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 thiolase precursors (types A and B) were reported to be cleavable signal peptides for peroxisomal protein translocation. In the present study, this was proven by immunoelectron microscopy of the cultured Chinese hamster ovary cells stably expressing fusion proteins of the amino-terminal sequences of the thiolase precursor and Escherichia coli dihydrofolate reductase. The fusion proteins were processed into mature forms of the apparently correct sizes. Site-directed mutagenesis studies of the 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 His-17 were also required in a longer presequence (36 amino acid residues) of the thiolase A, thereby suggesting that the signal can function in an internal position. When glutamic acid at position -11 was changed 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 newly defined type of peroxisomal targeting signal recognized by a mechanism presumably different from that for a known peroxisomal signal, the carboxy-terminal Ser-Lys-Leu-COOH motif.
Tsukamoto
T
T
Department of Life Science, Himeji Institute of Technology, Hyogo, Japan.
Hata
S
S
Yokota
S
S
Miura
S
S
Fujiki
Y
Y
Hijikata
M
M
Miyazawa
S
S
Hashimoto
T
T
Osumi
T
T
D000102:Acetyl-CoA C-Acyltransferase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D017931:DNA Primers; D004792:Enzyme Precursors / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D008830:Microbodies / Q000201:enzymology*; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D017354:Point Mutation; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000378:metabolism*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013347:Subcellular Fractions / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D017931:DNA Primers; D004792:Enzyme Precursors; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D000102:Acetyl-CoA C-Acyltransferase