PEX5 binds the PTS1 independently of Hsp70 and the peroxin PEX12.
Most peroxisomal enzymes are targeted to peroxisomes by virtue of a type-1 peroxisomal targeting signal (PTS1) at their extreme C terminus. PEX5 binds the PTS1 through its C-terminal 40-kDa tetratricopeptide repeat domain and is essential for import of PTS1-contining proteins into peroxisomes. Here we examined the PTS1-binding activity of purified, recombinant, full-length PEX5 using a fluorescence anisotropy-based assay. Like its C-terminal fragment, full-length tetrameric PEX5 exhibits high intrinsic affinity for the PTS1, with a K(d) of 35 nm for the peptide lissamine-Tyr-Gln-Ser-Lys-Leu-COO(-). The specificity of this interaction was demonstrated by the fact that PEX5 had no detectable affinity for a peptide in which the Lys was replaced with Glu, a substitution that inactivates PTS1 signals in vivo. Hsp70 has been found to regulate the affinity of PEX5 for a PTS1-containing protein, but we found that the kinetics of PEX5-PTS1 binding was unaffected by Hsp70, Hsp70 plus ATP, or Hsp70 plus ADP. In addition, we found that another protein known to interact with the PTS1-binding domain of PEX5, the PEX12 zinc RING domain, also had no discernable effect on PEX5-PTS1 binding kinetics. Taken together, these results suggest that the initial step in peroxisomal protein import, the recognition of enzymes by PEX5, is a relatively simple process and that Hsp70 most probably stimulates this process by catalyzing the folding of newly synthesized peroxisomal enzymes and/or enhancing the accessibility of their PTS1.
Harper
Courtney C
CC
Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Berg
Jeremy M
JM
Gould
Stephen J
SJ
D000255:Adenosine Triphosphate / Q000378:metabolism; D002850:Chromatography, Gel; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D000074435:Peroxisome-Targeting Signal 1 Receptor; D011485:Protein Binding; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*
D018840:HSP70 Heat-Shock Proteins; D008565:Membrane Proteins; C105251:PEX12 protein, human; C000614264:PEX5 protein, human; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D000255:Adenosine Triphosphate