We have identified a novel peroxisomal targeting sequence (PTS) at the extreme COOH terminus of human
catalase. The last four
amino acids of this
protein (-KANL) are necessary and sufficient to effect targeting to peroxisomes in both human fibroblasts and Saccharomyces cerevisiae, when appended to the COOH terminus of the reporter
protein,
chloramphenicol acetyl
transferase. However, this PTS differs from the extensive family of COOH-terminal PTS tripeptides collectively termed PTS1 in two major aspects. First, the presence of the uncharged
amino acid,
asparagine, at the penultimate residue of the human
catalase PTS is highly unusual, in that a basic residue at this position has been previously found to be a common and critical feature of PTS1 signals. Nonetheless, this
asparagine residue appears to constitute an important component of the
catalase PTS, in that replacement with
aspartate abolished peroxisomal targeting (as did deletion of the COOH-terminal four residues). Second, the human
catalase PTS comprises more than the COOH-terminal three
amino acids, in that COOH-terminal-ANL cannot functionally replace the PTS1 signal-SKL in targeting a
chloramphenicol acetyl
transferase fusion
protein to peroxisomes. The critical nature of the fourth residue from the COOH terminus of the
catalase PTS (
lysine) is emphasized by the fact that substitution of this residue with a variety of other
amino acids abolished or reduced peroxisomal targeting. Targeting was not reduced when this
lysine was replaced with
arginine, suggesting that a
basic amino acid at this position is required for maximal functional activity of this PTS. In spite of these unusual features, human
catalase is sorted by the PTS1 pathway, both in yeast and human cells. Disruption of the PAS10 gene encoding the S. cerevisiae
PTS1 receptor resulted in a cytosolic location of
chloramphenicol acetyl
transferase appended with the human
catalase PTS, as did expression of this
protein in cells from a
neonatal adrenoleukodystrophy patient specifically defective in PTS1 import. Furthermore, through the use of the two-hybrid system, it was demonstrated that both the PAS10 gene product (Pas10p) and the human
PTS1 receptor can interact with the COOH-terminal region of human
catalase, but that this interaction is abolished by substitutions at the penultimate residue (
asparagine-to-
aspartate) and at the fourth residue from the COOH terminus (
lysine-to-
glycine) which abolish PTS functionality. We have found no evidence of additional targeting information elsewhere in the human
catalase protein. An internal tripeptide (-SHL-, which conforms to the mammalian PTS1 consensus) located nine to eleven residues from the COOH terminus has been excluded as a functional PTS. Additionally, in contrast to the situation for S. cerevisiae
catalase A, which contains an internal PTS in addition to a COOH-terminal PTS1, human
catalase lacks such a redundant PTS, as evidenced by the exclusive cytosolic location of human
catalase mutated in the COOH-terminal PTS. Consistent with this species difference, fusions between
catalase A and human
catalase which include the
catalase A internal PTS are targeted, at least in part, to peroxisomes regardless of whether the COOH-terminal human
catalase PTS is intact.