Identification of Arabidopsis putative peroxisomal protein kinases (PPPKs)

Plant peroxisomal research so far has focused on peroxisomal metabolism, protein import into the organelle, and peroxisome biogenesis, whereas post-translational regulation of peroxisomal metabolism has not received much attention (Fukao et al., 2003; Reumann et al., 2004). Diverse signals may be transduced across the peroxisomal membrane to regulate processes, including peroxisome proliferation or the activity or turnover of key enzymes involved in photorespiration, fatty acid β-oxidation, and ROS detoxification. One or several of these processes may be regulated by reversible phosphorylation. Thiolase was identified as

a phosphorylated glyoxysomal protein in an in vitro phosphorylation assay (Fukao et al., 2003). Recent experimental data have provided evidence that a number of protein kinases may be targeted to plant peroxisomes (Yang and Poovaiah, 2002; Dammann et al., 2003;

Fukao et al., 2003). Four putative protein kinases were detected in leaf peroxisomes by mass spectrometry in a proteome study (Fukao et al., 2002). However, only one of them carried a PTS tripeptide, and about 2/3 of the unknown proteins identified in leaf peroxisomes in the same study lacked a PTS as well, raising doubts regarding the purity of the organelle fraction. More recently, a calcium-dependent protein kinase was reported to be anchored in the peroxisomal membrane by an acyl anchor (Dammann et al., 2003). Finally, a protein kinase, referred to as glyoxysomal protein kinase 1 (GPK1), has been found in glyoxysomes from Arabidopsis cotyledons (Fukao et al., 2003). This protein kinase is identical with PPPK7 investigated in this study and carries a PTS1-like motif (AKI>). The precise subcellular localization and physiological function of the protein are unknown (Fukao et al., 2003).

The study of post-translational regulatory mechanisms of peroxisomes by biochemical approaches is challenging because regulatory proteins are generally expressed at low levels and under specific environmental conditions and therefore difficult to be caught by traditional biochemical methods. In a bioinformatics approach the Arabidopsis genome sequence was screened for PPPKs with putative PTS peptides. This method allowed the identification of protein kinases that reside in the peroxisome matrix, but cannot be expected to indentify protein kinases attached to the cytosolic side or inserted into the peroxisomal membrane from the cytosol, because these proteins do not use the PTS1 or PTS2 import pathway. Also, protein kinases may interact with peroxisomal proteins in the cytosol shortly after polypeptide synthesis will be missed. However, matrix-targeted protein kinases were thought to play major roles in regulating the activity and/or the turnover of peroxisomal enzymes. Reversible phosphorylation of pre-existing polypeptides like those located in the peroxisome matrix enables quick adaptation of metabolism to drastically changing environmental and cellular conditions like light or cytosolic ROS concentration and thus is an ideal mechanism to complement enzyme regulation at the transcriptional level.

In total, seven PPPKs with five different putative PTS1s were identified in Arabidopsis. The tripeptides SKL> and SRL> had previously been classified together with seven further peptides as major PTS1 peptides, because these tripeptides were identified in a large number of plant EST sequences that were homologous to PTS1-targeted proteins

and in different orthologous groups (Reumann, 2004). These high-abundance PTS1 peptides were thought to indicate peroxisome targeting of unknown proteins with high probability. The remaining PTS1 peptides of the putative protein kinases (PKL>, SHL>, SNL>, and AKI>) have been defined as minor PTS1 peptides and indicated peroxisome targeting with lower accuracy (Reumann, 2004).

At the beginning of this study solid experimental evidence for peroxisomal targeting was lacking for all PPPKs. In addition, peroxisomal orthologs from other organisms have not been reported for any of these seven kinases. Two homologs of PPPK4 from Colletotrichum lindemuthianum (AAB61403, SRL>) and Caenorhabditis briggsae (CAE56822, SRI>) contained a putative PTS. The presence of these major PTS1 tripeptides in two PPPK4 homologs raised the question as whether homologs of these proteins were targeted to peroxisomes in some organisms.

Two protein kinases, PPPK2 and PPPK7, have been detected in Arabidopsis leaf peroxisomes and glyoxysomes, respectively (Fukao et al., 2002, 2003). However, some experimental and bioinformatics data regarding these proteins were discrepant. For instance, the predicted size of PPPK2 (At4g31230) is 84.5 kDa, but the protein spot on the 2-D gels identified as this protein had an apparent molecular mass of about 30 kDa (Fukao et al., 2002) and represented the protein kinase domain, which was at that time predicted to be encoded by an obsolete gene locus (At4g31220). Thus, this protein spot may have represented a degradation product of PPPK2 or falsely been assigned to PPPK2 according to flawed MALDI-TOF-MS data (matrix-assisted laser desorption/ionization-time of flight-mass spectrometry). The protein kinase PPPK7 was detected in glyoxysomes in a proteome study (Fukao et al., 2003). As for PPPK2, so far no in vivo data have been reported to support the postulated targeting of this protein to peroxisomes, nor have any downstream targets been identified (Fukao et al., 2003).

As outlined in chapter 4.1, the presence of a putative PTS1 peptide in unknown proteins is a strong yet insufficient indication for peroxisome localization in vivo. Therefore, the subcellular localization of the seven PPPKs was verified by experimental analyses. To this end, the cDNAs of PPPK1-4 were cloned. At the beginning of this project, the gene structure of none of these four PPPKs as predicted from the genome sequence had been confirmed by the identification of full-length cDNAs or overlapping ESTs. Therefore, gene prediction regarding the start codon of translation and exon-intron borders was first analyzed

manually by homology analysis and indeed had to be corrected for PPPK1, 3, and 4.

Appropriate cloning primers were designed according to the optimized gene structure. The primary structure of the cloned cDNAs was identical with that of our gene prediction and finally consistent with a more recent release of Arabidopsis gene structure prediction. cDNA clones for PPPK5 and PPPK7 were obtained from NASC (The Nottingham Arabidopsis Stock Centre, Nottingham, UK). For PPPK6, the full-length cDNA could not successfully be cloned in the course of this study.

4.2.2 Multiple factors are responsible for alternate subcellular targeting of PPPKs in