The 80 kDa cytosolic protein that binds the C-terminal part of rat acyl-CoA oxidase is not a peroxisomal import receptor but a prolyl-endopeptidase.
In an attempt to identify putative peroxisomal import receptors, we investigated the cross-linking of a radioiodinated peptide consisting of the 13 last amino acids of acyl-CoA oxidase and comprising the carboxy-terminal SKL-peroxisomal targeting motive, to proteins present in different subcellular fractions from rat liver. The radiolabeled peptide could be cross-linked to an 80 kDa protein present in the cytosol but not to proteins present in other subcellular fractions including highly purified peroxisomes. Binding was reversible, saturable and dependent on the presence of Mg2+ and ATP or GTP but hydrolysis of the nucleotides was not required. Binding was abolished by pretreatment of the cytosol--but not of the peptide--with N-ethylmaleimide. Binding was not specific for peptides containing the carboxy-terminal SKL-motive, since binding was competed for by the SKL-peptide from which the SKL-motive had been deleted, by the SKL-peptide with reversed sequence and by the SV40 T-antigen nuclear localisation signal peptide, but not by other peptides tested. The 80 kDa binding protein cross-reacted with a monoclonal antibody against hsp90. Purification and internal peptide sequencing of the binding protein revealed its identity as prolyl-endopeptidase. In retrospect, we realized that the SKL-peptide and all competing peptides contained a proline residue, which was not present in the non-competing peptides. In recent experiments in yeast McNew et al. (McNew, J.A., Sykes, K. and Goodman, J.M. (1993) Mol. Biol. Cell 4, 223-232) cross-linked a peroxisomal targeting peptide to a 20 kDa cytosolic protein that was identified as proline isomerase despite the fact that the peptide did not contain proline. The experiments by McNew et al. in yeast and our experiments in the rat suggest that the (peroxisomal) targeting sequence cross-linking approach may not be suited for the identification of (peroxisomal) import receptors.
Fransen
M
M
Katholieke Universiteit Leuven, Department of Pharmacology, Belgium.
Brees
C A
CA
de Béthune
B
B
Van Veldhoven
P P
PP
Mannaerts
G P
GP
D042962:Acyl-CoA Oxidase; D000595:Amino Acid Sequence; D000818:Animals; D003600:Cytosol / Q000378:metabolism*; D008297:Male; D008830:Microbodies / Q000378:metabolism*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000378:metabolism*; D000085822:Prolyl Oligopeptidases; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000378:metabolism*
D010088:Oxidoreductases; D042962:Acyl-CoA Oxidase; D012697:Serine Endopeptidases; D000085822:Prolyl Oligopeptidases