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First published online June 8, 2006
doi: 10.1242/10.1242/jcs.02979

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Targeting of the tail-anchored peroxisomal membrane proteins PEX26 and PEX15 occurs through C-terminal PEX19-binding sites

¹ Institut für Physiologische Chemie, Abt. Systembiochemie, Ruhr-Universität Bochum, 44780 Bochum, Germany
² Institut für Medizinische Immunologie, Universitätsklinikum Charité, 10115 Berlin, Germany
³ Institut für Biochemie, Universitätsklinikum Charité, 10115 Berlin, Germany

View larger version (18K): [in a new window]   Fig. 1. Prediction and identification of PEX19-binding sites in PEX26. (A) Binding site prediction. The prediction matrix for PEX19-binding sites (Rottensteiner et al., 2004) was applied to human PEX26 and the hits obtained are presented as dotted vertical lines. Two sites with a high probability score are clearly visible. The positions of the high-scoring peptides within these sites as well as the amino acid sequence of each peak scoring peptide are indicated. (B) Peptide scan for PEX26. 15-mer peptides with two amino acid shifts between neighboring peptides and covering the entire PEX26 protein were synthesized on a nitrocellulose membrane and tested for interaction with GST-HsPEX19. Bound PEX19 was visualized by monoclonal anti-GST antibodies in combination with the ECLTM reaction system. Arrows mark the peak interacting peptide within each binding site, the sequences of which are indicated. (C) Relative PEX19-binding affinity for the two binding sites in PEX26. Nine peptides per binding site were synthesized and assayed for PEX19 interaction as described in B. The sequences of the strongly interacting peptides are indicated. The right panel shows the relative intensities of the signals obtained for each spot. (D) Graphic representation of PEX26. Denoted are the positions of the PEX19-binding sites as determined in vitro and the postulated TMD (Matsumoto et al., 2003a).

View larger version (59K): [in a new window]   Fig. 2. Interaction of PEX26 with PEX19 in a yeast two-hybrid assay. The indicated PEX26 fragments were fused to the Gal4p binding domain (GAL4-BD) and expressed in combination with a HsPEX19 Gal4p activation domain fusion (GAL4-AD; left panel) or the Gal4p activation domain alone (right panel) in the yeast two-hybrid strain PJ69-4a. In this assay, protein-protein interactions were assessed by the ability of the double transformants to grow on histidine-adenine double-dropout medium plates. Two independent transformants are shown for each tested interaction.

View larger version (55K): [in a new window]   Fig. 3. Targeting of PEX26 is dependent on its PEX19-binding site. (A) Identification of an mPTS in PEX26. Human fibroblasts were transiently transfected with plasmids designed to express GFP fusions of the indicated PEX26 fragments. Thereafter, cells were processed for indirect immunofluorescence using polyclonal anti-PEX14 antibodies. Merged images reveal eventual colocalization of the GFP fusion proteins with peroxisomal PEX14 (Px). (B) The C-terminal PEX19-binding site of PEX26 functions as a PMP-targeting motif. A GFP fusion of a chimera of PEX26275-305 and ALDP87-164 was similarly analyzed for subcellular location. In this chimera, the targeting motif is contributed by the PEX26 fragment, whereas the transmembrane segment that is required for efficient membrane insertion is contributed by the ALDP fragment. GFP fusions of the individual fragments (PEX26275-305 and ALDP87-164) served as controls. (C) Function of the TMD-containing PEX19-binding site of PEX26 as a PMP targeting signal. A GFP fusion of PEX26245-274 was inspected for colocalization with PEX14. This transmembrane segment with PEX19 binding capability suffices for peroxisomal targeting. Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 4. The peroxisomally targeted PEX26 fragments are inserted into the peroxisomal membrane. Human skin fibroblasts expressing GFP fusions of PEX262-305, PEX26245-305, or PEX26275-305-ALDP87-164 were lysed in hypotonic buffer and membranes were separated from soluble proteins (SHyp) by centrifugation at 100,000 g. Membranes were then extracted with alkaline sodium carbonate buffer and again subjected to centrifugation to obtain a soluble fraction (SCarb) and a membrane pellet fraction (PCarb). Sample volumes were adjusted to that of the whole lysate (Lys) and each fraction was analysed by immunoblotting using antibodies against GFP, the integral PMP PEX14, and the soluble peroxisomal matrix enzyme catalase.

View larger version (81K): [in a new window]   Fig. 5. Targeting of PEX26 fragments to nonperoxisomal compartments. (A) Staining of mitochondria (Mc). Transiently transfected human fibroblasts were processed for indirect immunofluorescence using anti-TRAP1 antibodies to visualize mitochondria. The achieved staining pattern was compared with that caused by the GFP fluorescence of the indicated PEX26 fusion proteins. GFP-PEX262-274 and also some PEX26245-305 showed a congruent staining pattern with the mitochondrial marker protein. (B) Staining of the ER. The same kind of transfected cells were labeled with an anti-calreticulin antibody and were similarly analyzed for colocalization with the ER. The GFP-PEX26275-305 fusion protein revealed some overlap with the ER marker. Bar, 10 µm.

View larger version (37K): [in a new window]   Fig. 6. PEX19 is required for the targeting of PEX26. (A) PEX26 localizes to mitochondria in PEX19-deficient cells. A GFP-PEX262-305-expressing plasmid was transfected into PEX19-deficient human fibroblasts and the localization of the GFP fusion protein was determined. Mitochondria were labelled by indirect immunofluorescence using anti-TRAP1 antibodies (Mc). (B) Expression of PEX19 is downregulated in human fibroblasts treated with PEX19 siRNA. Wild-type fibroblasts were transfected with PEX19-specific siRNA or with PEX5 control siRNA and incubated for 5 days at 37°C. Samples were taken every 24 hours and whole-cell lysates were prepared. Equal amounts of protein were analyzed by immunoblotting using antibodies against PEX19 and PEX14. (C,D) Inhibition of PEX19 affects targeting of PEX26. After 60 hours, PEX19 siRNA-treated cells were transfected with a GFP-PEX262-305-expressing plasmid. 24 hours later, cells were analyzed for the distribution of the fusion protein. Peroxisomes (Px) and mitochondria (Mc) were visualized by indirect immunofluorescence using anti-PEX14 (C) and anti-TRAP1 (D) antibodies, respectively. Bar, 10 µm.

View larger version (32K): [in a new window]   Fig. 7. Prediction and determination of PEX19-binding sites in S. cerevisiae Pex15p. (A) Graphic representation of the mPTS of Pex15p as proposed by Elgersma et al. (Elgersma et al., 1997). Shown are the TMD as well as the proposed peroxisomal (Px-TS) and ER (ER-TS) targeting signal sequences. (B) Location of predicted PEX19-binding sites in the mPTS of yeast Pex15p. The two PEX19-binding sites as predicted by our program (Rottensteiner et al., 2004) are denoted. (C) Yeast two-hybrid assay for the interaction of Pex15p with Pex19p. The indicated fragments of Pex15p were tested as GAL4-BD fusions in combination with yeast Pex19p fused to the GAL4-AD (left panel) or the GAL4-AD alone (right panel) in the yeast strain PJ69-4a. Two independent colonies of each transformation were assessed for growth on histidine-adenine double-dropout medium plates.

View larger version (64K): [in a new window]   Fig. 8. The role of PEX19-binding sites in targeting of ScPex15p. S. cerevisiae wild-type strain yHPR251 harboring an integrated copy of the synthetic peroxisomal marker PTS2-DsRed (Px) was used to express the following protein fragments fused to GFP: the C-terminal Pex15p315-383 fragment containing both PEX19-binding sites (A); Pex15p361-383, comprising the single C-terminal PEX19-binding site (B); the ALDP87-164 fragment (C); Pex15p361-383 appended to the ALDP87-164 fragment (D); and PEX26275-305-ALDP87-164, the C-terminal PEX19-binding site of PEX26 fused to the same ALDP fragment (E). The transformed strains were grown on ethanol-containing plates for 2 days and inspected for GFP- and DsRed-derived fluorescence. The merged images of the two acquisitions reveal eventual colocalization of peroxisomal PTS2-DsRed with the GFP fusion proteins. DIC, differential interference contrast. Bar, 2 µm.

View larger version (51K): [in a new window]   Fig. 9. Peroxisomal redirection of the mitochondrial TA protein Fis1p by the luminal PEX19-binding site of Pex15p. A GFP fusion of Fis1p was coexpressed in the S. cerevisiae wild-type strain with the synthetic mitochondrial marker PrF0ATP9-DsRed (Mc, A) or PTS2-DsRed (Px, B). GFP- and DsRed-derived fluorescence was analyzed after strains had been grown on ethanol-containing plates for 2 days. Localization of GFP-Fis1p-Pex15p361-383 was similarly determined. DIC, differential interference contrast. Bar, 2 µm.

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