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.
Mullen
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Department of Botany, Arizona State University, Tempe 85287-1601, USA.
Lee
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Trelease
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002374:Catalase / Q000096:biosynthesis* / Q000737:chemistry*; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D000939:Epitopes / Q000032:analysis; D003368:Gossypium / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D021382:Protein Sorting Signals / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D012639:Seeds / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014026:Nicotiana
D017931:DNA Primers; D000939:Epitopes; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D002374:Catalase; D015500:Chloramphenicol O-Acetyltransferase