JCS03100 Supplementary Material

Files in this Data Supplement:

• Supplemental Table 1 - Adobe PDF

• Supplemental Figure 1 -

  Fig. S1. Cellular level of ectopically expressed Pex19p deletion mutants. Epitope-tagged Pex19p variants shown in Fig. 1 were expressed in pex19 ZP119 (A) and CHO-K1 (B) cells. The level of expressed proteins was assessed by immunoblotting with anti-HA antibody. Protein loading of respective cell lysates was likewise verified with anti-LDH antibody. Mock-transfected cell lysate was loaded in lane 1. (C) Ectopically expressed HA₂-Pex19p was compared with the level of endogenous Pex19p. Immunoblotting was done with anti-Pex19p antibody. Cell-types were indicated at the top.

• Supplemental Figure 2 -

  Fig. S2. Amino acid sequence alignment of N-terminal and C-terminal internal regions of Pex19p from various species. Alignment of amino acid sequences was done with a GENETYX-MAC program (Software Development, Tokyo, Japan). Prediction of secondary structure of Pex19p was done by Predict Protein service (http://cubic.bioc.columbia.edu/predictprotein/predictprotein.html). Hs, human; Cl, Chinese hamster; Ce, Caenorhabditis elegans; Sc, Saccharomyces cerevisiae. Identical and similar (D=E, K=H=R, L=M=F=V) residues are shaded in dark gray and gray, respectively. H and L shown at the top indicate amino acid residues predicted to form secondary structures, helix and loop, respectively. Dash designates a space.

• Supplemental Figure 3 -

  Fig. S3. Pex19p regions required for interaction with Pex14p and Pex16p. HA₂- or Myc₆-HA₂-tagged Pex19p variants were co-expressed in COS7 cells with Flag-Pex14p (A) and Flag-Pex16p (B). HA₂-Pex19p and Myc₆-HA₂-Pex19p variants were immunoprecipitated and analyzed as in Fig. 5A, except that Flag-Pex14p (A) and Flag-Pex16p (B) were detected using anti-Flag monoclonal antibody. Lanes: 1-10, 10% input; 11-20, immunoprecipitates. Solid arrowheads indicate Flag-Pex14p and Flag-Pex16p; open arrowheads show HA₂- or Myc₆-HA₂-Pex19p mutants. Flag-Pex14p was detected in the immunoprecipitates each of HA₂-tagged, full-length Pex19p, M12-261, M12-255, 1-131, and M40-131 (Fig. S3A, lanes 11-16). Shorter Myc₆-HA₂-Pex19p variants, 1-73, M12-73, M24-73, and M40-73 also showed weaker interaction with Flag-Pex14p (lanes 17-20), although the latter two were defective in localizing to peroxisome activity (see Fig. 3). Flag-Pex16p was likewise detected in the immunoprecipitate of normal Pex19p and M12-261, and less discernible in that of M12-255, whilst it was not detectable with 1-131, M40-131, 1-73, M12-73, M24-73, and M40-73 (Fig. S3B, lanes 11-20).