In vivo subcellular targeting analyses of newly predicted plant PTS2 nonapeptides
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Original versionConf. until june 2014
Peroxisomes are organelles bounded by a single membrane and are present in all major groups of eukaryotes. Peroxisomal proteins are synthesized in the cytosol and are targeted to the peroxisome by targeting signals present in the N-terminal or C-terminal region of the proteins. In the first part of this master thesis project, the relational database AraPerox, consisting of predicted and validated peroxisomal Arabidopsis proteins, was further developed and nearly brought to completion. The proteins consisting PTS1 tripeptide, PTS2 nonapeptide, PEX proteins and other peroxisomal proteins from Arabidopsis are uploaded in the database. The manual entry data is still pending along with the modification in the web based server. In the second part of thesis, three predicted PTS2 nonapeptides i.e. ([RTx5HL], [RMx5HL], [RAx5HL]) and the novel PTS2 [RIx5QL] detected in significant number of assembled positive example sequences of plant PTS2 proteins were analyzed for their ability to target a reporter protein EYFP to peroxisome. Indeed, the PTS2 nonapeptides RTx5HL] and [RMx5HL] were localized to peroxisome with moderate efficiency. The Novel PTS2 nonapeptide [RIx5QL], up to now H (pos 8) conserved in all plant PTS2s, was localized to some unknown punctuate subcellular structure whose identity with peroxisome remains to be demonstrated. Moreover, the effect of point mutations introduced at different positions of the two representative PTS2 domains (containing the nonapeptides [RTx5HL] and [RMx5HL]) were also analyzed for altered in vivo subcellular localization. L to G mutation at 5th position of the nonapeptide RTx5HL prevented reporter protein targeting to peroxisome, indicating leucine at 5th position, which was highly overrepresented in plant PTS2 nonapeptides, act as a targeting enhancing element in plant PTS2 domain. By contrast two point mutations introduced in to the PTS2 domain [RMx5HL] (R to G at pos -1 and P to I at pos 11) did not significantly alter the peroxisome targeting efficiency, questioning that the residues play a significant role in determining peroxisome targeting strength. The PTS2 nonapeptide [RLAALAQQL] from the N-terminal domain of AT1G28960.1 was found to be localized to peroxisome strongly suggesting that this protein has been correctly predicted as a novel PTS2 protein. However, the predicted PTS2 domain [RVNTVNDHL] from N-terminus of AT1G48500.3 and [RLAANHLHL] from N-terminal domain of AT2G25730.1 remained in cytosol. Alternative expression systems and new technologies of higher resolution capability of peroxisome targeting need to be applied in future studies to investigate in greater detail whether these predicted Arabidopsis PTS2 proteins indeed contain functional PTS2 domains. In summary, the worldwide unique, very comprehensive and user-friendly relational database AraPerox, which has been long-awaited by the scientific community, has been brought close to completion. The first plant PTS2 protein prediction algorithms developed by Dr. T. Lingner have been experimentally validated to correctly predict novel at least one Arabidopsis PTS2 protein and new residues have been experimentally verified in plant PTS2 nonapeptides for the first time.
Master's thesis in Biological chemistry