Skowyra ML , Rapoport TA .

R01 GM052586 NIGMS NIH HHS , Howard Hughes Medical Institute

	Graphical abstract.
	Figure 1. PEX5 recycling recapitulated in Xenopus egg extract (A) Xenopus eggs were lysed by centrifugation to generate an extract (the indicated layer between the lipid on top and cellular debris on bottom). The extract was treated with dimethylsulfoxide (DMSO) or the indicated E1 enzyme inhibitors dissolved in DMSO (UBA1, ubiquitin-activating enzyme; NAE, NEDD8-activating enzyme). Import activity was then assessed by incubating with the fluorescent cargo mScarlet-SKL and imaging on a spinning disk confocal microscope; repeated rounds of import into peroxisomes result in the appearance of bright puncta. The number of puncta in an imaged field was quantified relative to that in the untreated reaction (n = 9 fields per reaction; bars specify the median). (B) Upper scheme illustrates the strategy to deplete free ubiquitin from extract by inhibiting deubiquitinating enzymes (DUBs) with ubiquitin vinyl sulfone (UbVS); lower scheme shows the approach for restoring peroxisome import activity. On the right, extract was treated with buffer (mock) or UbVS and then supplemented either with buffer or wild-type ubiquitin, wild-type PEX5, or both components together. Import activity was then assessed as above (n = 16 fields per reaction). (C) As in (B), except that reactions were supplemented with the indicated components in the absence or presence of the E1 inhibitors. Import activity was assessed as above (n = 16 fields per reaction). (D) As in (C). Ubiquitin mutant (ΔGG) lacks both C-terminal glycines. Import activity was assessed as above (n = 16 fields per reaction). All scale bars, 5 μm. See also Figure S1.
	Figure 2. Sustained peroxisomal import requires monoubiquitination and the conserved cysteine in PEX5 (A) Xenopus egg extract was treated with UbVS and then supplemented either with buffer or PEX5 together with wild-type ubiquitin (Ub), methylated wild-type ubiquitin (meUb), or ubiquitin lacking all lysines (Ub ΔK). Import activity was assessed by incubating with mScarlet-SKL and imaging the formation of bright puncta on a spinning disk confocal microscope. The number of puncta in an imaged field was quantified relative to that in the mock reaction (n = 16 fields per reaction; bars specify the median). (B) As in (A), except that reactions were supplemented with ubiquitin together with wild-type PEX5 or PEX5 mutants in which Cys11 was converted to Ala (C11A) or Lys (C11K). Import activity was assessed as above (n = 16 fields per reaction). (C) Extract was depleted of endogenous PEX5 using beads conjugated to PEX5 antibodies and then supplemented either with buffer or wild-type PEX5, a PEX5 mutant lacking all lysines (ΔK), or PEX5 ΔK in which Cys11 was mutated to Lys (ΔK + C11K). Reactions 5 and 7 were performed with methylated versions of the indicated mutants. Import activity was assessed as above (n = 16 fields per reaction). All scale bars, 5 μm. See also Figure S2.
	Figure 3. Signals targeting PEX5 to peroxisomes (A) Diagram on top shows the locations of predicted α-helices (boxes) and key residues (numbered relative to the long isoform, see STAR Methods) in the N-terminal unstructured region of X. laevis PEX5 (AH1–AH4, amphipathic helices; W0–W7, WxxxF/Y motifs). The PEX7-binding region is enclosed by a gray dashed line. Helical wheels beneath the diagram illustrate the distribution of hydrophobic amino acids in each amphipathic helix. Gray arrows specify the N terminus of each helix; black dots denote residues that were mutated to alanines. (B) Sequence alignments of AH1 and AH2 in PEX5 homologs from the indicated organisms. Residue numbers refer to X. laevis PEX5 (long isoform); dashed line specifies the region shown in the helical wheels (see Figure S4A for alignments of AH3 and AH4). (C) Xenopus egg extract was depleted of endogenous PEX5 using beads conjugated to the PEX5-binding domain from PEX14 and then supplemented with wild-type PEX5 (WT) or the indicated mutants (C11A; ΔW, the specified WxxxF/Y motifs mutated to AxxxA; ΔAH1-ΔAH4, mutations in the hydrophobic face of each amphipathic helix as described above). Import activity was assessed by incubating with GFP-SKL and imaging the formation of bright puncta by spinning disk confocal microscopy. The number of puncta in an imaged field was quantified relative to that in the reaction with wild-type PEX5 (n = 16 fields per reaction; bars specify the median). Scale bars, 5 μm. (D) Quantification of experiments shown in (C). (E) Extract was incubated with the indicated PEX5 mutants and GFP-SKL, and peroxisomes were then isolated by flotation. Total extract (0.01%) and the peroxisome-containing fraction (2%) were immunoblotted for PEX5 and PEX14. Molecular weights (kD) are marked on the left. Amounts of PEX5 and PEX14 in the peroxisome fraction were quantified by densitometry (n = 3 independent experiments; ∗p ≤ 0.01 by Student's unpaired two-tailed t test). See also Figures S3–S7.
	Figure 4. PEX5 transits through the peroxisomal lumen (A) Protease protection analysis of X. laevis PEX14. Peroxisomes were isolated from Xenopus egg extract by flotation and treated with different concentrations of proteinase K, with or without Triton X-100 (TX). Diagram on the left depicts the orientation of PEX14 in the peroxisomal membrane, along with the predicted molecular weights of the indicated regions. Green dashed lines designate segments used to raise polyclonal antibodies, which were then used on the right to immunoblot the protease-treated reactions. Magenta arrow indicates the expected protease-protected fragment. (B) Extract was incubated with wild-type PEX5 or a mutant lacking the conserved cysteine (C11A) in the presence of GFP-SKL. The peroxisome-associated population was then isolated by flotation and treated with different concentrations of proteinase K, with or without Triton X-100 (TX), and immunoblotted for PEX5. Molecular weights (kD) are marked on the left. See also Figure S7C. (C) As in (B), except using PEX5 (C11A) mutants fused to a triple FLAG (3 × FLAG) tag at either terminus. (D) Scheme depicting the constructs containing cleavable PTS2 signals, which were used to assess whether PEX5 enters the lumen during import. Scissors indicate the site of cleavage by the peroxisomal lumenal protease TYSND1; PTS2∗ denotes an inactivated PTS2 motif. (E) Extract was incubated with GFP-SKL and PEX5 (C11A) fusions containing an N-terminal TYSND1 cleavage site from the matrix enzymes AGPS or PHYH (construct N in (D)). Note that the fusions were added at 20-fold excess over endogenous PEX5. Peroxisomes were then isolated by flotation, and total extract (0.01%) and the peroxisome-containing fraction (2%) were immunoblotted for PEX5. Scissors denote constructs with an intact TYSND1-cleavage site; ∅ denotes an inactivated site. Molecular weights (kD) are marked on the left. (F) As in (E), except using only the construct with the N-terminal TYSND1-cleavage site from PHYH. Peroxisomes isolated by flotation were treated with different concentrations of proteinase K, with or without Triton X-100 (detergent), and immunoblotted for PEX5. (G) As in (E), except using PEX5 (C11A) containing the TYSND1-cleavage site from PHYH at the indicated positions, as illustrated in (D). Arrows designate TYSND1-cleaved forms of each fusion protein. (H) Extract was depleted of endogenous PEX5 using beads conjugated to the PEX5-binding domain from PEX14 and then supplemented with a low concentration of the indicated TYSND1-cleavable constructs or the corresponding non-cleavable versions. Accumulation of the TYSND1-cleaved form was followed over time in total extract by immunoblotting. (I) As in (H), except that reactions were incubated in the presence or absence of a recombinant fragment corresponding to the PEX5-binding domain from PEX14, which inhibits import and retains PEX5 in the cytosol. See also Figure S7 and Table S1.
	Figure 5. A signal for PEX5 export (A) Xenopus egg extract was incubated with GFP-SKL and PEX5 lacking the conserved cysteine (C11A) or with mutants in which the residues along the hydrophobic face of AH1 or AH2 were mutated to alanine. The peroxisome-associated population was isolated by flotation and treated with different concentrations of proteinase K, with or without Triton X-100 (TX), and immunoblotted for PEX5. Molecular weights (kD) are marked on the left. (B) As in (A), except that the mutants were fused at the N terminus to a triple FLAG (3 × FLAG) tag. (C) As in (A), except that the constructs depicted in the scheme on the left were used: 3 × FLAG-tagged PEX5; 3 × FLAG-tagged PEX5 with an unstructured non-cleavable signal from PHYH; PEX5 fused to ubiquitin (Ub) via a non-hydrolyzable linker; and PEX5 fused to a non-hydrolyzable version of the ubiquitin-like protein SUMO. Blue arrows indicate protease-resistant fragments. (D) Extract was incubated with GFP-SKL and the indicated PEX5 variants. Peroxisomes were isolated by flotation and solubilized in digitonin, and the PEX5-associated population was isolated with anti-FLAG beads. Bound proteins were identified by mass spectrometry. The total number of peptides corresponding to the indicated peroxins recovered with each construct is shown on the right, from two independent replicates. See also Figure S7 and Table S2.
	Figure 6. Unfolding of PEX5 during export (A) Scheme depicting the constructs used to evaluate whether PEX5 is unfolded during its export from peroxisomes. NB denotes a nanobody against GFP that was fused either to the N-terminal unstructured region of PEX5 (ΔTPR + NB) or to the full-length protein (PEX5 + NB). Pink bars designate WxxxF/Y motifs; blue bars indicate amphipathic helices AH1 and AH2. (B) Xenopus egg extract was depleted of endogenous PEX5 using beads conjugated to the PEX5-binding domain from PEX14 and then supplemented either with wild-type PEX5, PEX5 lacking the conserved cysteine (C11A), or PEX5 (ΔTPR + NB) with or without the C11A mutation. Import activity was assessed by incubating with GFP-SKL or GFP and imaging the formation of bright puncta by spinning disk confocal microscopy. (C) As in (B), except that the PEX5 + NB construct was used, with or without the C11A mutation. (D) The number of puncta in an imaged field, for the numbered reactions in (B) and (C), was quantified relative to that in the reaction with GFP-SKL and wild-type PEX5 (n = 24 fields per reaction; bars specify the median). All scale bars, 5 μm.
	Figure 7. Model of PEX5 shuttling Step 1: PTS1 cargo binds to the TPR domain of PEX5 in the cytosol. Step 2: cargo-bound PEX5 is recruited to the docking complex in the peroxisomal membrane, composed of PEX13 and PEX14, using WxxxF/Y pentapeptide motifs (pink). Only the first 5 motifs are shown. Step 3: PEX5 translocates into the lumen along with cargo. Diffusion back into the cytosol is prevented by the high-affinity interaction between the pentapeptide motifs and the lumenal domain of PEX14 (crimson square). Step 4: export of PEX5 is initiated by binding to the ligase complex through an amphipathic helix (blue). The unstructured N terminus then inserts into the ligase pore and emerges in the cytosol. Step 5: the conserved cysteine in PEX5 is monoubiquitinated. Step 6: PEX5 is pulled out of the lumen through the ligase pore by the PEX1/PEX6 AAA ATPase. Extraction is accompanied by unfolding of the TPR domain and release of bound cargo. Step 7: the TPR domain refolds in the cytosol and ubiquitin is removed by deubiquitinating enzymes, resetting PEX5 for a subsequent import cycle.
	Supplemental Figure S1. Restoration of peroxisomal protein import in DUB-inhibited extract by ubiquitin and PEX5 (related to Figure 1) (A) Xenopus egg extract was treated with buffer (mock) or ubiquitin vinyl sulfone (UbVS), and the UbVS-treated reactions were then supplemented with buffer or wild type ubiquitin at the indicated concentrations. Import activity was assessed by incubating the reactions with mCherry-SKL and imaging the formation of bright puncta on a spinning disk confocal microscope. The number of puncta in an imaged field was quantified relative to that in the mock reaction (n = 17 fields per reaction; bars specify the median). (B) Extract was incubated with mCherry-SKL in the presence of buffer (mock) or 100 μM wild type ubiquitin, and import activity was quantified as above (n = 17 fields per reaction). (C) As in (A), except that extract was pre-treated with the DUB inhibitors ubiquitin aldehyde (Ubal) or PR-619, a small molecule, then supplemented with buffer or ubiquitin together with PEX5. Import activity was quantified as above (n = 17 fields per reaction). (D) Coomassie-stained gels of select purified recombinant PEX5 proteins used in this study. Lanes 1-7 correspond to proteins in Figs. 1 and 2; lanes 8-17 to Fig. 3C-E; lanes 18-19 to Fig. 4C; lanes 20-21 to Fig. 5B; lanes 22-24 to Fig. 5D; lanes 25-35 to Fig. 4E-I and supplemental fig. S7D-E; lanes 36-39 to Fig. 6; and lanes 40-41 to Fig. 5C. See also supplemental table S3. All scale bars equal 5 μm.
	Supplemental Figure S2. Ubiquitin surface residues required for peroxisomal protein import (related to Figure 2) (A) Structural model of ubiquitin (PDB: 1UBQ), depicting key surface residues that were mutated to evaluate their role in peroxisomal import. Mutants lacking the underlined residues did not support import. On the right, the ubiquitin mutants were used in peroxisomal import assays. Xenopus egg extract was treated with buffer (mock) or ubiquitin vinyl sulfone (UbVS), and the UbVS-treated reactions were then supplemented with buffer or wild type PEX5 together with the indicated Ub mutants. Import activity was assessed by incubating the reactions with mCherry-SKL and imaging the formation of bright puncta on a spinning disk confocal microscope. The number of puncta in an imaged field was quantified relative to that in the mock reaction (n = 16 fields per reaction; bars specify the median). Scale bar equals 5 μm. (B) Purified mouse UBA1 (E1), X. laevis UBCH5C (E2), and wild type human Ub (WT) or the indicated Ub mutants, were incubated in the presence of ATP and Mg2+ for the times shown. Reactions were quenched in SDS-sample buffer with or without the reducing agent b- mercaptoethanol (BME). E1~Ub and E2~Ub denote thioester-linked adducts between Ub and either the E1 or E2, respectively.
	Supplemental Figure S3. Role of Pex5's conserved asparagine (N15) and amphipathic helices AH3 and AH4 in peroxisomal protein import (related to Figure 3) (A) Diagram of the N-terminal unstructured region of X. laevis PEX5, showing the locations of predicted α-helices (boxes) and key residues (numbered relative to the long isoform, see Methods). Amphipathic helices are labeled AH1-AH4; WxxxF/Y pentapeptide motifs are labeled W0-W7. The PEX7-binding region is enclosed by a gray dashed line. Sequence alignments underneath correspond to the region between the conserved cysteine and the C- terminal end of the first amphipathic helix (AH1) in PEX5 homologs from the indicated organisms. (B) Xenopus egg extract was depleted of endogenous PEX5 using beads conjugated to the PEX5-binding domain from PEX14, then supplemented either with buffer, wild type PEX5 (WT), or a PEX5 mutant in which Asn15 was converted to alanine (N15A). Import activity was assessed by incubating the reactions with mCherry-SKL and imaging the formation of bright puncta on a spinning disk confocal microscope. The number of puncta in an imaged field was quantified relative to that in the reaction with wild type PEX5 (n = 8 fields per reaction; bars specify the median). (C) As in (B). C11A refers to a PEX5 mutant in which Cys11 was mutated to Ala; ∆W (all) refers to a mutant in which all WxxxF/Y motifs were mutated to AxxxA; ∆204-297 lacks the indicated residues (shown in the diagram on top, numbered relative to the long isoform); and AH3 + ∆204-297 additionally has the residues along the hydrophobic face of AH3 mutated to alanines. In the quantitation on the right, n = 16 fields per reaction. All scale bars equal 5 μm.
	Supplemental Figure S4. Conservation of amphipathic helices AH2-AH4 in the N- terminal unstructured region of PEX5 (related to Figure 3) (A) Diagram on top shows the locations of predicted α-helices (boxes) and key residues (numbered relative to the long isoform, see Methods) in the N-terminal unstructured region of X. laevis PEX5. Amphipathic helices are labeled AH1-AH4; WxxxF/Y pentapeptide motifs are labeled W0-W7. The PEX7-binding region is enclosed by a gray dashed line. Helical wheel diagrams illustrate the distribution of hydrophobic amino acids along AH3 and AH4 (gray arrows specify the N-terminus of each helix; black dots denote residues that were mutated to alanines). Underneath are sequence alignments of AH3 and AH4 in PEX5 homologs from the indicated organisms. Residue numbers refer to X. laevis PEX5 (long isoform); dashed line specifies the region shown in the helical wheels. (B) As in (A), except that the N-terminal unstructured regions of PEX5 from the indicated organisms are shown. Amphipathic helices that might fulfill a similar role to AH2 in animals are labeled with an asterisk (*). Helical wheel diagrams illustrate the distribution of hydrophobic residues along each putative AH2 helix in PEX5 from the above organisms. The conservation of the hydrophobic residues is shown in the sequence alignments underneath. Dashed line indicates the regions used to construct the helical wheel diagrams; residue numbers refer to PEX5 from the organism in bold type.
	Supplemental Figure S5. Import activity of PEX5 splice variants and mutants lacking individual WxxxF/Y motifs (related to Figure 3) (A) Diagram on top shows the locations of predicted α-helices (boxes) and key residues (numbered relative to the long isoform, see Methods) in the N-terminal unstructured region of X. laevis PEX5. Amphipathic helices are labeled AH1-AH4; WxxxF/Y pentapeptide motifs are labeled W0-W7. The PEX7-binding region is enclosed by a gray dashed line. Underneath, Xenopus egg extract was depleted of endogenous PEX5 using beads conjugated to the PEX5- binding domain from PEX14, then supplemented either with buffer, or with the long or short splice variants of PEX5. Import activity was assessed by incubating the reactions with GFP- SKL and imaging the formation of bright puncta on a spinning disk confocal microscope. (B) As in (A), except using the short PEX5 isoform (WT) or the short isoform containing the following mutations: Cys11 converted to alanine (C11A); all WxxxF/Y motifs mutated to AxxxA (∆W all); or individual WxxxF/Y motifs mutated to AxxxA (∆W0 through ∆W7). The number of puncta in an imaged field was quantified relative to that in the reaction with wild type PEX5 (n = 7, 8, 13, 9, 18, 18, 18, 18, 17, 17, 19 fields per reaction, numbered in the order shown; bars specify the median). All scale bars equal 5 μm.
	Supplemental Figure S6. Import activity of PEX5 mutants containing different WxxxF/Y motifs (related to Figure 3) (A) Diagram on top shows the locations of predicted α-helices (boxes) and key residues (numbered relative to the long isoform, see Methods) in the N-terminal unstructured region of X. laevis PEX5. Amphipathic helices are labeled AH1-AH4; WxxxF/Y pentapeptide motifs are labeled W0-W7. The PEX7-binding region is enclosed by a gray dashed line. Underneath, Xenopus egg extract was depleted of endogenous PEX5 using beads conjugated to the PEX5- binding domain from PEX14, then supplemented either with buffer, wild type PEX5 (WT), a PEX5 mutant in which all WxxxF/Y motifs were converted to AxxxA (∆W all), or PEX5 mutants containing only individual WxxxF/Y motifs. Import activity was assessed by incubating the reactions with GFP-SKL and imaging the formation of bright puncta on a spinning disk confocal microscope. The number of puncta in an imaged field was quantified relative to that in the reaction with wild type PEX5 (n = 17 fields per reaction; bars specify the median). (B) As in (A). C11A denotes a PEX5 mutant in which the conserved cysteine was mutated to alanine. Reactions in the bottom row were supplemented with PEX5 mutants containing only the indicated pairs of WxxxF/Y motifs. In the quantitation on the right, n = 17 fields per reaction. All scale bars equal 5 μm.
	Supplemental Figure S7. Validation of peroxisome isolation procedure and import activity of TYSND1-cleavable forms of PEX5 as well as FLAG-tagged PEX5 (related to Figures 3, 4, and 5) (A) Scheme on top depicts the procedure for isolating peroxisomes from Xenopus egg extract. Peroxisome identification was facilitated by pre-incubating extract with mScarlet-SKL. Reactions were then diluted into a dense sucrose buffer and loaded on the bottom of a discontinuous sucrose gradient. Following centrifugation, peroxisomes accumulate on top of the gradient. On the right, fractions harvested from the top of the gradient were imaged by spinning disk confocal microscopy to confirm the separation of fluorescently-labeled peroxisomes (which appear as bright puncta) from cytoplasmic material containing unimported cargo (which appears diffuse). The starting material (input) is boxed in red. On the bottom left, the same fractions were immunoblotted for the indicated peroxins. Arrow indicates the peroxisome fraction. (B) Extract was incubated with buffer (mock) or 1 μM recombinant wild type PEX5 in the presence of the endogenous receptor and GFP-SKL, and formation of puncta was imaged on a spinning disk confocal microscope. The number of puncta in an imaged field was quantified relative to that in the mock reaction (n = 17 fields per reaction; bars specify the median). (C) Quantitation of PEX5 density in lanes 3 and 6 of Fig. 4B, normalized to the density of PEX14 (n = 3 independent experiments; *, p ≤ 0.01 by Student's unpaired two-tailed t-test). (D) As in Fig. 4G, except using wild type PEX5 (with the conserved cysteine intact) containing the TYSND1-cleavage site from PHYH at the indicated positions, as illustrated in Fig. 4D. (E) Extract was depleted of endogenous PEX5 using beads conjugated to the PEX5-binding domain from PEX14, and supplemented with wild type PEX5, PEX5 (C11A), or wild type PEX5 containing the PHYH TYSND1-cleavage site at the indicated positions (as illustrated in Fig. 4D). Import activity was assessed and quantified as in (B), relative to the reaction with wild type PEX5 (n = 24, 24, 24, 24, 8, and 9 fields per reaction, in the order shown; bars specify the median; *, p ≤ 0.0001 by Student's unpaired two-tailed t-test). (F) As in (E), except that the PEX5-depleted extract was supplemented either with buffer, wild type PEX5, or PEX5 fused at the C-terminus to a single FLAG tag. All scale bars equal 5 μm.

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