RESEARCH PAPER |
Improved prediction of peroxisomal PTS1 proteins from genome sequences based on experimental subcellular targeting analyses as exemplified for protein kinases from Arabidopsis
Department of Plant Biochemistry, Georg-August-University of Goettingen, Albrecht-von-Haller-Institute for Plant Sciences, Justus-von-Liebig-Weg 11, D-37077 Goettingen, Germany
To whom correspondence should be addressed. E-mail: sigrun.reumann{at}uis.no
Received 3 July 2008; Revised 25 July 2008 Accepted 5 August 2008
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Abstract |
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Due to current experimental limitations in peroxisome proteome research, the identification of low-abundance regulatory proteins such as protein kinases largely relies on computational protein prediction. To test and improve the identification of regulatory proteins by such a prediction-based approach, the Arabidopsis genome was screened for genes that encode protein kinases with predicted type 1 or type 2 peroxisome targeting signals (PTS1 or PTS2). Upon transient expression in onion epidermal cells, the predicted PTS1 domains of four of the seven protein kinases re-directed the reporter protein, enhanced yellow green fluorescent (EYFP), to peroxisomes and were thus verified as functional PTS1 domains. The full-length fusions, however, remained cytosolic, suggesting that PTS1 exposure is induced by specific signals. To investigate why peroxisome targeting of three other kinases was incorrectly predicted and ultimately to improve the prediction algorithms, selected amino acid residues located upstream of PTS1 tripeptides were mutated and the effect on subcellular targeting of the reporter protein was analysed. Acidic residues in close proximity to major PTS1 tripeptides were demonstrated to inhibit protein targeting to plant peroxisomes even in the case of the prototypical PTS1 tripeptide SKL>, whereas basic residues function as essential auxiliary targeting elements in front of weak PTS1 tripeptides such as SHL>. The functional characterization of these inhibitory and essential enhancer-targeting elements allows their consideration in predictive algorithms to improve the prediction accuracy of PTS1 proteins from genome sequences.
Key words: Kinases, metabolism, peroxisomes, phosphorylation, targeting prediction
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Peroxisomes are ubiquitous eukaryotic cell organelles that compartmentalize a large variety of oxidative metabolic reactions. Plant peroxisomes play essential roles in glycolate recycling and amino acid biosynthesis during photosynthesis and in fatty acid degradation during lipid mobilization in germinating seeds (Graham and Eastmond, 2002; Reumann and Weber, 2006). Peroxisomes of higher plants also house enzymes that are involved in nitrogen and sulphur metabolism (Eilers et al., 2001; Verma, 2002; Hansch et al., 2006), the degradation of branched amino acids (Zolman et al., 2001), and the biosynthesis of the plant hormone jasmonic acid (Stintzi and Browse, 2000; Schneider et al., 2005; Koo et al., 2006). Plant peroxisomes differentiate in a tissue-specific and developmentally regulated manner into metabolically specialized microbodies, referred to as leaf peroxisomes in photosynthetically active green tissue, glyoxysomes in oil-depositing seeds and cotyledons, glyoxysome-related gerontosomes in senescent tissue, nodule-specific peroxisomes in symbiotic plants, and as yet uncharacterized unspecialized peroxisomes (Graham and Eastmond, 2002; Verma, 2002; Reumann and Weber, 2006).
Apart from classical enzymes that catalyse metabolic reactions, the current knowledge on peroxisomal matrix proteins with regulatory or chaperone function is rather limited (Ma et al., 2006). In fact, our understanding of the physiological function of plant peroxisomes reveals probably the largest gaps in post-translational regulation of peroxisomal metabolism. Rapid adaptation to changing environmental conditions is often achieved by fine-tuning the kinetic properties of key enzymes by reversible phosphorylation, as studied in detail, for instance, for nitrate reductase and sucrose-6-phosphate synthase (for a review see Huber, 2007). The regulation of key enzymes of peroxisomal metabolism by reversible phosphorylation is reasonable to postulate, but has not been conclusively demonstrated yet, nor have signal transduction pathways been described. Only phosphorylation of Ricinus malate synthase has been reported (Yang et al., 1988). Preliminary evidence for the existence of protein kinases and phosphatases in plant peroxisomes has recently emerged, as a calcium-dependent membrane-anchored protein kinase and glyoxysomal protein kinase 1 (GPK1) have been described (Dammann et al., 2003; Fukao et al., 2003). Several leaf peroxisomal enzymes exhibit isoelectric focusing and electrophoretic migration patterns on two-dimensional gels that are typical for post-translationally modified proteins (Reumann et al., 2007). Also, two putative Arabidopsis protein phosphatases carry putative peroxisome targeting signals (PTSs) and are prime candidates for the dephosphorylation of matrix enzymes (Reumann et al., 2004).
Most known peroxisomal enzymes are targeted to the matrix either by a PTS1, the C-terminal so-called ‘SKL-motif’, or by a PTS2, which is a conserved nonapeptide of the prototype RLx5HL located in the N-terminal domain (Gould et al., 1989; Swinkels et al., 1991; see also references in Reumann, 2004). Both targeting pathways to peroxisomes are conserved throughout the eukaryotic kingdom. The cytosolic receptors Pex5p and Pex7p for PTS1- and PTS2-targeted proteins, respectively, share high sequence similarity between different organisms and largely carry out a conserved function. Both receptor proteins, however, differ between fungi, mammals, and plants in some aspects regarding, for instance, their affinity for specific targeting peptides, which is reflected by experimentally determined kingdom-specific PTS consensus motifs. Plant-specific PTS motifs have been deduced from experimental targeting studies in vivo (Hayashi et al., 1997; Mullen et al., 1997; Flynn et al., 1998; Kato et al., 1998; Kragler et al., 1998). From an in silico study of plant expressed sequence tag (EST) databases, it was concluded that only a small number of stringently defined PTS1 and PTS2 peptides are widespread in nature and indicate peroxisome targeting of unknown proteins with moderate to high probability (Reumann, 2004).
In the post-genomic era of plant research, proteome analyses have emerged as an important experimental strategy to identify novel peroxisomal proteins on a large scale. A few proteome analyses of plant peroxisomes have been reported (Fukao et al., 2002, 2003; Reumann et al., 2007; Arai et al., 2008). The identification of low-abundance proteins such as regulatory proteins by this approach, however, remains challenging due to the predominance of metabolic enzymes, which requires the isolation of extremely pure Arabidopsis peroxisomes. Additionally, protein kinases are often targeted to multiple cell compartments at selected developmental stages or under very specific physiological conditions.
Protein targeting prediction of putative peroxisomal proteins applied to whole genomes in combination with experimental subcellular targeting analyses of candidate proteins are a powerful alternative to proteomics. The prediction of plant peroxisomal matrix proteins is currently based on the presence of PTS1 tripeptides and PTS2 nonapeptides at the extreme C-terminus and the N-terminal 40-amino acid residue domain, respectively, in the proteins of interest. This strategy applied to Arabidopsis revealed 220 putative PTS1 and 60 PTS2 proteins listed in the database AraPerox (Reumann et al., 2004; www3.uis.no/AraPeroxV1). Despite their potential, these protein predictions include some false positives due to three principal constraints: (i) limited knowledge of the dependence of peroxisome targeting by the short PTS peptides on essential enhancer elements located in close proximity; (ii) possible dominance of N-terminal non-peroxisomal targeting signals over C-terminal PTS1s; and (iii) unpredictable masking of PTS surface exposure. The amino acid residues upstream of PTS1 tripeptides have been noticed to be atypical in amino acid distribution (Reumann, 2004). A few studies indicated the dependence of peroxisome targeting by specific PTS1 tripeptides on upstream residues (Distel et al., 1992; Kragler et al., 1998; Bongcam et al., 2000; Brocard and Hartig, 2006), but these data have currently only an illustrative character and cannot be generalized across kingdoms for implementation in predictive algorithms.
To test and improve the prediction accuracy of PTS1 proteins by characterizing targeting-inhibitory and essential enhancing elements in PTS1 domains, the family of protein kinases was selected for this case study. Among the 
1000 putative Arabidopsis protein kinases (Wang et al., 2003), seven putative peroxisomal protein kinases carrying predicted PTS1s were identified. The full-length cDNAs of several Arabidopsis kinases were isolated by RT-PCR, and subcellular targeting of full-length proteins and C-terminal domain constructs fused with the reporter protein enhanced yellow fluorescent protein (EYFP) was analysed in vivo. Four out of seven protein kinases were shown to possess functional PTS1 domains. Acidic and basic residues located in close proximity to PTS1 tripeptides were demonstrated to play essential roles as inhibitory and enhancing elements, respectively, in protein targeting to plant peroxisomes. Consideration of these novel targeting elements of PTS1 domains will help improve the prediction accuracy of protein targeting to peroxisomes by the PTS1 pathway.
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Materials and methods |
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Genomic identification of putative protein kinases
Arabidopsis candidate proteins that are possibly targeted to the matrix of plant peroxisomes were identified based on the presence of a putative major or minor PTS following a previous definition (Reumann, 2004) or [SA]HL> and are accessible through the database AraPerox (www3.uis.no/AraPeroxV1; Reumann et al., 2004). The prediction of open reading frames and exon–intron borders was verified by cross-wise homology analysis using BlastP against the non-redundant database at the National Center of Biological Research (NCBI, http://www.ncbi.nlm.nih.gov/). Targeting prediction was performed as described earlier (Reumann et al., 2004).
Gene cloning by RT-PCR
For none of the four A. thaliana protein kinases (AtPKs) had the gene structure predicted from the genome sequence at that time been confirmed by the identification of full-length or overlapping ESTs present in public cDNA collections of Arabidopsis. Therefore, gene prediction regarding the start codon of translation and exon–intron borders was verified manually by homology analysis and corrected for AtPK1, AtPK3, and AtPK4. Total RNA was isolated from different tissues of Arabidopsis thaliana cv. Columbia using the Invisorb Spin Plant Mini Kit (Invitek GmbH, Berlin, Germany). The RNAs for cDNA isolation of AtPK3 (At4g18950) and AtPK4/ATG1a (At3g61960) were isolated from rosette leaves, whereas the RNA of AtPK1 (At3g20530) and the C-terminal domain of AtPK2 (At4g31220, At4g31230: full-length cDNA) were isolated from flowers using appropriate oligonucleotide primers (Supplementary Table 1 available at JXB online). Total RNA was converted to single-stranded cDNA by reverse transcriptase (Superscript II, Invitrogen, Karlsruhe, Germany) and used as a template for PCR employing a proofreading DNA polymerase (Thermozyme, Invitrogen). Amplification products were subcloned into pGEMT using the pGEM®-T Easy Vector System (Promega, Madison, WI, USA) and sequenced. The primary structure of these cDNAs was identical to that of improved manual gene prediction and consistent with the gene models deposited under AGI accession numbers. For AtPK1, a single amino acid exchange occurred (V362M). The full-length cDNAs of AtPK5/RPK1 (At1g69270) and AtPK7/GPK1 (At3g17420) were ordered from the Arabidopsis Biological Research Center (ABRC, Columbus, OH, USA). A full-length cDNA of AtPK6/S6K1 (At3g08720) was not available in the course of this study, but peroxisome targeting enabled by the putative PTS1 domain was investigated by addition of the C-terminal 10 residues to EYFP by PCR (Supplementary Table 1 at JXB online).
Subcellular localization analysis in Allium cepa L.
To study the subcellular targeting of full-length protein kinases in plant cells, fusion proteins with N-terminally located EYFP were generated. The full-length cDNAs of AtPK1, AtPK3–5, and AtPK7, and the C-terminal domains of AtPK1 (AtPK1C, residues 294–386), AtPK2 (AtPK2C, At4g31220 or residues 521–764 of At4g31230), and AtPK7/GPK1
AKI lacking the C-terminal tripeptide AKI> (residues 1–464) were amplified from pGEMT or pUNI51 with primers containing appropriate restriction sites (Supplementary Table 1 at JXB online) and subcloned in-frame into the plant expression vector pCAT-YFP-N-fus (Fulda et al., 2002) under control of a double 35S cauliflower mosaic virus (CaMV) promoter.
For analysis of the peroxisome targeting efficiency of putative PTS domains, the C-terminal 10 residues of AtHPR and AtPK2–AtPK7 (Fig. 1) were fused to the C-terminus of EYFP by PCR using an extended reverse primer, and subcloned into the plant expression vector pCAT. Due to its unusual stretch of eight acidic residues at position –5 to –12 in AtPK1, the C-terminal domain fused to EYFP was extended for this protein kinase to 15 residues (VEGEEEEEEDERSKL>, Fig. 1). To define targeting-inhibitory elements within the C-terminal domain of AtPK1, the stretch of eight acidic residues was removed from the putative PTS domain of AtPK1 (residues 375–382) and the six residues of the endogenous protein upstream of the putative PTD were added to RSKL> (GQTVEG, residues 369–374), yielding the construct EYFP–PTDEAtPK1 (EYFP–GQTVEGRSKL>). To analyse if acidic residues added to the PTS1 domain of AtHPR inhibit peroxisome targeting, four neutral residues of the PTS1 targeting domain of AtHPR (KALGLPVSKL>) were exchanged for acidic residues (A378
D, G380E, P382D, and V383E) and subcellular targeting of the corresponding EYFP fusion protein, referred to as EYFP–PTD+EAtHPR (EYFP–KDLELDESKL>), was investigated (Supplementary Table 1 at JXB online). To study the role of basic residues in SHL> domains, two arginine residues located in front of SHL> in AtPK4 (SNLQHRRSHL>) were exchanged for neutral residues to yield EYFP–PTDRAtPK4 (SNLQHGASHL>). Conversely, two arginine residues were introduced into the C-terminal domain of AtPK3 (HDGSSSGSHL> to GSSSGRRSHL>).
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For labelling of peroxisomes in double transformants, a fusion protein of the N-terminal 50 residues of glyoxysomal malate dehydrogenase (CsgMDH) from Cucumis sativus L. comprising the PTS2 targeting domain and enhanced cyan fluorescent protein (ECFP) was used (CsgMDH–ECFP, Fulda et al., 2002; Ma et al., 2006). For labelling of mitochondria in double transformants, a fusion protein of the mitochondrial pre-sequence of the cytochrome c oxidase subunit IV from Saccharomnyces cerevisiae and ECFP was applied (ScCOX–ECFP, Fulda et al., 2002). Onion epidermal cells were transformed biolistically as described (Fulda et al., 2002). A 5 µg aliquot of plasmid DNA was precipitated on gold particles (1.0 µm, Biorad, Hercules, CA, USA). Commercial onions (Allium cepa L.) were cut into slices and bombarded using a particle gun (Biolistic PDS 1000/He Biolistic Particle Delivery System, Biorad) using 1100 psi rupture discs and a vacuum of 0.1 bar. The onion slices were placed on wet paper in Petri dishes and stored on a benchtop for 16–24 h. For analysis by fluorescence microscopy, the onion skin epidermal cell layer was peeled and transferred to a glass slide.
Fluorescence microscopy
Analysis of yeast and onion epidermal cells was performed using a fluorescence microscope (Olympus BX51) with the following filter sets: EYFP (F41-028; excitation filter HQ500/20, barrier HQ535/30), ECFP (F31-044; excitation filter D436/20, barrier D480/40). Digital images were captured using a CCD camera (colorViewII) with analySIS3.1 Imaging software (Soft imagine system GMDH).
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In silico prediction of peroxisomal protein kinases
To identify putative protein kinases that are targeted to the matrix of plant peroxisomes, the Arabidopsis genome was screened for predicted protein kinases containing putative PTSs. In our initial screen, application of the conservative PTS1 consensus motif [SAPC][KR][LMI]> (Hayashi et al., 1997) and two histidine-containing tripeptides (SHL> and AHL>, Mullen et al., 1997; Kragler et al., 1998) led to the identification of four AtPKs (Fig. 1). Improved prediction of exon–intron borders and the recognition of other functional PTS peptides by an in silico approach (Reumann, 2004) allowed the detection of three further candidate proteins (Fig. 1). For the search for protein kinases targeted to peroxisomes by the PTS2 pathway, the 11 PTS2 nonapeptides defined for spermatophyta were used (Reumann, 2004). However, protein kinases with putative PTS2s within the N-terminal 40 amino acid residues were not found. Two kinases with predicted PTS1 tripeptides each carry major (AtPK1, At3g20530, SKL>; AtPK5/RPK1, At1g69270, SRL>) or minor PTS1 tripeptides (AtPK2, At4g31230, PKL>; AtPK6/S6K1, At3g08720, SNL>) of high and moderate peroxisomal targeting probability, respectively, whereas three kinases possess rare PTS1 tripeptides that had previously not been detected in plant peroxisomal ESTs in a significant number (AtPK3, At4g18950, AtPK4/ATG1a, At3g61960, both SHL>; AtPK7/GPK1, At3g17420, AKI>; Reumann, 2004). Apart from the PTS1s, some protein kinases are predicted to possess N-terminal targeting signals for mitochondria (AtPK1) or the endoplasmic reticulum (AtPK5/RPK1, Osakabe et al., 2005), or nuclear localization signals (NLSs, AtPK3; Fig. 1).
Subcellular targeting analysis of full-length protein kinases in vivo
To analyse predicted peroxisome targeting of the seven protein kinases experimentally, subcellular targeting of the full-length fusion proteins was first investigated. The cDNAs were subcloned into the plant expression vector pCAT-YFP-Nfus under control of a 2-fold version of the CaMV 35S promotor and fused at the 5' end in-frame to EYFP. Upon biolistic bombardment with plasmid-coated gold particles, the genes were transiently expressed in the single epidermal cell layer of A. cepa L. (Fulda et al., 2002; Ma et al., 2006).
In summary, three N-terminal full-length fusion proteins (EYFP–PK1, EYFP–PK4, and EYFP–PK5) were detected in the cytosol, while one was detected in the nucleus (EYFP–PK3) and one in punctuate subcellular structures (EYFP–PK7, Supplementary Fig. S1 at JXB online). A C- and N-terminally shortened fragment of PK1 (PK1N–EYFP and EYFP–PK1C) also remained cytosolic, indicating that the predicted N-terminal mitochondrial pre-sequence was functionally inactive (Supplementary Fig. S1B, C). The partial fusion protein of PK2 remained cytosolic as well (Supplementary Fig. S1D). EYFP–PK3 was detected in the nucleus, consistent with the prediction of two NLSs. Although PK7/GPK1 had previously been identified in Arabidopsis glyoxysomes (Fukao et al., 2003), the subcellular fluorescent structures detected did not coincide with either peroxisomes or mitochondria (Supplementary Fig. S1H). Upon deletion of the predicted PTS1 AKI>, the fusion protein was still targeted to unknown subcellular structures that were not identical to peroxisomes or mitochondria (Supplementary Fig. S1J, K, data not shown). As a whole these data suggested that some proteins were either falsely predicted to be peroxisome targeted and/or some proteins were dual-targeted to both the cytosol and peroxisomes with a cytosolic default localization upon expression from the strong constitutive promoter in the transient standard expression system of onion epidermal cells.
Functional analysis of predicted PTS1 domains
False-positive predictions of peroxisomal PTS1 proteins carry non-functional PTS1 domains that are not able to redirect EYFP from the cytosol to peroxisomes, if fused as short 10-amino acid domains with the reporter protein. In contrast, dual-targeted kinases whose PTS1 domain is internally buried in the cytosolic default conformation carry PTS1 domains that only become surface exposed and functionally active by signal-induced conformational changes under very specific conditions, thereby mediating protein targeting to peroxisomes. If the PTS1 domains of such dual-targeted kinases are expressed independently of the default conformation of the full-length kinase, they redirect a reporter protein from the cytosol to peroxisomes.
To discriminate between false-positive protein predictions and dual-targeted protein kinases, we fused short 10-amino acid peptides comprising the predicted PTS1 domains of the kinases to EYFP. When the corresponding construct of AtPK2 terminating with PKL> [EYFP–PTD(PKL>)AtPK2] was transiently expressed in onion epidermal cells, punctate cell structures were fluorescently labelled in single transformants (Fig. 2B). Even though these organelles moved quickly along cytoplasmic strands, they were shown to coincide with peroxisomes identified as such by the peroxisomal marker CsgMDH–CFP (Fig. 2C). In contrast, mitochondria labelled by ScCOX–CFP were not identical to these yellow fluorescent organelles (Fig. 2D, E). Likewise, the 10 C-terminal residues of AtPK4/ATG1a and AtPK5/RPK1 containing the PTS1 peptides SHL> and SRL>, respectively, were sufficient to target EYFP to peroxisomes (Fig. 2G–K). AtPK4/ATG1a is thus the first plant protein shown to carry the PTS1 tripeptide SHL>. The fusion protein EYFP–PTD(SNL>)AtPK6/S6K1 with the non-canonical PTS1 SNL> was the fourth protein kinase with a functional PTS1 domain able to target EYFP to punctate structures in onion epidermal cells that coincided with peroxisomes (Fig. 2L, M).
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In contrast, when the C-terminal domains of AtPK1 and AtPK3, which possessed the tripeptides SKL> and SHL>, respectively, were appended to EYFP, the reporter proteins remained cytosolic (Fig. 2A, F). Likewise, the C-terminal domain of AtPK7/GPK1 with the putative PTS1 AKI> was not able to target EYFP to peroxisomes (Fig. 2N). In summary, the experimental data demonstrated that four out of
1000 Arabidopsis protein kinases possess functional peroxisome targeting domains, whereas three proteins were identified as false-negative predictions carrying non-functional PTS1 domains.
Characterization of targeting-inhibitory elements in PTS1 domains
Non-peroxisomal targeting of EYFP extended C-terminally by the SKL> domain of AtPK1 in particular was completely unexpected based on previous understanding of protein targeting by the PTS1 pathway. SKL> is the prototypical PTS1 and the targeting signal of many plant peroxisomal proteins including several Arabidopsis homologues (e.g. Arabidopsis HPR, long-chain acyl-CoA synthetase isoform 7, and sterol carrier protein isoform 2). Additionally, the tripeptide SKL> alone is reported to be sufficient to target different reporter proteins to plant peroxisomes (Banjoko and Trelease, 1995; Hayashi et al., 1996).
Non-peroxisomal targeting of EYFP extended C-terminally by the SKL> domain of AtPK1 suggested that specific amino acid residues in front of the tripeptide may take on the role of as yet unknown inhibitory elements that are incompatible with protein targeting to plant peroxisomes. The C-terminal tripeptide SKL> of AtPK1 is preceded by a basic residue at position –4 and eight acidic residues closely upstream (EEEEEEDERSKL>, residues 375–386) that provide this domain with a pronounced negative net charge at physiological pH (Fig. 1), while plant PTS1 domains generally lack acidic residues (Reumann, 2004). When the stretch of eight acidic residues was eliminated from the targeting domain of AtPK1, the peptide [PTDE(SKL>)PK1; GQTVEGRSKL>, Table 1] gained the ability to target EYFP to peroxisomes and was thus converted to a functional PTS1 domain (Fig. 3A, B). These results support the previous computational hypothesis that acidic amino acid residues located in close proximity to PTS1 peptides are incompatible with protein targeting to peroxisomes (Reumann, 2004).
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To strengthen this hypothesis further, four neutral residues of the PTS1 domain of AtHPR (KALGLPVSKL>) were exchanged with acidic residues by site-directed mutagenesis (KDLELDESKL>). In contrast to the wild type (Table 1, Fig. 3C, D), the mutated PTS1 domain of AtHPR, referred to as EYFP–PTD+E(SKL>)AtHPR, was no longer able to cause peroxisome targeting of EYFP (Fig. 3E). These results conclusively demonstrate that acidic residues located closely in front of PTS1 tripeptides abolish peroxisome targeting even in the case of the major and presumably one of the strongest PTS1 tripeptides, SKL>.
Characterization of essential targeting-enhancing elements in PTS1 domains
Even though both peptides terminated with SHL>, the C-terminal domain of AtPK4/ATG1a, but not that of AtPK3, was able to target EYFP to peroxisomes (Fig. 2F–I). These results suggested either the presence of inhibitory elements in the C-terminal domain of AtPK3 or the presence of targeting-enhancing elements in the domain of AtPK4/ATG1a that are essential for peroxisome targeting by SHL>. Two arginine residues are located in front of SHL> in AtPK4/ATG1a (SNLQHRRSHL>), whereas mostly neutral amino acids precede SHL> in AtPK3 (HDGSSSGSHL>; Fig. 1, Table 1). To investigate whether basic residues are essential in front of SHL> for protein targeting to plant peroxisomes, both arginine residues of the C-terminal domain of AtPK4/ATG1a were changed to neutral residues [PTDR(SHL>)AtPK4: SNLQHGASHL>, Table 2]. In contrast to the wild-type PTS1 domain (EYFP–PTDAtPK4/ATG1a) that labelled a multitude of brightly fluorescent peroxisomes without giving rise to any cytosolic or nuclear staining (Fig. 2G–I), the majority of the cells (about two-thirds) expressing the deletion construct EYFP–PTDR(SHL>)AtPK4/ATG1a lacked fluorescent peroxisomes; instead, EYFP largely localized to the nucleus and the cytosol (Fig. 3F, Table 1). In the remaining cells (about one-third), the number of fluorescent spots and their fluorescence intensity were strongly reduced (Fig. 3G), indicating that elimination of two Rs in the PTS1 domain of AtPK4 significantly reduced the targeting efficiency of the reporter protein to peroxisomes.
In contrast, the introduction of two basic residues into the C-terminal domain of AtPK3 [PTD+R(SHL>)AtPK3, GSSSGRRSHL>] directed the reporter protein to peroxisomes (Fig. 3H, I, compare with Fig. 2F). It was concluded that basic residues in front of SHL> play an important role in enhancing protein targeting to plant peroxisomes. In summary, the characterization of acidic amino acid residues as targeting inhibitory and basic residues as essential enhancing elements for peroxisome targeting provides novel information on discriminative properties upstream of PTS1 tripeptides and will, upon consideration in predictive algorithms, allow improvement of the prediction accuracy of protein targeting to peroxisomes from genome sequences.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Toward an improvement of predictive algorithms: the characterization of targeting-inhibitory elements
The identification of low-abundance and inducible organellar proteins often relies on subcellular protein targeting prediction. Proteins following the PTS1 pathway are not only the largest group of peroxisomal proteins but can also currently be predicted with the highest accuracy. Apart from the seven Arabidopsis protein kinases with predicted PTS1s analysed in the present study, another protein terminating with ANL> was not pursued experimentally because the peroxisome targeting probability of its C-terminal domain was estimated to be below threshold (At2g18530, DVEVEYEANL>). The number of eight protein kinases with predicted PTS1s versus none with a predicted PTS2 is largely consistent with the higher number of PTS1 proteins predicted for Arabidopsis (see Introduction, Reumann et al., 2004).
Except for SNL> and AKI>, all PTS1 tripeptides of the kinases had previously been shown to be sufficient to target a reporter protein to plant peroxisomes (Hayashi et al., 1997; Mullen et al., 1997; Kragler et al., 1998). The presence of a putative PTS1 peptide is currently the best indicator, albeit still an insufficient criterion to conclude definitively that protein targeting to plant peroxisomes occurs (see Introduction) owing to its minimal size, which bears insufficient discriminative information. Some auxiliary targeting-enhancing elements such as basic residues at position –4 have been experimentally characterized (Purdue and Lazarow, 1996; Neuberger et al., 2003a, b, 2004). These elements, however, have only vaguely been defined regarding, for instance, their identity, number, and precise localization in PTS domains and their essential function for peroxisome targeting. Detailed characteristic patterns of the entire PTS1 domains remain to be deduced for plants in particular.
The experimental characterization of three protein kinases with predicted PTS1s as non-peroxisomal proteins revealed the current limitations in predicting protein targeting to plant peroxisomes by the PTS1 pathway. The PTS1s of protein kinases with functional PTS1 domains (SRL>, PKL>, SNL>, and SHL>) were identical or very similar to those with non-functional PTS domains (SKL>, SHL>, and AKI>) and did not correlate with the previous classification into major (e.g. SRL>, SKL>), minor (PKL>, SNL>), and ambiguous and rare PTS1 peptides still absent from known plant PTS1 homologues (SHL>, AKI>, Reumann, 2004).
Non-peroxisomal targeting of EYFP–PTDAtPK1 terminating with SKL> was most unexpected, because SKL> is the prototypical PTS1 and well known to be sufficient to target various native and reporter proteins across organisms of the three major eukaryotic kingdoms (Gould et al., 1989; Banjoko and Trelease, 1995; Hayashi et al., 1996). Elimination of the unusual stretch of acidic residues in front of SKL> in AtPK1 converted the domain to a functional PTS1 domain. Likewise, the exchange of four neutral residues with acidic residues in the SKL> domain of AtHPR prevented import of the reporter protein into peroxisomes. These results demonstrate for the first time that acidic residues located upstream of putative plant PTS1 tripeptides act as targeting inhibitory elements that suppress protein targeting to plant peroxisomes. Presumably, the high local negative net charge reduces the interaction between SKL> and Pex5p below threshold.
Remarkably, the inhibitory effect of acidic residues on peroxisome targeting has not been observed for low-abundance and presumably ‘weak’ PTS peptides (e.g. SKM>, ARM>, and PKL>), but for the major prototypical PTS1 peptide SKL>. Hence, in contrast to previous understanding, SKL> can no longer be regarded as sufficient to target any reporter protein to plant peroxisomes. Instead, peroxisome targeting by SKL> depends on the absence of targeting-inhibitory amino acid residues located upstream, such as acidic residues. It is reasonable to predict that acidic residues interfere with peroxisome targeting by all PTS1 peptides and that their inhibitory effect is even more pronounced on rare and presumably weaker PTS1 peptides, i.e. any of the 11 minor PTS1 tripeptides (Reumann, 2004) and rare peptides such as SHL>. The results demonstrate that about four acidic residues located in close proximity to SKL> are incompatible with peroxisome targeting. Future studies need to address more detailed questions regarding (i) the minimum number of acidic residues that are required to inhibit peroxisome targeting by SKL>; (ii) a postulated interdependency between the inhibitory effect of acidic amino acid residues and the strength of the PTS1 tripeptide; (iii) possible position-dependent effects of these residues within the targeting domain; and (iv) to what extent the inhibitory effect can be compensated, for instance, by neighbouring basic residues.
Targeting-enhancing elements that are essential for specific PTS1 tripeptides
In addition, it was also possible to define upstream residues that are essential for peroxisome targeting. The exchange of the two basic residues for neutral residues in AtPK4 (SHL>) significantly reduced import of EYFP into peroxisomes, while the introduction of two arginines into the SHL> domain of AtPK3 converted this domain into a functional PTS1 domain. It can thus be generalized that protein targeting to plant peroxisomes by SHL> is strongly enhanced by and, probably in most proteins, essentially dependent on the presence of basic residues closely upstream of the PTS1. It is reasonable to predict that peroxisome targeting by other minor PTS1 peptides also depends on auxiliary basic residues. Similar to the precise definition of inhibitory elements, future studies need to determine in more detail to what extent peroxisome targeting is affected by the number of basic residues, their identity (R or K), and their position in the PTS1 domain.
Plant proteins with the PTS1 SHL> have not been reported nor been identified by EST database searches (Reumann, 2004). Even in a more general context, any plant PTS1 tripeptides carrying H at position –2 (e.g. AHL>) are not reported, while SHL> is an abundant PTS1 in fungi (Elgersma et al., 1996; Lametschwandtner et al., 1998). Position –2 of PTS1 tripeptides thus appears to bear the most significant differences between plants and yeast, further supporting the idea that the properties of PTS1 peptides reveal major differences between the three eukaryotic kingdoms. The shape and surface charge of the binding pocket of plant Pex5p orthologues appear to reveal significant differences as compared with yeast Pex5p and to be less suitable to accommodate SHL> peptides, unless they are preceded by basic residues. The pronounced dependency of the SHL> domain on upstream basic residues determined here thus provides an explanation for the general rare abundance of SHL> in plant peroxisomal proteins. Random single point mutations from S[RK]L> to SHL> only appear to maintain peroxisome targeting if basic residue(s) were located in front of the PTS1 or introduced independently in parallel, which happens with extremely low probability.
Proposed mechanism for peroxisome targeting of protein kinases
The question of whether matrix enzymes are regulated by reversible phosphorylation is central to our understanding of plant peroxisomal metabolism but has remained largely unanswered. Peroxisome-targeted protein kinases are suspected to mediate signal transduction across the membrane to fine-tune the activity and/or the turnover of key metabolic enzymes in response to altered biotic and abiotic environmental conditions. The important enzyme catalase, for instance, is inactivated by high light (Feierabend and Engel, 1986; Grotjohann et al., 1997). Sessile plants thus probably evolved a fast adaptive mechanism to protect themselves against H2O2 accumulation by instantly reducing the activities of H2O2-producing flavin oxidases (e.g. acyl-CoA oxidase and glycolate oxidase under CAT-inactivating conditions). Also, the activities of serine–glyoxylate and glutamate–glyoxylate aminotransferase (Liepman and Olsen, 2001; Liepman and Olsen, 2003) are expected to be fine-tuned to each other because glycolate recycling during photorespiration is most efficient when the aminotransferases convert glyoxylate at a stoichiometric ratio of 1:1. Finally, not only the enzymes of the photorespiratory C2 cycle but also those of fatty acid β-oxidation are expressed in leaf peroxisomes, and both pathways share identical intermediates and cofactors, for instance NADH, malate, and oxaloacetate. The flux of intermediates into these alternative pathways is expected to be regulated according to the physiological product requirements by as yet unknown mechanisms that could be of a post-translational nature.
To identify dual-targeted protein kinases, the C-terminal 10–15 residues of the candidate proteins were fused to the reporter protein and thus expressed independently of the default conformation of the full-length protein. Co-crystallization of the tetratricopeptide repeat domain of human Pex5p with a PTS1 peptide and bioinformatics analyses indicated that few residues upstream of the PTS1 are in contact with surface residues of the receptor and/or determine the secondary structure of the PTS1 domain (Gatto et al., 2000; Emanuelsson et al., 2003; Neuberger et al., 2003a, b, 2004; Reumann, 2004). The genome screen conducted here identified four protein kinases with functional PTS1 domains (AtPK2, AtPK4/ATG1a, AtPK5/RPK1, and AtPK6/S6K1). Upon expression as full-length proteins, however, these kinases remained cytosolic, suggesting that the functional PTS1 domains were either constitutively or transiently masked by the proteins’ conformation. In light of (i) the low number of protein kinases with functional PTS1s domains among 1000 Arabidopsis protein kinases (Wang et al., 2003); (ii) the pronounced dependence of functional plant PTS1 peptides on the presence of targeting-enhancing elements and the absence of inhibitory elements upstream of the tripeptide, requiring co-evolution to constitute functional PTS1 domains; and (iii) the well-known dynamic conformation of protein kinases, the functional PTS1 domains are highly unlikely to have evolved in Arabidopsis by chance. Additionally, a dozen ‘classical’ metabolic enzymes with predicted PTS1 tripeptides have been localized to peroxisomes in the same experimental system, suggesting that the cytosolic default localization observed in this study is typical for protein kinases (Reumann et al., 2007).
It is hypothesized that specific as yet unknown signals trigger activation of the kinases by altering their polypeptide conformation and inducing surface exposure of the PTS1 and targeting to the peroxisome matrix. This idea is further supported by the lack of non-peroxisomal targeting signals in three kinases (AtPK2, AtPK4/ATG1a, and AtPK6/S6K1); only AtPPPK5/RPK1 possesses a functional signal peptide that may over-rule the PTS1. Moreover, many kinases contain long terminal domains that probably have a regulatory function (Fig. 1). Nevertheless, conclusive evidence for peroxisome targeting of all four kinases with functional PTS1 domains needs to be provided by the creation of stable transgenic plants that express tagged versions of the kinases preferentially from their endogenous promoter and screening of these plants immunocytochemically for conditions, under which the kinases are transiently targeted to peroxisomes.
The identity of four kinases with functional PTS1 domains
Characterization of a functional PTS1 domain in AtPK2 is consistent with its detection in isolated Arabidopsis leaf peroxisomes (Fukao et al., 2002). The lower apparent molecular mass of the protein spot identified (At4g31220, 30 kDa) as compared with the revised molecular mass of the gene model At4g31230 (85 kDa) may have been caused by artificial proteolytic degradation during organelle isolation. AtPK5/RPK1 is a leucine-rich repeat receptor-like protein kinase that is induced by abscisic acid and abiotic stress conditions (Hong et al., 1997). The kinase was localized to the plasma membrane in Arabidopsis roots (AtPK5/RPK1–GFP, Osakabe et al., 2005). The characterization of a functional PTS1 domain in AtPK5/RPK1 suggests dual protein targeting regulated by alternative transcriptional, translational, or post-translational mechanisms, and needs to be addressed in future studies in more detail.
AtPK4/ATG1a is one of three Arabidopsis homologues of the yeast protein kinase ATG1 (Hanaoka et al., 2002), but experimental data on ATG1 homologues from plants are not reported yet. Yeast ATG1 is a protein kinase essential for the cytosol to vacuole pathway and the catabolic process of autophagy that allows protein turnover under nutrient starvation (Straub et al., 1997; Kamada et al., 2000; Abeliovich et al., 2003). Interestingly, AtPK6/S6K1 is one of two Arabidopsis homologues of ribosomal protein S6 kinase (S6K, Lee et al., 2007; for a review, see Ruvinsky and Meyuhas, 2006), which is, similarly to ATG1, also regulated by the target of rapamycin (TOR) signal transduction pathway. Future studies need to address in more detail under which conditions AtPK4/ATG1a and AtPK6/S6K1 enter the matrix of plant peroxisomes and elucidate their physiological function.
Kinases with non-functional PTS1 targeting domains
The C-terminal domains of three AtPKs (AtPK1, AtPK3, and AtPK7/GPK1) were not able to direct EYFP to peroxisomes (Fig. 2). It was thus concluded that the kinases are not targeted to plant peroxisomes by the PTS1 pathway under any physiological conditions, thereby identifying them as false-positive PTS1 proteins of the genome screen. AtPK1 is probably cytosolic because the C-terminal fusion construct with accessible predicted mitochondrial pre-sequence was not organelle-targeted (Supplementary Fig. S1C at JXB online). Regarding AtPK3, the present data indicate nuclear targeting of the protein kinase.
AtPK7/GPK1 has been identified in a proteome study of Arabidopsis glyoxysomes, and peroxisome targeting has been supported by immunochemical subfractionation (Fukao et al., 2003). The kinase was thought to follow the PTS1 pathway based on its PTS1-like tripeptide AKI> and owing to its topology with the kinase domain facing the matrix side (Fukao et al., 2003). The low abundance of AKI> in the set of 400 plant PTS1 proteins indicates a weak targeting function of the tripeptide and a pronounced dependence on targeting enhancing elements (Reumann, 2004). Arabidopsis monodehydroascorbate reductase isoform 1 (AtMDAR1) has recently been characterized as the first plant peroxisomal protein carrying the PTS1 AKI> (Leterrier et al., 2005; Lisenbee et al., 2005). The presence of three acidic residues in front of AKI> in AtPK7/GPK1 (DNDITTDAKI>) was predicted to reduce the peroxisome targeting ability of its C-terminal domain below threshold (Reumann, 2004). Although full-length EYFP–AtPK7/GPK1 and the AKI> deletion construct were targeted to punctate subcellular structures, these organelles were not identical either to peroxisomes or to mitochondria. Moreover, EYFP extended by the C-terminal 10 residues of AtPK7/GPK1 remained cytosolic, strongly arguing against the idea that the protein kinase is targeted to peroxisomes by AKI>. The organelle-like nature of the subcellular structures labelled by EYFP–AtPK7/GPK1 may suggest that this as yet unknown compartment was accidentally co-purified along with glyoxysomes in the corresponding proteome study (Fukao et al., 2003). Future studies need to address the identity of the compartment labelled by EYFP–AtPK7/GPK1.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Supplementary data can be found at JXB online. All oligonucleotides are listed in Supplementary Table S1. Supplementary Figure S1 shows the in vivo subcellular targeting analysis of full-length protein kinases.
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Acknowledgements |
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We are grateful for cDNA sequencing by the Göttingen Genomics Center (Professor Gottschalk, Department of Microbiology), the Medical Faculty (Professor Pieler, Department of Developmental Biochemistry), and cDNA provision by ABRC. We thank Professor K Pawlowski, Dr E Hornung, and especially Dr I Heilmann for critical reading of the manuscript, and Professor I Feussner for the support for our research. The research is supported by grants from the Deutsche Forschungsgemeinschaft (RE1304/2 and RE1304/4) and by a Dorothea-Erxleben stipend from the government of Lower Saxony (to SR).
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* Present address: Section of Molecular Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA, USA
Present address: Centre for Organelle Research, Faculty of Science and Technology, University of Stavanger, N-4036 Stavanger, Norway
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Abbreviations |
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ATG, autophagy-related protein; COX, cytochrome c oxidase; EYFP, enhanced yellow fluorescent protein; GPK1, glyoxysomal protein kinase 1; HPR, hydroxypyruvate reductase; NLS, nuclear localization signal; PTD, peroxisome targeting domain; PTS1/2, peroxisome targeting signal type 1/2; S6K, ribosomal protein S6 kinase; TOR, target of rapamycin.
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