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
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
* 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
Received 3 July 2008; Revised 25 July 2008 Accepted 5 August 2008
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