were subjected to sucrose gradient centrifugation. After fractionation, equivalent aliquots were subjected to SDS-PAGE/Western blotting. Note that the sedimentation coefficient of [³⁵S]SCPx increases in the presence of PEX5, indicating that the two proteins interact. Autoradiographs are shown. Protein standards used were: ovalbumin (Ova; 45 kDa), bovine serum albumin (BSA; 66 kDa), and aldolase (Ald; 140 kDa). C, organelles from an in vitro import reaction were resuspended in import buffer and treated with proteinase K (PK) in the absence (lane 2) or presence of Triton X-100 (TX-100; lane 3). Samples were analyzed as in A. The behaviors of endogenous SCPx and catalase (Cat) are shown. D, [³⁵S]SCPx preincubated with recombinant PEX5 was subjected to a standard import assay. Aliquots of the reaction were withdrawn at the indicated time points, treated with proteinase K, and processed for SDS-PAGE/autoradiography (upper panel). The Ponceau S-stained membrane is also shown (lower panel). E, [³⁵S]SCPx was preincubated in the absence (−) or presence (+) of the indicated recombinant proteins. Protein mixtures were then subjected to standard import assays and analyzed as described above. Note that PEX19 is involved in a different aspect of peroxisomal biogenesis (59) and was used here just as a negative control. F, a protease-treated import reaction was subjected to Nycodenz gradient centrifugation. The behaviors of [³⁵S]SCPx, endogenous SCPx, catalase (Cat), cytochrome c (Cyt c; a mitochondrial marker), and two endoplasmic reticulum proteins (KDEL; recognizes GRP72 and GRP98) are shown. The fraction of catalase detected at the top of the gradient represents mostly catalase that has leaked from peroxisomes during PNS preparation. Unlike soluble SCPx, soluble mouse catalase is quite resistant to proteinase K (see C). Lanes 1 in C, D, and E, 5% of the [³⁵S]SCPx protein used in each reaction. Numbers to the left indicate the molecular masses of protein standards in kDa.

FIGURE 2.

Import of a PTS1 protein into peroxisomes does not require cytosolic ATP. A, [³⁵S]SCPx was preincubated in the presence of either recombinant PEX5 (lanes 1 and 2) or PEX5(C11A) (lane 3). An aliquot of the PEX5-containing [³⁵S]SCPx was further treated with apyrase (lane 2). These samples were then subjected to import assays, as follows: lane 4, assay containing ATP and [³⁵S]SCPx preincubated with PEX5; lane 5, the same as in lane 4 but in the presence of AMP-PNP instead of ATP; lane 6, assay containing [³⁵S]SCPx preincubated with PEX5 and PNS, both pretreated with apyrase; lane 7, import assay containing ATP and [³⁵S]SCPx preincubated with PEX5(C11A); lane 8, the same as in lane 4 but also containing recombinant NDPEX14. Samples were processed as described in the legend for Fig. 1A. Lanes 1–3, 5% of the [³⁵S]SCPx samples used in the assays. B, ³⁵S-labeled PEX5(C11K) was subjected to in vitro import reactions containing ubiquitin aldehyde and either ATP (lanes 2 and 3) or AMP-PNP (lanes 4 and 5). After 7 min at 37 °C, the import reactions were centrifuged to obtain an organelle pellet (P) and a supernatant (S), and both fractions were analyzed by SDS-PAGE/Western blotting. Note that AMP-PNP allows PEX5(C11K) ubiquitination but not its export into the soluble phase of the import reaction (39). C, ³⁵S-labeled PEX5(C11K) was incubated in the absence (−) or presence (+) of apyrase (lanes 1 and 2, respectively). The first of these samples (minus apyrase) was subjected to an in vitro import assay in the presence of ATP (lane 3); the apyrase-treated ³⁵S-labeled PEX5(C11K) was subjected to an import assay using apyrase-treated PNS (lane 4). Both reactions also contained 15 μm GST-Ub. The organelles were then isolated by centrifugation and analyzed by SDS-PAGE/Western blotting/autoradiography. Note that GST-Ub is efficiently used by the machinery that monoubiquitinates PEX5 but, in contrast to ubiquitin, results in a PEX5 species that is not exported from the DTM (32, 38). Also, monoubiquitinated PEX5(C11K) is more stable than monoubiquitinated PEX5 upon SDS-PAGE. This property increases the sensitivity of the ubiquitination assays (38). D, [³⁵S]DECR2 was subjected to import assays exactly as described in A for [³⁵S]SCPx. The asterisk indicates an unspecific radiolabeled band produced during the in vitro translation reaction. Lane 1 in B and lanes 1 and 2 in C, 5% of the ³⁵S-labeled PEX5(C11K) used in the assays.

FIGURE 3.

In vitro imported [³⁵S]SCPx behaves as endogenous SCPx upon fractionation of peroxisomes. [³⁵S]SCPx preincubated with the indicated PEX5 proteins was subjected to in vitro import assays under different energetic conditions, as specified. At the end of the import reaction, the organelles were treated with proteinase K, isolated by centrifugation, and sonicated. One-half of these samples was kept on ice (lanes T), whereas the other half was ultracentrifuged to obtain membrane (P) and soluble (S) fractions. Samples were analyzed by SDS-PAGE/Western blotting/autoradiography. The behaviors of endogenous SCPx, catalase (Cat), PEX14, and cytochrome c (Cyt c) are also shown. Note that PEX14 is converted into small 16–18-kDa fragments (PEX14′) upon proteinase K treatment (see also Ref. 62). Lane 1, 5% of the [³⁵S]SCPx protein used in each reaction.

FIGURE 4.

Temperature dependence of the docking/insertion of PEX5 into the DTM and SCPx import. A, [³⁵S]SCPx preincubated with a mixture of recombinant and ³⁵S-labeled PEX5(C11A) was subjected to import assays at different temperatures. After 15 min, the samples were halved and treated (lanes +) or not (lanes −) with proteinase K (PK). The organelles were analyzed by SDS-PAGE/autoradiography (upper panel). The Ponceau S-stained membrane is also shown (lower panel). B, [³⁵S]SCPx preincubated as described above was subjected to import assays at 0 or 37 °C in the presence of either recombinant ΔC1PEX5 or PEX19 (5 μm each), as indicated. Protease-treated and untreated organelles were then analyzed as above. C, organelles from an import assay performed at 0 °C (lane 2) were resuspended in import buffer containing either recombinant ΔC1PEX5 or PEX19, incubated for 15 min, and reisolated by centrifugation. Organelle pellets (P) (lanes 3 and 5) and the corresponding supernatants (S) (lanes 4 and 6) were analyzed as above. Lanes 1 in A, B, and C, 5% of the radiolabeled proteins used in the assays.

FIGURE 5.

Working model for the PEX5-mediated protein import pathway. After binding a PTS1 protein in the cytosol, PEX5 docks at the DTM in a reversible manner. PEX5 then becomes inserted into the DTM, pushing the cargo protein across the organelle membrane. In contrast to PEX5 recycling, which includes monoubiquitination and PEX1/PEX6-catalyzed extraction of the receptor from the DTM, the docking and translocation steps do not require cytosolic ATP.