Pex5p, the peroxisomal cycling receptor, is a monomeric non-globular protein.
In mammals, targeting of newly synthesized peroxisomal matrix proteins to the organelle requires Pex5p, the peroxisomal cycling receptor. Pex5p is a multidomain protein involved in a complex network of transient protein-protein interactions. Besides interacting directly with most peroxisomal proteins en route to the organelle, Pex5p has also binding domains for several components of the peroxisomal docking/translocation machinery. However, our knowledge of how binding of a cargo protein to Pex5p influences its properties is still rather limited. Here, we describe a protease assay particularly useful for identifying and characterizing protein-protein interactions involving human Pex5p. Binding of a PTS1-containing peptide/protein to Pex5p as well as the interaction of this peroxin with the Src homology domain 3 of Pex13p could be easily demonstrated using this assay. To address the possible effects of these Pex5p-interacting peptides/proteins on the assumed quaternary structure of Pex5p, we have analyzed the hydrodynamic properties of human Pex5p using size exclusion chromatography, sucrose gradient centrifugation, and sedimentation equilibrium centrifugation. Our results show that Pex5p is a monomeric protein with an abnormal shape. The implications of these findings on current models of protein translocation across the peroxisomal membrane are discussed.
Costa-Rodrigues
João
J
Instituto de Biologia Molecular e Celular, Rua do Campo Alegre, 823, 4150-180 Porto, Portugal.
Carvalho
Andreia F
AF
Fransen
Marc
M
Hambruch
Eva
E
Schliebs
Wolfgang
W
Sá-Miranda
Clara
C
Azevedo
Jorge E
JE
D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D002845:Chromatography; D004591:Electrophoresis, Polyacrylamide Gel; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry; D013395:Sucrose / Q000494:pharmacology
D008565:Membrane Proteins; C101884:PEX13 protein, human; D010455:Peptides; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D011994:Recombinant Proteins; D013395:Sucrose; D005982:Glutathione Transferase