Figure 3. Miro1-var2 and Miro1-var4 are translocated to peroxisomes in a manner dependent on their specific insertion 1 and the PMP receptor Pex19p. (A) FLAG-EGFP–fused C-terminal regions of Miro1 variants (FL-EGFP-MVCs) indicated at the top were synthesized in RRL in the presence of RRL-synthesized HA2-Pex19p and subjected to immunoprecipitation (IP) with anti-FLAG agarose beads. Immunoprecipitates and input (10%) were analyzed by immunoblotting using antibodies to HA and FLAG. Solid and open arrowheads indicate unmodified and farnesylated HA2-Pex19p, respectively. (B) Partial amino acid sequences near the C terminus of insertion 1 of human Miro1-var2 and Miro1-var4 are aligned with those of known Pex19p binding sites such as those of S. cerevisiae Pex13p and Pex11p and human adrenoleukodystrophy protein (ALDP). Underlined and bold letters indicate experimentally identified Pex19p binding sites and the core 9-aa residues in typical Pex19p-binding sites in three representative PMPs (Rottensteiner et al., 2004; Halbach et al., 2005) and putative corresponding ones in human Miro1 variants, respectively. The dashed line shows the C terminus of insertion 1. Asterisks and blue letters show highly conserved amino acid residues of putative Pex19p binding sites and leucine608, respectively, in Miro1-var2 and Miro1-var4. An amino acid sequence of the C-terminal region of Miro1-var2 and Miro1-var4 containing a TMD (shaded) and two lysine and arginine residues mutated in KR2S mutants (colored in red) are also shown. (C) FL-EGFP-MVC variants and their L608P mutants were synthesized in RRL in the presence of HA2-Pex19p and immunoprecipitated with anti-FLAG agarose beads as in A. Solid and open arrowheads indicate unmodified and farnesylated HA2-Pex19p, respectively. (D) L608P mutation in insertion 1 impairs peroxisomal localization of Miro1-var2 and Miro1-var4. HeLa cells were transiently transfected with FLAG-EGFP-MV4C-L608P (a–d) and FLAG-EGFP-MV2C-L608P (e–h) and immunostained using antibodies to Pex14p (b and f, red) and Tom20 (c and g, blue) as in Fig. 1 C. EGFP fluorescence of FL-EGFP-MVCs (a and e, green) and merged views (d and h) are shown. (E) In vitro import assay of Miro1 variants was performed by incubating semipermeabilized HeLa cells at 26°C for 1 h with cytosolic fractions each containing FLAG-Pex19p plus HA2–Miro1-var4 (a and b), HA2–Miro1-var2 (c and d), or HA2-Pex26p (e and f). Cells were immunostained with antibodies against HA and Pex14p. Bars, 10 µm. (F) Cells shown in E were treated with 0.1 M Na2CO3 and separated into soluble (supernatant, S) and membrane (pellet, P) fractions. Equal aliquots of respective fractions were analyzed by immunoblotting with the indicated antibodies.
Figure 3.

Miro1-var2 and Miro1-var4 are translocated to peroxisomes in a manner dependent on their specific insertion 1 and the PMP receptor Pex19p. (A) FLAG-EGFP–fused C-terminal regions of Miro1 variants (FL-EGFP-MVCs) indicated at the top were synthesized in RRL in the presence of RRL-synthesized HA2-Pex19p and subjected to immunoprecipitation (IP) with anti-FLAG agarose beads. Immunoprecipitates and input (10%) were analyzed by immunoblotting using antibodies to HA and FLAG. Solid and open arrowheads indicate unmodified and farnesylated HA2-Pex19p, respectively. (B) Partial amino acid sequences near the C terminus of insertion 1 of human Miro1-var2 and Miro1-var4 are aligned with those of known Pex19p binding sites such as those of S. cerevisiae Pex13p and Pex11p and human adrenoleukodystrophy protein (ALDP). Underlined and bold letters indicate experimentally identified Pex19p binding sites and the core 9-aa residues in typical Pex19p-binding sites in three representative PMPs (Rottensteiner et al., 2004; Halbach et al., 2005) and putative corresponding ones in human Miro1 variants, respectively. The dashed line shows the C terminus of insertion 1. Asterisks and blue letters show highly conserved amino acid residues of putative Pex19p binding sites and leucine608, respectively, in Miro1-var2 and Miro1-var4. An amino acid sequence of the C-terminal region of Miro1-var2 and Miro1-var4 containing a TMD (shaded) and two lysine and arginine residues mutated in KR2S mutants (colored in red) are also shown. (C) FL-EGFP-MVC variants and their L608P mutants were synthesized in RRL in the presence of HA2-Pex19p and immunoprecipitated with anti-FLAG agarose beads as in A. Solid and open arrowheads indicate unmodified and farnesylated HA2-Pex19p, respectively. (D) L608P mutation in insertion 1 impairs peroxisomal localization of Miro1-var2 and Miro1-var4. HeLa cells were transiently transfected with FLAG-EGFP-MV4C-L608P (a–d) and FLAG-EGFP-MV2C-L608P (e–h) and immunostained using antibodies to Pex14p (b and f, red) and Tom20 (c and g, blue) as in Fig. 1 C. EGFP fluorescence of FL-EGFP-MVCs (a and e, green) and merged views (d and h) are shown. (E) In vitro import assay of Miro1 variants was performed by incubating semipermeabilized HeLa cells at 26°C for 1 h with cytosolic fractions each containing FLAG-Pex19p plus HA2–Miro1-var4 (a and b), HA2–Miro1-var2 (c and d), or HA2-Pex26p (e and f). Cells were immunostained with antibodies against HA and Pex14p. Bars, 10 µm. (F) Cells shown in E were treated with 0.1 M Na2CO3 and separated into soluble (supernatant, S) and membrane (pellet, P) fractions. Equal aliquots of respective fractions were analyzed by immunoblotting with the indicated antibodies.

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