Characterization of the mammalian peroxisomal import machinery: Pex2p, Pex5p, Pex12p, and Pex14p are subunits of the same protein assembly.
Although many of the proteins involved in the biogenesis of the mammalian peroxisome have already been identified, our knowledge of the architecture of all this machinery is still very limited. In this work we used native gel electrophoresis and sucrose gradient sedimentation analysis in combination with immunoprecipitation experiments to address this issue. After solubilization of rat liver peroxisomes with the mild detergent digitonin, comigration of Pex5p, Pex14p, and a fraction of Pex12p was observed upon native electrophoresis and sucrose gradient sedimentation. The existence of a complex comprising Pex2p, Pex5p, Pex12p, and Pex14p was demonstrated by preparative coimmunoprecipitation experiments using an antibody directed to Pex14p. No stoichiometric amounts of Pex13p were detected in the Pex2p-Pex5p-Pex12p-Pex14p complex, although the presence of a small fraction of Pex13p in this complex could be demonstrated by Western blot analysis. Pex13p is also a component of a high molecular mass complex. Strikingly, partial purification of this Pex13p-containing complex revealed Pex13p as the major (if not the only) component. Taken together, our data indicate that Pex2p, Pex5p, Pex12p, and Pex14p, on one side, and Pex13p, on the other, are subunits of two stable protein complexes that probably interact with each other in the peroxisomal membrane.
Reguenga
C
C
Instituto de Biologia Molecular e Celular and Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, 4150-180 Porto, Portugal.
Oliveira
M E
ME
Gouveia
A M
AM
Sá-Miranda
C
C
Azevedo
J E
JE
D000818:Animals; D001692:Biological Transport; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry; D000074428:Peroxisomal Biogenesis Factor 2; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D051381:Rats; D018160:Receptors, Cytoplasmic and Nuclear / Q000737:chemistry; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions; D014357:Trypsin / Q000494:pharmacology
D002352:Carrier Proteins; D008565:Membrane Proteins; D000074435:Peroxisome-Targeting Signal 1 Receptor; C491336:Pex12 protein, rat; C499194:Pex14 protein, rat; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D012097:Repressor Proteins; D000074428:Peroxisomal Biogenesis Factor 2; D014357:Trypsin