Involvement of Pex13p in Pex14p localization and peroxisomal targeting signal 2-dependent protein import into peroxisomes

Abstract

Pex13p is the putative docking protein for peroxisomal targeting signal 1 (PTS1)-dependent protein import into peroxisomes. Pex14p interacts with both the PTS1- and PTS2-receptor and may represent the point of convergence of the PTS1- and PTS2-dependent protein import pathways. We report the involvement of Pex13p in peroxisomal import of PTS2-containing proteins. Like Pex14p, Pex13p not only interacts with the PTS1-receptor Pex5p, but also with the PTS2-receptor Pex7p; however, this association may be direct or indirect. In support of distinct peroxisomal binding sites for Pex7p, the Pex7p/Pex13p and Pex7p/ Pex14p complexes can form independently. Genetic evidence for the interaction of Pex7p and Pex13p is provided by the observation that overexpression of Pex13p suppresses a loss of function mutant of Pex7p. Accordingly, we conclude that Pex7p and Pex13p functionally interact during PTS2-dependent protein import into peroxisomes. NH2-terminal regions of Pex13p are required for its interaction with the PTS2-receptor while the COOH-terminal SH3 domain alone is sufficient to mediate its interaction with the PTS1-receptor. Reinvestigation of the topology revealed both termini of Pex13p to be oriented towards the cytosol. We also found Pex13p to be required for peroxisomal association of Pex14p, yet the SH3 domain of Pex13p may not provide the only binding site for Pex14p at the peroxisomal membrane.

Figure 1

The two-hybrid interaction of the PTS receptors Pex5p and Pex7p requires the presence of Pex14p. Two-hybrid interactions between Pex5p and Pex7p were monitored by a filter assay for β-galactosidase activity and test for His auxotrophy in wild-type strains PCY2 and HF7c, respectively, or isogenic strains deleted for PEX17, PEX13, and PEX14. The Pex5p/Pex7p interaction is still observed in the strains lacking either Pex17p or Pex13p, but not in strains lacking Pex14p. Pex14p may be required for the activation of either receptor, or it may function as a bridging molecule between Pex5p and Pex7p. Three representative independent transformants are shown (1–3).

Figure 2

A portion of mycPex7p is associated with a membrane sediment in pex14Δ cells. Immunological detection of mycPex7p and peroxisomal membrane markers Pex13p and Pex14p in 200 K supernatant and pellet fractions of homogenates from indicated pex mutants. The cytosolic marker Fbp1p served as a control for nonspecific presence of proteins in the membrane sediment. Note the residual amount of Pex7p in the membrane pellet of pex14Δ cells. Equal portions of membrane and supernatant fractions were analyzed.

Figure 3

Pex7p is associated with Pex13p in the absence of Pex5p and Pex14p. (A) Whole cell extracts of wild-type (UTL-7A) cells, and of wild-type, pex5Δ, pex14Δ, pex5/14Δ, pex13Δ, and pex17Δ cells expressing mycPex7p, were immunoprecipitated using antibodies against the c-myc epitope. A significant amount of Pex13p coimmunoprecipitated with Pex7p in the absence of Pex5p and Pex14p. Note the decreased amount of Pex5p in the precipitate from pex14Δ cells while the amount of Pex13p remained nearly unchanged. Equal amounts of immunoprecipitates (10% of total) were separated by SDS-PAGE and subjected to immunoblot analysis using antibodies against c-myc, Pex5p, Pex13p, Pex14p, and Pex17p. (B) Immunoblot of equal portions of the whole cell extracts (1% of total) for mycPex7p, Pex13p, Pex14p, Pex5p, and Fox3p.

Figure 4

Pex13p interacts with Pex7p in the yeast two-hybrid system independent of Pex5p or Pex14p. Interaction of (A) Pex13p with Pex7p and (B) Pex13pE320K with Pex7p and Pex14p. An interaction of Pex13p and Pex7p is indicated by the β-galactosidase expression and the histidine prototrophy of the transformants and is still observed in strains lacking either Pex14p or Pex5p. Note that Pex13pE320K lacks an interaction with Pex14p, but still interacts with Pex7p in strains lacking Pex5p. Assays were carried out in the yeast reporter strain PCY2 and HF7c, or isogenic strains deleted for PEX14 or PEX5 as indicated. Three representative independent transformants are shown (1–3).

Figure 8

The Pex13p/Pex14p interactions require a proline-rich motif in Pex14p, as well as the RT loop of Pex13p. (A) Two- hybrid interaction of Pex14p and Pex14pAXXA with Pex5p, Pex7p, Pex13p, and Pex14p. Pex14pAXXA contains the mutation of Pro87 and Pro90 of a putative SH3 ligand motif of Pex14p. The two-hybrid interactions were monitored by determination of the β-galactosidase expression and of the histidine prototrophy of pex14Δ deletion strains of PCY2 and HF7c, respectively. The Pex14pAXXA still interacted with Pex5p, Pex7p, and Pex14p, but an interaction with the SH3 domain of Pex13p is no longer observed. (B) The SH3 domain of Pex13pE320K still interacts with the PTS1 receptor Pex5p but not with Pex14p. The Pex13pE320K contains the substitution of Glu320 by Lys320 within the RT loop of the SH3 domain. The interaction of Pex13p(SH3) and Pex13p(SH3)E320K with Pex5p and Pex14p was analyzed by the β-galactosidase expression and the histidine prototrophy in PCY2 and HF7c, respectively. Three representative independent transformants are shown (1–3).

Figure 5

Suppression of the reduced complementing activity of an HA-tagged Pex7p by overexpression of Pex13p. Expression from a low copy plasmid of a Pex7p that is COOH-terminally fused to a triple hemagglutinin tag (PEX7HA) results in only partial complementation of the pex phenotype of pex7Δ cells. Upon overexpression, the inability of Pex7p-HA3 to fully complement the pex7Δ phenotype is suppressed by Pex13p, but not by Pex14p. (A) Growth behavior of wild-type and pex7Δ mutant cells, as well as indicated single and double transformants on oleic acid medium. Note the weak growth of pex7Δ which express the PEX7HA in comparison to the nearly normal growth behavior of these cells upon additional overexpression of Pex13p. The spots to the left correspond to 2 × 10⁴ cells. (B) Immunofluorescence microscopical localization of thiolase (Fox3p) in wild-type and pex7Δ mutant cells, as well as indicated single and double transformants. The import defect for PTS2 proteins in pex7Δ cells, pex7Δ cells (b) expressing Pex7p-HA₃ from pRSPEX7HA alone (d) or in conjunction with PEX14 (f) is indicated by overall cell labeling. Suppression of the import defect for PTS2 proteins by overexpression of Pex13p in pex7Δ cells expressing pRS- PEX7HA is indicated by the punctate fluorescence pattern (e) that is comparable to the pattern observed in wild-type cells (a) or fully complemented pex7Δ cells (c). Bar, 5 μm.

Figure 6

The NH₂ terminus and the COOH-terminal SH3 domain of Pex13p are exposed to the cytoplasm. Protease protection analysis of isolated peroxisomes. Peroxisomes were prepared from pex13Δ cells expressing mycPex13p (see Materials and Methods). Equal amounts of the organelles were incubated with increasing concentrations of proteinase K in the absence of detergent for 10 min on ice. Equal amounts were separated by SDS-PAGE and analyzed by immunoblotting using specific antibodies for the SH3 domain of Pex13p, the myc-epitope, Pex14p, and the peroxisomal matrix protein thiolase (Fox3p). While the intraperoxisomal thiolase was stable throughout the experiment, the COOH and NH₂ termini of Pex13p, as well as Pex14p, were rapidly degraded.

Figure 7

Pex13p is required for the peroxisomal membrane targeting of Pex14p. (A) Oleic acid–induced wild-type, pex13Δ, pex14Δ, and pex17Δ cells, expressing HA-tagged Pex11p were processed for double immunofluorescence microscopy localization of Pex14p and HA-Pex11p. The congruent fluorescence patterns in wild-type and pex17Δ cells indicate a peroxisomal localization of Pex14p. No congruent fluorescence pattern was observed in pex13Δ cells, suggesting that the majority of Pex14p is mislocalized. Detection was performed with rabbit polyclonal antibodies against Pex14p and mouse mAb against the HA-epitope. Secondary antibodies were FITC-conjugated anti–mouse IgG and CY3-conjugated anti–rabbit IgG. Bar, 5 μm. (B) Flotation of peroxisomal membranes in wild-type, pex13Δ, and pex17Δ cells. Cell-free extracts were separated on 50–25% (wt/wt) sucrose step gradients. Localization of Pex14p as well as peroxisomal membrane markers Pex3p and Pex11p, and peroxisomal catalase and mitochondrial fumarase in fractions were monitored by immunoblot analysis and enzyme activity measurements. The peroxisomal membrane ghosts were detected at the top of the gradient while intact peroxisomes and cytosolic proteins predominantly remained in the loading zone. In pex13Δ cells, no Pex14p was detected in the ghost fractions, but was found mislocalized to fractions of lighter density.

Figure 9

Binding to the SH3 domain of Pex13p does not seem to be required for the peroxisomal membrane targeting of Pex14p. (A) Double immunofluorescence microscopy localization of Pex14p and HA-Pex11p. Oleic acid–induced pex13Δ cells coexpressing HA-tagged Pex11p either with Pex13p or Pex13pE320K, as well as oleic acid–induced pex14Δ cells coexpressing HA-tagged Pex11p either with Pex14p or Pex14pAXXA, were processed for double immunofluorescence microscopy localization of Pex14p and HA-Pex11p. Rabbit antibodies against Pex14p and mouse antibodies against the HA-epitope were used for the detection. Neither mutation in the proline-rich motif of Pex14p nor mutation of the RT loop of the SH3 domain of Pex13p resulted in the mislocalization of Pex14p. Secondary antibodies were FITC-conjugated anti–mouse IgG and CY3-conjugated anti–rabbit IgG. Bar, 5 μm. (B) Flotation of peroxisomal membranes in pex13/17Δ cells expressing wild-type or mutated Pex13p and pex14/17Δ cells expressing wild-type or mutated Pex14p. Cell-free extracts were separated on 50–25% (wt/wt) sucrose step gradients and localization of Pex3p, Pex11p, and Pex14p was monitored by immunoblot analysis. Peroxisomal membrane ghosts were detected at the top of the gradient. A colocalization of Pex14p with the peroxisomal membrane markers suggests the association of the protein with peroxisomal membrane ghosts independent of the interaction of Pex14p with the SH3 domain of Pex13p.