CHARACTERIZATION OF THE SIGNAL PEPTIDE AT THE AMINO-TERMINUS OF THE RAT PEROXISOMAL 3-KETOACYL-COA THIOLASE PRECURSOR

The amino-terminal presequences of rat peroxisomal 3-ketoacyl-CoA thio lase precursors (types A and B) were reported to be cleavable signal p eptides for peroxisomal protein translocation. In the present study, t his was proven by immunoelectron microscopy of the cultured Chinese ha mster ovary cells stably expressing fusion proteins of the amino-termi nal sequences of the thiolase precursor and Escherichia coli dihydrofo late reductase. The fusion proteins were processed into mature forms o f the apparently correct sizes. Site-directed mutagenesis studies of t he charged residues in the B-type presequence (26 amino acid residues) revealed that arginine at position -24 and histidine at position -17 were both indispensable. Even replacement of these residues with other basic amino acids abolished the import activity. Both Arg(-24) and Hi s(-17) were also required in a longer presequence (36 amino acid resid ues) of the thiolase A, thereby suggesting that the signal can functio n in an internal position. When glutamic acid at position -11 was chan ged to amino acids other than aspartic acid, the signal peptide became apparently effective in both peroxisomal and mitochondrial targeting. All of these data indicate that the thiolase signal peptide is a newl y defined type of peroxisomal targeting signal recognized by a mechani sm presumably different from that for a known peroxisomal signal, the carboxyl-terminal Ser-Lys-Leu-COOH motif.