Rat liver catalase is sorted to peroxisomes by its C-terminal tripeptide Ala-Asn-Leu, not by the internal Ser-Lys-Leu motif.
The molecular signal for targeting catalases to peroxisomes has not been defined. In this study, a plant in vivo import system (tobacco BY-2 suspension culture cells) was used to test the current postulate that the peroxisome targeting signal (PTS) for mammalian catalases is the internal Ser-Lys-Leu (SKL) motif found approximately eight amino acid residues from the C-terminus. Elucidation of the catalase PTS has been hampered previously by the ubiquitous presence of catalase in peroxisomes. The current study was possible because antibodies to mammalian catalases did not recognize endogenous, tobacco peroxisome catalase. Rat and mouse liver catalases (Rcat and Mcat), with an internal Ser-His-Ile (SHI) and Ser-His-Met (SHM), respectively, and both with a C-terminal Ala-Asn-Leu (ANL), were expressed transiently in BY-2 cells and targeted to the peroxisomes. Sorting was demonstrated by double-label immunofluorescence colocalization of these catalases with tobacco catalase. Peroxisome targeting of Rcat was abolished as expected when the internal SHI residues were removed by deletion of three C-terminal portions (28, 16, or 11 residues). Surprisingly, peroxisome targeting was still abolished when SHI (or SHL produced by site-directed mutagenesis) were at the extreme C-terminus as a consequence of deleting eight residues. However, when SHL was at the C-terminus in full-sized Rcat via a mutation of ANL-COOH, the enzyme sorted to peroxisomes indicating that the position of the PTS is significant in Rcat. The importance of the internal context of the SHI (or SHL) was examined further by changing ANL-COOH to a non-SKL motif, AGS-COOH. This Rcat did not sort to the peroxisomes, nor did Rcat with its ANL-COOH deleted; these data indicated the necessity of the C-terminal tripeptide. Sufficiency of ANL was demonstrated when chloramphenicol acetyltransferase with an appended ANL-COOH was redirected from the cytosol to peroxisomes. Collectively, these results do not support the internal PTS hypothesis, but indicate that a type 1 PTS slightly divergent from the typical SKL motif serves as the necessary and sufficient PTS for rat liver and probably other eukaryotic catalases.
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Department of Botany, Arizona State University, Tempe 85287-1601, USA.
Xie
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Lee
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Mullen
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D000818:Animals; D002374:Catalase / Q000378:metabolism*; D002478:Cells, Cultured; D008099:Liver / Q000201:enzymology* / Q000648:ultrastructure; D051379:Mice; D008830:Microbodies / Q000201:enzymology*; D008856:Microscopy, Fluorescence; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000378:metabolism*; D021382:Protein Sorting Signals / Q000378:metabolism*; D051381:Rats; D013329:Structure-Activity Relationship
D009842:Oligopeptides; D021382:Protein Sorting Signals; C088449:seryl-lysyl-leucine; D002374:Catalase