Identification of the peroxisomal targeting signal for cottonseed catalase

Catalase is a ubiquitous peroxisomal matrix enzyme, yet the molecular targeting signal(s) for sorting it in plant cells has not been defined. The most common peroxisome targeting signal (PTS) is a C‐terminal tripeptide composed of a conserved SKL motif (type 1 PTS). The PTS for cottonseed catalase (Ccat) was elucidated in this study from immunofluorescence microscopic analyses of tobacco BY‐2 suspension cells serving as an in vivo import system. To distinguish biolistically introduced Ccat from endogenous tobacco catalase, Ccat was hemagglutinin (HA) epitope‐tagged at its N‐terminus. Bombardment with HA‐Ccat resulted in the import of Ccat into glyoxysomes, the specialized type of peroxisome in BY‐2 cells. The C‐terminal tripeptide of Ccat, PSI, is necessary for import. Evidence for this were mislocalizations to the cytosol of PSI‐truncated Ccat and AGV‐substituted (for PSI) Ccat. PSI‐COOH, however, was not sufficient to re‐route chloramphenicol acetyltransferase (CAT) from the cytosol to glyoxysomes, whereas the Ccat tetrapeptide RPSI‐COOH was sufficient. Surprisingly, substitution of K (common at the fourth position in other plant catalases) for the R (CAT‐KPSI) decreased import efficiency. However, substitution of K did not affect import, when additional upstream residues in Ccat were included (e.g. CAT‐NVKPSI). Other evidence for the importance of upstream residues comprised abolishment of Ccat import due to substitutions with non‐conserved residues (e.g. ‐AGVNVRPSI for ‐SRLNVRPSI). These data indicate that Ccat is sorted to plant peroxisomes by a degenerate type 1 PTS (PSI‐COOH) whose residues are functionally dependent on a strict context of adjacent C‐terminal amino acid residues.