Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal peroxisomal targeting sequence.

Abstract

Abstract. 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 PASIO 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 PASIO gene product (Pasl0p) and the human PTS1 receptor can interact with the COOH-terminal region of human catalase, but that ithis interaction is abolished by substitutions at the penultimate residue (asparagine-toaspartate) 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 mutat+d in the COOH-terminal PTS. Consistent wi...

Peroxisomal Targeting Signal Receptor Pex5p Interacts with Cargoes and Import Machinery Components in a Spatiotemporally Differentiated Manner: Conserved Pex5p WXXXF/Y Motifs Are Critical for Matrix Protein Import

“…In contrast, catalase with C-terminal tetrapeptide KANL deleted was not detectable in any of the fractions (lanes 8 to 10). These results strongly suggested that catalase bound to Pex5p via C-terminal KANL, consistent with the report that KANL is the PTS of human catalase (32). Thus, a mode of recognition of catalase PTS by Pex5p resembled that of PTS1 but the import mechanisms of these cargoes were apparently distinguishable in the requirement for Pex5p-Pex13p interaction (Fig.…”

“…PEX5 and PEX7 encode the receptors for PTS1 and PTS2, respectively. Of note, catalase with the Cterminal sequence KANL is also imported into peroxisomes by the Pex5p-dependent pathway (29,32). However, the underlying molecular mechanism for catalase import remains unclear.…”

Getting a camel through the eye of a needle: the import of folded proteins by peroxisomes

“…A range of C-terminal tripeptides have since been shown to function as PTS1s in various organisms in vivo (Gould et al, 1988;Aitchison et al, 1991;Purdue and Lazarow, 1996;Kragler et al, 1998). These generally fit the consensus sequence of , although a number of variants have been identified which only conform to this consensus in two out of three positions (Reumann et al, 2007).…”

“…Mammalian catalase has been shown to be targeted to the peroxisome by a four-amino-acid sequence of KANL at its C-terminus (Purdue and Lazarow, 1996). Alanine:glyoxylate aminotransferase is targeted by two signals (Huber et al, 2005); a C-terminal KKKL motif and an internal eightamino-acid sequence, both of which are predicted to be separately located on the surface of the protein (Ikeda et al, 2008).…”

Peroxisomal Import of Human Alanine:glyoxylate Aminotransferase Requires Ancillary Targeting Information Remote from Its C Terminus

“…Whereas in the case of the latter, amino acids in close proximity (i.e. within 10 -20 residues) to the C-terminal tripeptide can markedly influence the properties of, and allowable residues within, the PTS1 (16,17,(32)(33)(34)(35). The importance of specific residues near the C terminus appears to be particularly important if the C-terminal tripeptide deviates from the conservative consensus PTS1 (16).…”