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Plant Physiology 135:783-800 (2004) © 2004 American Society of Plant Biologists Specification of the Peroxisome Targeting Signals Type 1 and Type 2 of Plant Peroxisomes by Bioinformatics Analyses1,[w]Albrecht-von-Haller-Institute for Plant Sciences, Department for Plant Biochemistry, D–37077 Goettingen, Germany
To specify the C-terminal peroxisome targeting signal type 1 (PTS1) and the N-terminal PTS2 for higher plants, a maximum number of plant cDNAs and expressed sequence tags that are homologous to PTS1- and PTS2-targeted plant proteins was retrieved from the public databases and the primary structure of their targeting domains was analyzed for conserved properties. According to their high overall frequency in the homologs and their widespread occurence in different orthologous groups, nine major PTS1 tripeptides ([SA][RK][LM]> without AKM> plus SRI> and PRL>) and two major PTS2 nonapeptides (R[LI]x5HL) were defined that are considered good indicators for peroxisomal localization if present in unknown proteins. A lower but significant number of homologs contained 1 of 11 minor PTS1 tripeptides or of 9 minor PTS2 nonapeptides, many of which have not been identified before in plant peroxisomal proteins. The region adjacent to the PTS peptides was characterized by specific conserved properties as well, such as a pronounced incidence of basic and Pro residues and a high positive net charge, which probably play an auxiliary role in peroxisomal targeting. By contrast, several peptides with assumed peroxisomal targeting properties were not found in any of the 550 homologs and hence play—if at all—only a minor role in peroxisomal targeting. Based on the definition of these major and minor PTS and on the recognition of additional conserved properties, the accuracy of predicting peroxisomal proteins can be raised and plant genomes can be screened for novel proteins of peroxisomes more successfully.
Peroxisomes are ubiquitous small cell organelles that are involved in a variety of oxidative metabolic processes. Plant peroxisomes participate in recycling of P-glycolate produced by the oxygenase activity of Rubisco during photorespiration and they are the site of fatty acid  -oxidation. Some enzymes of the main metabolic pathways of peroxisomes have been cloned only recently, such as the photorespiratory enzymes Ala-glyoxylate aminotransferase (AGT) and Glu-glyoxylate aminotransferase (GGT; Liepman and Olsen, 2001 , 2003; Igarashi et al., 2003), two isoforms of peroxisomal acyl-CoA synthetase (LACS; Fulda et al., 2002; Hayashi et al., 2002; Shockey et al., 2002) as well as four isoforms of acyl-CoA oxidase with distinct substrate specificity (ACX; Hayashi et al., 1998, 1999; Hooks et al., 1999; Froman et al., 2000). Cloning of several other genes in the past few years and localization of the encoded proteins to plant peroxisomes support the idea that the metabolic function of plant peroxisomes is more diverse than expected and far from being understood in its complexity. For instance, plant peroxisomes do also play an important role in metabolism of reactive oxygen species (Kliebenstein et al., 1998; Lopez-Huertas et al., 2000; del Rio et al., 2002), in the biosynthesis of the hormone jasmonic acid (Sanders et al., 2000; Stintzi and Browse, 2000; Strassner et al., 2002) and the osmoprotectant Glybetaine (Nakamura et al., 1997), in Val catabolism (Zolman et al., 2001), and possibly sulfur metabolism (Eilers et al., 2001; Nakamura et al., 2002). In line with the functional diversity of plant peroxisomes, the proteome of plant peroxisomes seems to be larger than in mammals and fungi (Emanuelsson et al., 2003), which most likely is a result of the large number of metabolically specialized plant microbodies, i.e. leaf peroxisomes, glyoxysomes, and senescent- and nodule-specific peroxisomes as well as hardly characterized unspecialized peroxisomes.
Peroxisomal proteins are encoded in the nucleus and synthesized in the cytoplasm on free ribosomes with targeting signals that specify their delivery to peroxisomes (Lazarow and Fujiki, 1985
Experimental studies revealed a functional degeneracy of the PTS1 motif: a small uncharged residue at position –3, a basic residue at position –2, and a nonpolar residue at position –1 ([SAC][KRH]L>; Gould et al., 1989
The PTS2 of mammalian and yeast proteins has been defined as a nonapeptide with four conserved amino acid residues separated by five variable amino acid residues ([RK][LVI]x5[HQ][LA]; Swinkels et al., 1991
Prediction of the subcellular localization of unknown proteins is a challenge of the postgenomic era but requires targeting motifs of high specificity to maximize the number of true positives and minimize that of false positives. Major difficulties in predicting PTS1-targeted peroxisomal proteins are (1) the small size of the signal, (2) the missing cleavage site that would provide additional diagnostic information, (3) the lower hierarchy of the PTS1 as compared to N-terminal signals (Neuberger et al., 2003b
We aimed to identify novel genes encoding putative peroxisomal proteins in the Arabidopsis genome (Arabidopsis Genome Initiative, 2000
Identification of Sequences Homologous to PTS1- and PTS2-Targeted Proteins
Thanks to large-scale sequencing projects of whole plant genomes (Arabidopsis, Oryza sativa) and ESTs (as of August 2003, 27 plant species with >20,000 ESTs each) the large number of DNA sequences in the public databases provides an enormous amount of biological information that can possibly be used to specify the signals that are indicative of targeting proteins to plant peroxisomes. According to our current knowledge, the targeting mechanism of specific peroxisomal proteins is conserved within the plant kingdom. No peroxisomal protein has been reported that contains a PTS1 in one plant species while carrying a PTS2 or another peroxisomal targeting signal in an ortholog in another plant species or vice versa. Thus, if a specific plant protein is targeted to peroxisomes by a PTS1 (or PTS2), all plant orthologs of this protein are expected to possess a PTS1 (or PTS2). About two-thirds of the currently known matrix proteins from plant peroxisomes contain a C-terminal canonical tripeptide of the PTS1 motif defined by Hayashi et al. (1997)
The starting point of the database search for homologs of PTS1/2-targeted peroxisomal proteins was a retrieval of the Arabidopsis orthologs of all PTS1/2-targeted plant proteins from the protein database (Fig. 1). The genes that were cloned from Arabidopsis were supplemented by the Arabidopsis orthologs of known PTS1/2-targeted matrix proteins, which were identified by sequence similarity. In case of unusually large gene families in Arabidopsis, such as that of the glycolate oxidase (GOX)-related proteins comprising five genes (Reumann, 2002
Bioinformatics Specification of the PTS1 Motif for Higher Plants In total, 391 sequences were retrieved that are homologous to PTS1-targeted peroxisomal matrix proteins and derive from various higher plant species. Of these sequences, 73 represented full-length homologs and 318 homologous ESTs (81%). About one-fourth of the sequences were derived from monocotyledons and three-quarters from dicotyledons and in total from about 80 different plant species. The total number of different C-terminal tripeptides was 39, one-half of which were found several times and represented 95% of the sequences. The remaining 19 tripeptides were not found in any other homologous sequence. Considering the large number of possible tripeptides, these 19 sequences with unique C-terminal tripeptides (5%) are estimated to represent the maximum number of false positives, i.e. homologs corresponding to nonperoxisomal isoforms, incorrectly annotated genes, or incorrectly sequenced ESTs. It can be concluded that the criteria to select the ESTs were reasonably chosen and that the statistical analysis was not considerably disturbed by sequencing errors or an unwanted extraction of sequences derived from nonperoxisomal isoforms.
Within each orthologous group, on average 30.1 ± 14.5 homologs from different plant species were found, reflecting the high sensitivity of the homology analysis as well as the fact that most known proteins of plant peroxisomes are abundant enzymes of primary metabolism, the cDNAs of which are represented in many EST collections. The homologs of each enzyme contained on average 6.5 ± 2.5 different C-terminal tripeptides (all sequences included) and at least 5.2 ± 1.9 different true PTS1 tripeptides (sequences with unique tripeptides excluded). This number indicates that the sequence of the PTS1 is medium conserved within an orthologous group and that a considerable number of different PTS1 tripeptides are produced if summarized over all 13 orthologous groups. The large majority of PTS1 sequences of an orthologous group contained a canonical C-terminal tripeptide included in the plant-specific PTS1 motif defined by Hayashi (93% ± 4%, SOX homologs excluded, [SACP][KR][LMI]>; Hayashi et al., 1997
The possible 24 tripeptides of the conservative Hayashi motif ([SACP][KR][LMI]>) turned out to differ largely in the frequency at which they occur in the homologs of PTS1-targeted proteins. Nine major PTS1 tripeptides can be defined, each of them comprising at least 10 ESTs and occurring in at least 3 different orthologous groups (Fig. 3). They are headed by far by SRL> (91 sequences, 23.3%), which accordingly represents the prototypical plant PTS1, followed by SRM> and SKL>, PRL>, ARL>, and SRI>, and finally AKL>, SKM>, and ARM>. These major PTS1 tripeptides ([SA][RK][LM]> without AKM> plus SRI> and PRL>) are thought to represent the most reliable indicators for peroxisomal localization and covered altogether 84% of the homologous sequences of PTS1-targeted proteins (Fig. 3).
A smaller number of sequences contained any of the tripeptides PKL>, PRM>, SKI>, CKL>, or CRL>, most of which were found in ESTs and have not been reported in plant peroxisomal matrix proteins before. These tripeptides are defined as minor PTS1 tripeptides and considered functional but low-abundant PTS1. If only a single sequence with a particular C-terminal tripeptide per orthologous group is considered, the overall result is similar even though the frequency of some tripeptides is lower (e.g. SRL>, SRM>, SRI>, SKL>, and PRL>), indicating their dominance in particular orthologous groups (Fig. 2), whereas that of others is higher (e.g. SKI>, PRM>, and CRL>), reflecting the fact that these tripeptides occur in a number of orthologous groups above average. The tripeptides AKI>, CRM>, and CRI> were found in single sequences and probably also represent low-abundant variations of the plant PTS1. By contrast, no homologs of PTS1-targeted proteins were detected that contained any of the remaining tripeptides included in the most conservative pattern of the plant-specific PTS1 (ARI>, AKM>, PRI>, PKM>, PKI>, CKM>, and CKI>; Hayashi et al., 1997 ). The data imply that these undetectable tripeptides do not indicate accurately targeting to plant peroxisomes. The enzyme sulfite oxidase (SOX), which contains a noncanonical PTS1 tripeptide in Arabidopsis itself (SNL>), is unusual because the majority of its homologs (14 out of 19) contain a noncanonical PTS1 as well, such as SNM>, SSM>, SAM>, or ANL>. Because SNL> is found in several PTS1 proteins from yeast (e.g. D-Asp oxidase; Amery et al., 1998) and because a few SOX homologs contained a canonical PTS1 (e.g. SKL> and SKM>; Supplemental Table SI), the targeting pathway of SOX is not expected to differ substantially from the well-characterized PTS1 pathway. In total, 6 noncanonical tripeptides were found in several ESTs and considered low-abundant minor PTS1 (Fig. 3). Of these the tripeptide SRV> was most widespread, being found in 6 ESTs and 4 different orthologous groups. If particular amino acid combinations are by far more widespread as PTS1 tripeptides than other closely related combinations, the question arises whether rules can be deduced about which combinations yield a functional PTS1. The frequency of each amino acid residue at its specific position within the tripeptide was calculated and the amino acids classified accordingly (Fig. 4A). Highly abundant amino acids in PTS1 are S, R, and L of the tripeptide SRL>, medium abundant K and M, and the remaining low abundant (Fig. 4A). The amino acids differed also in the number of different PTS1 tripeptides in which they occurred. The residues S, R, and L were found in a large number of different tripeptides (9–11) and seem to reveal a broad tolerance with respect to the residues at the remaining positions, whereas other amino acids, such as P, C, N, and I, were restricted to a small number of very specific tripeptides and mostly to combinations with two residues of the motif S[RK]L> (Fig. 4B). Combinations exclusively of amino acids none of which belongs to the prototypical plant PTS1 SRL> were not found in any plant protein (e.g. A, P, and C at position –3; K, N, M, and S at position –2; and M, I, and V at position –1).
Conserved Properties of the PTS1 Domain Sequences upstream of the PTS1 may have an auxiliary function as accessory elements in targeting proteins to peroxisomes and can provide additional indications for peroxisomal localization of unknown proteins. To investigate whether PTS1-targeted proteins contain conserved sequences upstream of the PTS1 tripeptide, the C-terminal 18 amino acids of the PTS1 targeting domains were analyzed successively in groups of tripeptides both per orthologous group (data not shown) and per group of identical PTS1 tripeptides (Fig. 5). The content of basic residues, R and K, was found to increase significantly from an average value in the core protein (about 10%, position –18 to –7) to 24% in the tripeptide in front of the PTS1 and to 32% in the PTS1 tripeptide itself (Fig. 5A). Thus, most PTS1-targeted proteins carry a second basic residue closely in front of the PTS1 tripeptide. The rise in the content of basic residues is accompanied by a decrease in the content of acidic residues toward the C terminus (Fig. 5A). The uneven distribution of charged residues leads to an increasing positive net charge toward the C-terminal end with a total positive charge of the C-terminal 6-mer of 1.6 and results in a significant increase of the pI to 12 (Fig. 5, B and C). Because of the low SD, the pI in particular is a useful additional criterion to confirm the postulated peroxisomal localization of unknown proteins. In addition the PTS1 domain is characterized by a high probability of P occurring in front of the PTS1 (Fig. 5D), showing that plant PTS1 proteins contain on average 0.70 ± 0.61 P residues within the 6-mer preceeding the PTS1 tripeptide.
For proteins with an unusually low positive charge directly in front of the PTS1 peptide, such as those carrying AKL> and SKL> (Fig. 6, A and B), the positive charge seems to be spread over a larger region of 9 to 12 residues upstream of the PTS1 tripeptide, leading to similar values for the entire domain compared with the other PTS1 groups. In parallel, the position of P also is shifted further upstream (Fig. 6B). Closer inspection of sequence variations in the targeting domain within an orthologous group revealed that changes of the PTS from the prototypical targeting peptide SRL> or SKL> to low-abundant peptides of presumably weaker targeting efficiency (see "Discussion") are often accompanied by a simultaneous addition of further basic and/or P residues in close proximity to the PTS1 peptide (Fig. 7, A–C). Overall, the second basic residue was mostly located at position –4 or at position –6. Only for proteins with M at position –1 of the PTS1 tripeptide, such as ARM>, PRM>, SKM>, and SRM>, which all contain a second basic residue in close proximity to the PTS1 tripeptide except for one-half of the GGT homologs, a preferential localization of the second basic residue at position –4 was found (Fig. 7D).
In light of targeting prediction an important question is whether different conserved properties function independently of each other or can compensate for each other. In case of an auxiliary function of specific amino acids in front of the PTS1 tripeptide in facilitating recognition by the cytosolic receptor Pex5, basic and P residues seem to be able to complement the accessory targeting function of each other. Most proteins with M at position –1 of the PTS1 peptide lack a P residue in front of the PTS1 tripeptide but are characterized by a high positive charge above average in the 3-mer in front of the PTS1 tripeptide (Fig. 6, C and D). Vice versa, proteins terminating with SRI>, most of which are AGT homologs, do not carry a pronounced positive net charge outside of the PTS1 tripeptide but contain about 2 P residues in the 6-mer adjacent to the PTS1 (Fig. 6, E and F). In summary, the PTS1 targeting domain comprises about 12 to 15 residues with characteristic properties, and the 3-mer directly in front of the PTS1 tripeptide is characterized by a positive net charge and a high P content. At least one of these properties seems to be required in plant PTS1 proteins to enhance targeting to peroxisomes.
About one-third of the proteins from plant peroxisomes contain an N-terminal PTS2 (Supplemental Table SI). Except for the peroxisomal Hsp70 homolog (Wimmer et al., 1997
Upon analysis of the neighboring regions of the PTS2 nonapeptide for conserved properties, the size of the PTS2 targeting domain was defined as a region of approximately 15 residues surrounding roughly symmetrically the PTS2 nonapeptide (position –3 to 12). Common features of the PTS2 domain are the following (Fig. 10): (1) a high incidence of R, (2) a low content of acidic residues (Fig. 10A), (3) a high positive charge of the targeting domain of on average 2.2 ± 0.7 (Fig. 10B), (4) a pronounced increase in pI, of which the low SD within the x5 region is most noteworthy (Fig. 10C), (5) a strict absence of P in front of the nonapeptide and within the x5 region, contrasting with a frequent presence immediately downstream of the PTS2, and (6) a high incidence of A, L, and V in front of the nonapeptide and within the x5 region (Fig. 10D). In addition, the sequences imply a conserved secondary structure of the PTS2 domain because in the large majority of sequences examined, the nonapeptide seemed to be located in the end of a short  -helix (data not shown).
The large amount of biological information in the public databases opens the possibility to apply bioinformatics tools to build up novel hypotheses and to answer biological questions. The complete genome sequences of Arabidopsis and Oryza allow now the extraction of genes encoding unknown proteins with a putative PTS1 or PTS2. To increase the prediction accuracy of peroxisomal localization, an exact definition of the PTS is crucial. Experimental studies have provided valuable information on plant-specific PTS motifs but suffer from three important drawbacks. First, the number of experimentally analyzed PTS peptides is limited, and solid support for the assumption that the amino acids of different functional PTS can freely be combined is currently lacking. For instance, inclusion of G and T (position –3) in the permissive PTS1 motif is solely based on positive targeting results of three tripeptides (GRL>, GKL>, and TKL>; Mullen et al., 1997a ; Kragler et al., 1998), and data are lacking on whether the remaining 28 tripeptides constitute functional PTS1 ([GT][HNRK][IMY]> and [GT][HN]L>). Second, targeting efficiency is difficult to quantify experimentally, resulting in mostly qualitative results. The yeast-two-hybrid system can partially overcome these limits but restricts the multi-step targeting pathway to the binding affinity between the PTS1 tripeptide and Pex5 and thus relies for plant proteins on a heterologous system (Kragler et al., 1998). Third, the use of different expression systems (constitutive or transient expression) and a negligence of accessory sequences may have accounted for some discrepant experimental targeting results, for instance, those of the tripeptides SHL>, SLL>, and GRL> (Hayashi et al., 1997; Mullen et al., 1997a; Kragler et al., 1998).
Bioinformatics analyses can possibly provide additional information to specify targeting motifs. Complete plant genome sequences provide novel essential information on the size of gene families, the subcellular localization of different isoforms, and on sequence similarity shared between orthologs and paralogs, all of which are prerequisite for an unambiguous identification of homologous sequences that correspond to specific isoforms in species lacking a sequenced genome. Especially kingdom-specific variations of targeting signals can be deduced from such bioinformatics analyses. The frequency at which PTS1 and PTS2 peptides occur in plant proteins reflects a close to final stage of the ongoing evolutionary optimization of targeting signals and reflects semiquantitatively the targeting efficiency of these peptides (see below). The large data set of PTS-targeted sequences also allows a study of the targeting signals within their native context and facilitates the detection of yet unrecognized accessory sequences with an auxiliary targeting function by sequence conservation. Critical to the specificity of targeting motifs deduced from such analyses is a reasonable compromise between the identification of a maximum number of homologous sequences and a minimum number of false positives. A major factor to increase the size of the data set to about 550 sequences (catalase homologs excluded) and thereby more than 10-fold as compared to the number of cloned genes was the use of EST databases, which contributed about 80% of the sequences. To prevent that the higher rate of sequencing errors of ESTs led to an erroneous identification of PTS1 tripeptides particularly because of their localization next to a stop codon, reasonable requirements for the selection of ESTs were chosen and all sequences manually inspected. In cases of a problematic differentiation between homologs corresponding to peroxisomal or nonperoxisomal isoforms due to the small length of ESTs, further bioinformatics tools were applied to identify the homologs of interest but three peroxisomal proteins had to be excluded beforehand.
The number of false PTS peptides determines the minimum number of sequences required to judge a particular tri- or nonapeptide a functional PTS and was estimated. Considering that the number of different C-terminal tripeptides is 8,000 and that the number of noncanonical tripeptides that can by created by single amino acid substitutions from canonical PTS1 is 448, it is not expected that 2 identical tripeptides can derive from canonical PTS1 by random point mutations within a total number of 39 different detected tripeptides and 23 sequences that contained a tripeptide found only once or twice. Therefore, those sequences containing unique targeting peptides are considered to represent the maximum number of false positives, which is relatively low (PTS1, 5%; PTS2, 0.6%). Because many of these unique peptides are either included in the conservative PTS1 motif defined by Hayashi et al. (1997)
A certain degree of sequence variation of the PTS within an orthologous group is required to allow the identification of PTS peptides rather independently of accessory elements specific to particular orthologous groups and to reach high coverage of all PTS1/2 peptides present in higher plants despite the relatively low number of input proteins as compared to the large size of the estimated peroxisomal proteome (Fukao et al., 2002
The high number of homologous sequences identified, the low number of false positives, and the medium degree of sequence variability of the PTS within orthologous groups allowed a statistically significant analysis of the frequency of particular PTS tripeptides in the plant kingdom. Regarding first the PTS1 proteins, not expected and most important is the result that only a small specific subset of PTS1 tripeptides is widespread in plant proteins and seems to constitute functional PTS peptides. Nine PTS1 tripeptides ([SA][RK][LM]> without AKM> plus SRI> and PRL>) have been defined as major PTS, of which PRL> and SRI> had shown only weak targeting efficiencies in experimental studies (Hayashi et al., 1997
From a position-specific quantitative analysis of the amino acids, it can be concluded which amino acid residues are tolerated in PTS1 peptides. Overall, the eleven amino acids of the restrictive PTS1 motif were indeed present in functional PTS ([CASP][KR][ILM]>; Hayashi et al., 1997
The strikingly different abundance of PTS1 peptides in plant proteins is interpreted from an evolutionary point of view in the following way: (1) a strong selection pressure has optimized the primary structure of PTS1 tripeptides with the result that the abundance of specific peptides in nature differs drastically nowadays, and (2) the current abundance of specific peptides in plant proteins correlates roughly with their targeting efficiency. In line with experimental results, highly abundant peptides, defined as major PTS peptides in this study, can be regarded as peptides with strong peroxisomal targeting properties (Kragler et al., 1998
The region upstream of the PTS1 tripeptide was also characterized by conserved properties. According to our analysis, the PTS1 targeting domain with characteristic and diagnostic properties comprises about 12 to 15 amino acids, similar to recent results for fungi and mammals (Neuberger et al. 2003a
Taken together, conserved properties upstream of the PTS1 tripeptide represent important information to further increase the prediction accuracy and shed new light on previous experimental results. The negative charge present upstream of the PTS1 in
As for the PTS1, only a specific subset of PTS2 nonapeptides seems to be widespread in the plant kingdom and to constitute functional PTS. Two major (R[LI]x5HL) and nine minor PTS2 nonapeptides were defined. In support of the conservative variant of the plant PTS2 motif (R[LIQ]x5HL, Kato et al., 1998
The PTS2 RQx5HL is unusual in several aspects. The nonapeptide was present in all plant thiolase homologs and exclusively present in this orthologous group. Moreover, to the best of our knowledge, Q (position 2) is absent in all known PTS2-targeted proteins from mammals and fungi including thiolase homologs. The fact that no stable single point mutation [LITMAV]
Strikingly similar to PTS1-targeted proteins, the PTS2 targeting domain was characterized by a high content of basic residues accompanied by a lack of acidic residues, resulting in a basic pI and a high positive charge. In this case, the positive net charge was concentrated upstream of the nonapeptide and within the x5 region, whereas P was predominantly present downstream of the PTS2. In addition, the PTS2 domain was significantly enriched in three hydrophobic residues, namely A, L, and V, and mostly forms a short
Application of the major PTS peptides to genome screens are expected to lead to the identification of a large number of interesting novel proteins from plant peroxisomes. By contrast, those peptides that have been included in experimentally determined plant PTS motifs but not been defined as functional PTS in this study are not recommended for any genome screens. For minor PTS peptides, true positives are expected, but an elevated number of nonperoxisomal proteins needs to be anticipated as well. It needs to be pointed out that the indicative value of peroxisomal targeting differs significantly within the group of minor PTS peptides. The tripeptide PKL>, for instance, is relatively close to the empirical threshold for peptides defined as major PTS peptides and close to the frequency of the major PTS1 ARM>, whereas other minor PTS1 tripeptides were rare and only found in single orthologous groups (e.g. GOX, SML>; SOX, SNL>, ANL>, SNM>, SSM>). In this regard, unknown structural properties of SOX are suspected to be responsible for the unusual tolerance of these noncanonical PTS1 peptides as compared to other PTS1-targeted proteins and need to be investigated experimentally. Taken together, targeting to peroxisomes should only be predicted if a minor PTS is detected in at least two to three different orthologous groups and present in combination with other conserved properties of peroxisomal targeting domains (Table I). As a general rule of thumb, peroxisomal targeting of atypical putative PTS1 can be predicted in the following way: First, the higher the abundance of each single amino acid of the tripeptide of interest in all PTS1 proteins examined in this study, the higher the probability that this tripeptide targets a protein to plant peroxisomes. Second, a low-abundant amino acid is only found in a functional tripeptide in the presence of one to two high-abundant amino acids with presumably high targeting properties. To improve targeting prediction, the amino acids of the plant-specific motif are now given in order of abundance and deduced targeting strength (PTS1, [SAPC][RKNMS][LMIV]>). For PTS2 proteins, R (position 1), L (position 2, 9), H (position 8), and, to a lesser degree, I (position 2, 9) represent residues of presumably strong targeting properties; at least three of these residues need to be present to constitute a functional PTS2 nonapeptide (R[LIQTMAV]x5H[LIF]).
Before starting time-consuming experiments, confirmation of the postulated peroxisomal localization of unknown proteins by further bioinformatics analyses is recommended. Postulated targeting to plant peroxisomes can sometimes be supported by results provided by new subcellular prediction software for peroxisomes despite their lack of plant-specific algorithms (PeroxiP; Emanuelsson et al., 2003 ; PTS1 predictor, http://mendel.imp.univie.ac.at/mendeljsp/sat/pts1/PTS1predictor.jsp; Neuberger et al., 2003a, 2003b). Dual subcellular localization of peroxisomal proteins has been reported (Hayashi et al., 1996b; Wimmer et al., 1997; Kunze et al., 2002) but is expected to represent an exception from the general rule of an exclusive localization of peroxisomal proteins in microbodies. Thus, the presence of accurately predicted plastidic transit peptides, mitochondrial presequences, or ER signal peptides can mostly be considered as evidence against a localization of the same protein in peroxisomes. The peroxisomal predictor PeroxiP even excludes proteins with secretory signals as potential peroxisomal matrix proteins (Emanuelsson et al., 2003). Because the prediction of a protein being peroxisomal is strengthened if its orthologs in other organisms possess a PTS1/2 as well, strongest support for the predicted peroxisomal localization can be provided by identification of homologous proteins with a PTS in other plant species similar to the strategy of this study. Finally, the prediction accuracy of PTS1/2-targeted proteins can be further raised by analyzing the neighboring region for accessory elements found to be conserved in PTS1/2-targeted proteins. These properties are more pronounced in proteins carrying a minor PTS and should be considered as positive complimentary indications for peroxisomal localization because we are only in the beginning of understanding the structure of these accessory elements and because not all properties are strictly conserved in the orthologous groups.
The number of plant genomes, the number and size of EST collections, and—most important—the number of known proteins of plant peroxisomes will increase in the near future and affect the results provided in this study. Therefore, this definition of functional PTS peptides represents an intermediate result. Whereas the frequency and the strong indicative properties of major PTS are not expected to change significantly, new database entries will affect the frequency of minor and currently undetected PTS. The tripeptide CRM> found in a single sequence, for instance, was currently excluded but is expected to represent a rare but functional PTS1 (Kragler et al., 1998
In summary, the major and minor PTS deduced from this study are recommended for genome screens for novel peroxisomal matrix protein. In the Arabidopsis genome, about 170 proteins with a major PTS and 110 with a minor PTS are detected (S. Reumann, C. Ma, S. Lemke, and L. Babujee, unpublished data). We are currently setting up a database of Arabidopsis proteins with a putative PTS1 or PTS2 that will be presented in a forthcoming article and publicly available in the near future.
Selection of the Arabidopsis Proteins Suitable for the Bioinformatics Analysis
All peroxisomal sequences that were cloned from Arabidopsis and encode PTS1/2-targeted proteins were retrieved. The Arabidopsis orthologs of PTS1/2-targeted proteins that were only cloned from other plant species were identified based on sequence similarity using blastp (http://www.ncbi.nlm.nih.gov/BLAST/; nonredundant database; E value threshold: 10; matrix BLOSUM62, gap costs: existence: 11, extension: 1; Supplemental Table SI). Proteins were used for subsequent bioinformatics analyses if they fulfilled the following criteria: (1) localization of one protein of an orthologous group to plant peroxisomes by biochemical studies using native proteins or by in vivo localization studies using fusion proteins; (2) identification of the PTS by redirection of the protein with deleted PTS to the cytosol as compared to the full-length protein; and (3) possible differentiation between homologs corresponding to peroxisomal isoforms or to isoforms from other cell compartments by sequence similarity. With respect to the enzyme CHY1 (Zolman et al., 2001
To identify the plant homologs of known PTS1-targeted proteins from various plant species, the entire amino acid sequence of the Arabidopsis orthologs was blasted against the nonredundant database and the database of ESTs of GenBank at the National Center for Biotechnology Information using blastp and tblastn, respectively (http://www.ncbi.nlm.nih.gov/; E value threshold: 10; matrix BLOSUM62, gap costs: existence: 11, extension: 1; Supplemental Table SI). All blast searches were updated in August and September 2003, when the nonredundant database contained about 1,500,000 sequences and the database dbEst at NCBI the following number of sequences (for different species of the same genus, only the species with the largest EST collection is listed): 200,000 EST clones were selected for further analysis if they met the following requirements: (1) sufficient size (mostly longer than 150 bp), (2) sufficient sequence quality (no undefined nucleotides "x" in the sequence) and no stop codon within the C-terminal 20 amino acids (PTS1 homologs) or the N-terminal 50 amino acids (PTS2), (3) homology throughout the entire domain, and (4) identification as the homolog corresponding to the peroxisomal isoforms based on sequence similarity or analysis by multiple sequence alignments. Some ESTs were excluded because an abrupt reduction of sequence conservation in the C-terminal region suggested an upstream sequencing error leading to a frame shift. For the selected ESTs, the nucleotide sequence was then translated (http://www.expasy.org/tools/dna.html). Regarding the homologs of PTS1-targeted proteins, EST sequences were only included if the peptide that aligned with the C terminus of the open reading frame was followed by a stop codon (about 5 amino acids up- or downstream of the PTS1 of the query). Regarding the homologs of PTS2-targeted proteins, EST sequences were only included if the peptide that aligned with the N terminus of the peroxisomal protein was preceded by an M (about 20 amino acids upstream of the PTS2 of the query). The amino acid sequence of the C-terminal 20 amino acids (PTS1 proteins) or that of the N-terminal 50 amino acids (PTS2 proteins) was saved for later analysis of the targeting domain for conserved properties. For plants with large EST collections, several homologous cDNAs or ESTs from the same plant species or the same genus were present in the database that differed only slightly (e.g. Sorghum bicolor and S. propinquum, Triticum aestivum and T. monococcum, Oryza sativa and O. minuta). In an attempt to distinguish sequences with sequencing errors from sequences of closely related genes, an empirical threshold was chosen. Regarding homologs of PTS1-targeted proteins, additional sequences were excluded unless the C-terminal 20 amino acids differed by at least three amino acids (<90% sequence identity) compared to another homologous sequence from the same plant species or genus. The N-terminal 20 amino acids of PTS2-targeted proteins were handled analogously. To facilitate detection of these ESTs, homologous ESTs of the plant species or genus of interest were often used as query. For peroxisomal proteins that are encoded by several genes in Arabidopsis (e.g. ACX1, HIBCH, thiolase, citrate synthase, multi-functional protein, etc.), the application of this empirical rule resulted in a number of homologous sequences in large EST collections that was close to the size of the gene family in Arabidopsis.
For analysis of amino acid composition, charge, and pI of the targeting domain, the sequences were grouped according to their PTS peptide. Groups of canonical PTS1 tripeptides containing less than five sequences (CKL>, CRL>, SKI>) and the noncanonical tripeptides (ANL>, SML>, SNL>, SNM>, SRV>, and SSM>) were combined and analyzed together. Similarly, groups of PTS2 nonapeptides containing less than five sequences (RLx5HF, RLx5HI, RIx5HI, RAx5HL, RAx5HI, and RVx5HL) were analyzed together. For PTS1 sequences, the C-terminal 18 amino acids were analyzed in groups of three amino acids and provided with position-specific numbers relative to the PTS1 tripeptide (PTS1, position –1 to –3; 3-mer preceeding the PTS1, position –4 to –6). For PTS2 sequences, the residues of the nonapeptide were numbered according to the traditional system (PTS2, position 1 to 9) and residues in front and behind the nonapeptide were provided with negative and positive numbers, respectively. The amino acid composition of PTS2 proteins was analyzed in two groups of three amino acids in front of the nonapeptide (position –6 to –4 and position –3 to –1), the first two conserved residues of the nonapeptide (Rx, position 1, 2), the five residues of the unspecific x5 region (position 3 to 7), the other two conserved residues of the nonapeptide (Hx, position 8, 9), as well as two groups of three amino acids behind the nonapeptide (position 10 to 12 and position 13 to 15). For these groups, the amino acid composition, the absolute content of basic and acidic residues, the charge (difference of basic and acidic residues), and the pI were calculated (http://us.expasy.org/tools/protparam.html) and mean values and SD determined. Secondary structure was analyzed using PredictProtein (http://www.embl-heidelberg.de/predictprotein/predictprotein.html).
I thank Martin Fulda, Ivo Feussner, Hans-Walter Heldt, and the students of my group for stimulating discussions and critical comments on the manuscript with special thanks to Katharina Pawlowski. Received November 2, 2003; returned for revision January 22, 2004; accepted January 22, 2004.
1 This work was supported by the Deutsche Forschungsgemeinschaft (Re1304/2–1). 
[w] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.103.035584. * E-mail sreuman{at}gwdg.de; fax 49–551–395749.
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Zolman BK, Monroe-Augustus M, Thompson B, Hawes JW, Krukenberg KA, Matsuda SPT, Bartel B (2001) chy1, an Arabidopsis mutant with impaired This article has been cited by other articles:
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15%; A, P, and C at position –3; N, M, and S at position –2, and I and V at position –1). A, The position-specific frequency of amino acids roughly correlates with the number of different PTS1 peptides in which they are present (B). Sequences with unique C-terminal tripeptides were not considered.
10 sequences and 
Q has occurred in any of the 110 PTS2 sequences of nonthiolase proteins strongly suggests that yet unrecognized features of the PTS2 domain of thiolase need to act synergistically with this nonapeptide to evoke targeting to peroxisomes. This hypothesis is further supported by the result that the mutation R(L
