FIG. 7.
Pex5 translocation machinery on peroxisomal membranes. (A) Pex5-containing complexes on peroxisomal membrane were analyzed by BN-PAGE. The 35S-Pex5 import reaction was carried out with PNS factions each from CHO-K1, pex5 ZP105, pex14 ZP161, pex2 Z65, and pex12 ZP109, as for Fig. 1A. Organelle membrane fractions were isolated, solubilized with 1% digitonin, and analyzed by BN-PAGE. 35S-Pex5 was detected by a Fujix autoimaging analyzer. The figure represents a composite of two separate experiments. Arrowheads indicate two distinct complexes containing 35S-Pex5. Molecular mass markers are on the left. (B) AAA ATPase peroxin, Pex1, is not involved in the two types of import complexes. 35S-Pex5 import was done using PNS from fibroblasts from a normal control (lane 1) and a PEX1-defective PBD patient of CG1 (CG-E) (lane 2), as for Fig. 3B. Organelle fractions were analyzed as for panel A. (C) Assembly of the two import complexes does not require ATP. 35S-Pex5 import was done with PNS from ZP105 in the presence of 1 mM ATP plus ARS (lane 1) or after preincubation with apyrase (lane 2), as for Fig. 2A. Organelle fractions were analyzed as for panel A. (D) Antibody shift assays using antibodies against Pex14 and Pex2 were performed. 35S-Pex5 import was done as for panel A. Solubilized organelle fractions of CHO-K1, Z65, and ZP109 were incubated with rabbit preimmune serum (lanes 1, 5, and 8) or antibodies to Pex14 (lanes 2, 6, and 9) or Pex2 (lanes 3, 7, and 10) or the mixture of antibodies to Pex14 and Pex2 (lane 4) and were analyzed as for panel A. Arrowheads indicate positions of 35S-Pex5-containing complexes.