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First published online 8 August 2006
doi: 10.1242/jcs.03100

Journal of Cell Science 119, 3539-3550 (2006)
Published by The Company of Biologists 2006
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Functional domain mapping of peroxin Pex19p: interaction with Pex3p is essential for function and translocation

Yuji Matsuzono, Takashi Matsuzaki and Yukio Fujiki*

Department of Biology, Faculty of Sciences, Kyushu University Graduate School, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan


Figure 1
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  		Fig. 1. Schematic representation of human Pex19p constructs. (A-C) Representative Pex19p constructs of deletions from C-terminus, N-terminus, and both terminal regions, respectively, used in this study. Numbers designate the positions in the amino acid sequence. Two tandem epitopes of hemagglutinin (HA2) were tagged to the N-terminus of respective Pex19p variants. Several mutants with shorter sequences were fused to hexa-Myc tag, Myc6, and HA2. Farnesylation CAAx motif is shaded. The activities of the constructs in restoring peroxisomes in pex19 ZP119 cells and translocating to peroxisomal membranes in CHO-K1 are summarized on the right: +++, strong; ++, medial; +, positive; +/-; weakly positive; -, negative.

 

Figure 2
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  		Fig. 2. Analysis of the region of Pex19p required for re-establishing peroxisome assembly. (A) HA2-Pex19p variants shown in Fig. 1A were expressed in CHO pex19 ZP119 cells: full-length Pex19p (a,b), 1-295 (the same as {Delta}C4) with deletion of four amino acid residues from the C-terminus (c,d), 1-261 ({Delta}C38) (e,f), 1-255 ({Delta}C44) (g,h), and {Delta}256-260 with internal deletion of amino-acid residues 256-260 (i,j). Cells were dual-stained with antisera against PTS1 and Pex14p for verifying peroxisome-restoring activity of Pex19p mutants. (B) HA2-Pex19p deletion mutants in Fig. 1B,C were likewise expressed in ZP119 cells: 12-299 ({Delta}N11) lacking 11 amino-acid residues from the N-terminus (a,b), 24-299 ({Delta}N23) (c,d), {Delta}12-23 (e,f), M12-261 with deletion of 11 and 38 amino acid residues from the N- and C-termini (g,h) and M12-255 (i,j). Cells were stained as in A. Bars, 20 µm.

 

Figure 3
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  		Fig. 3. Pex19p translocates to peroxisome membranes without N- and C-terminal parts. Full-length HA2-Pex19p and its deletion mutants: 1-73 ({Delta}C226), 40-299 ({Delta}N39), 70-299 ({Delta}N69), M40-131, M12-73, M24-73, and M40-73 were expressed in wild-type CHO-K1 cells. Cells were double-stained with antisera to HA (left panels) and Pex14p (right panels). Bar, 20 µm.

 

Figure 4
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  		Fig. 4. Interacting region of Pex19p with Pex3p and Pex26p. Pex19p interaction was assessed by yeast two-hybrid assay. Host strain MaV203 was transformed with two plasmids, one encoding the DNA-binding domain (BD) of Gal4p fused to the full-length rat Pex3p (A) or human Pex26p (B) and the other encoding the Gal4p transactivation domain (AD) fused to Pex19p variants indicated on the left. Transformants were assayed for ß-galactosidase expression (A, left panels) and growth on synthetic complete medium lacking His in the presence of 10 mM 3-aminotriazole (3AT) (A, right panels; B). BD-fused peroxins without Pex19p and Pex19p-AD with mock vector showed no self-activation.

 

Figure 5
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  		Fig. 5. A region of Pex19p encompassing amino acid residues 12-73 and 40-131 is sufficient for binding to Pex3p. (A) HA2- or Myc6-HA2-tagged Pex19p truncation mutants were co-expressed with FL-Pex3p-EGFP in COS7 cells. HA2-Pex19p and Myc6-HA2-Pex19p variants were immunoprecipitated using rabbit anti-HA antibody and analyzed by SDS-PAGE. HA2-Pex19p and Myc6-HA2-Pex19p proteins were detected by immunoblot with mouse anti-HA antibody (lower panel). FL-Pex3p-EGFP co-immunoprecipitated with Pex19p was detected with anti-GFP antibody (upper panel). Lanes 1-10, 10% input used for immunoprecipitation; lanes 11-20, immunoprecipitates. Solid arrowhead indicates FL-Pex3p-EGFP and open arrowheads, HA2- and Myc6-HA2-Pex19p variants. (B) Direct interaction of Pex19p N-terminal region with Pex3p. 35S-labeled, HA2- and Myc6-HA2-tagged Pex19p mutants and [35S]FL-Pex3p-EGFP were separately synthesized in a cell-free system. [35S]HA2-Pex19p and [35S]Myc6-HA2-Pex19p variants (2.5 µl each) were incubated with [35S]FL-Pex3p-EGFP (2.5 µl) and immunoprecipitated with anti-HA antibody. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography. Lanes 1-10, 10% input used for immunoprecipitation; lanes 11-20, immunoprecipitates. Solid arrowhead indicates [35S]FL-Pex3p-EGFP and open arrowheads indicate [35S]HA2-Pex19p and [35S]Myc6-HA2-Pex19p mutants.

 

Figure 6
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  		Fig. 6. Pex19p translocates to peroxisomes in a PMP-independent manner. (A) HA2-Pex19p was expressed in CHO-K1 cells (a,b) and CHO mutants, pex14 ZP161 (c,d), pex26 ZP167 (e,f) and pex3 ZPG208 (g,h). Cells were stained with antisera to HA (left panels) and PMP70 (right panels). (B) Fibroblasts from a normal control (a,b) and PBD patients defective in PEX12 (CG3) (c,d), PEX3 (CG-G, CG12) (e,f), and PEX16 (CG-D, CG9) (g,h) were transfected with HA2-PEX19. Cells were stained with antibodies against HA (left panels) and Pex14p (right panels). Bar, 20 µm.

 

Figure 7
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  		Fig. 7. Pex19p functions as a chaperone and transports PMPs in a temporally differentiated manner in peroxisome biogenesis. (A) Flag-tagged PMPs including Pex14p (FL14, a-h), Pex16p (FL16, i-p), and Pex26p (FL26, q-x) were expressed (two left panels each) or co-expressed with HA2-Pex19p (middle and right panels) in pex19 ZP119 cells. Cells were stained with antibodies to Flag and HA and with MitoTracker, at the times indicated at the top after the transfection. ZP 119 cells co-transfected with Flag-PEX26 and HA2-PEX19 were also stained with antibodies to Pex14p and HA at 36 hours post transfection (y,z). (B) ZP119 cells were likewise transfected with HA2-PEX19 and were assessed for endogenous Pex14p by immunostaining with specific antibody, mitochondria by MitoTracker and Pex19p by HA staining. Note that Pex14p and HA2-Pex19p were detectable in the cytosol at 12 hours post transfection. (C) ZP119 cells expressing Pex3p-EGFP alone (3EGFP, left panels) or with HA2-Pex19p (right) were verified at 12 hours post transfection for Pex3p by EGFP, mitochondria and HA2-Pex19p. Note that Pex3p-EGFP and HA2-Pex19p were detectable in numerous particles, apparently mitochondria. Bar, 20 µm.

 




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