In vivo subcellular targeting analyses of newly predicted plant PTS2 nonapeptides
Master thesis
Permanent lenke
http://hdl.handle.net/11250/182596Utgivelsesdato
2012Metadata
Vis full innførselSamlinger
Originalversjon
Conf. until june 2014Sammendrag
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.
Beskrivelse
Master's thesis in Biological chemistry