A Conserved Tripeptide Sorts Proteins to Peroxisomes

The firefly luciferase protein contains a peroxisomal targeting signal at its extreme COOH terminus (Gould et al., 1987). Site-directed mutagenesis of the luciferase gene reveals that this peroxisomal targeting signal consists of the COOH-terminal three amino acids of the protein, serine-lysine-leucine. When this tripeptide is appended to the COOH terminus of a cytosolic protein (chloramphenicol acetyltransferase), it is sufficient to direct the fusion protein into peroxisomes. Additional mutagenesis experiments reveal that only a limited number of conservative changes can be made in this tripeptide targeting signal without abolishing its activity. These results indicate that peroxisomal protein import, unlike other types of transmembrane translocation, is dependent upon a conserved amino acid sequence. I N eukaryotic cells, the sorting of proteins to their proper subcellular locations is often mediated by cis-acting sequences present in the transported polypeptide (Blobel, 1980). This mode of protein segregation has been demonstrated for protein import into mitochondria (Hase et al., 1984), chloroplasts (Van den Broek et al., 1985), lysosomes (Johnson et al., 1987), the nucleus (Kalderon et al., 1984), and the endoplasmic reticulum (ER)Vsecretion pathway (for review, see Walter and Lingappa, 1986) as well as for retention of proteins in the ER (Munro and Pelham, 1987). Only recently has this observation been extended to the process of peroxisomal protein import (Gould et al., 1987, 1988; Small et al., 1988). Proteins destined to reside in the peroxisome are transported into the organelle posttranslationally after synthesis on free polysomes. Unlike import of proteins into mitochondria, chloroplasts, or the ER/secretion pathway, peroxisomal import is not associated with removal ofa presequence or with any other detectable modification of the imported polypeptide (for a review of peroxisomal protein biogenesis, see Lazarow and Fujiki, 1985). We have previously demonstrated that the peroxisomal protein luciferase (Keller et al., 1987) contains a peroxisomal targeting signal (PTS) within its COOH-terminal twelve amino acids (Gould et al., 1987). More recently, we identified additional PTSs at or near the COOH terminus of the peroxisomal proteins human catalase, pig D-amino acid oxidase, rat acylcoenzyme A (CoA) oxidase, and rat enoylCoA/hydratase-3-hydroxyacyl CoA dehydrogenase (Gould et al., 1988), and also in the Candida boidinii PMP-20 protein (Gould et al., manuscript in preparation). Miyazawa et al. (1989) have recently demonstrated that the COOH terminus of rat acyl-CoA oxidase, which had been previously shown 1. Abbreviations used in this paper: CAT, chloramphenicol acetyltransferase; CoA, coenzyme A; PTS, peroxisomal targeting signal. to contain a PTS (Gould et al., 1988), is also necessary for its own import into rat liver peroxisomes in vitro. To further investigate the nature of the PTS, we have created mutations that alter the amino acid sequence of luciferase at or near the COOH terminus of the protein. The mutant proteins were tested for their ability to be imported into peroxisomes in vivo. The results of these experiments indicate that a tripeptide (serine, alanine, or cysteine at the first position; lysine, histidine, or arginine at the second; leucine at the third) is capable of acting as a PTS when present at the COOH terminus of a protein. A search of known peroxisomal protein sequences reveals that this tripeptide is a common feature of many peroxisomal proteins. These data suggest that a consensus three-amino-acid targeting signal is responsible for the import of a large number of peroxisomal proteins. In contrast, other processes that transport proteins across membranes (ER, mitochondrial, or chloroplast transport) are usually mediated through larger NH2-terminal signals that are cleaved upon import and lack sequence conservation. The unique properties of peroxisomal protein import indicate that it may involve a fundamentally different type of protein translocation mechanism. Materials and Methods