Investigation of Dinoflagellate Plastid Protein Transport using Heterologous and Homologous in vivo Systems
Expressed Sequence Tag (EST) data from Ceratium horridum generated and analyzed in this thesis conform to the general parameters of dinoflagellate EST libraries. Comparison with diatom and plant transit peptides, revealed that transit peptides from peridinin-containing dinoflagellate conform to gene...
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|Summary:||Expressed Sequence Tag (EST) data from Ceratium horridum generated and analyzed in this thesis conform to the general parameters of dinoflagellate EST libraries. Comparison with diatom and plant transit peptides, revealed that transit peptides from peridinin-containing dinoflagellate conform to general trends for transit peptides but are relatively deficient in hydroxylated amino acids, have a slight net positive charge, and contain N-terminal basic amino acids among the most N-terminal amino acids. Like transit peptides in the alveolate Plasmodium falciparum, dinoflagellate transit peptides contain positively charged amino acids, have a depleted acidic residue content, and mostly contain one or more chaperone binding sites. The feature of dinoflagellate transit peptides that has gone unnoticed heretofore is the low overall positive charge, in addition to the significant division of charge between C- and N-termini.
Despite its overwhelming prominence in dinoflagellate transit peptides, C-terminal negative charge clearly had no impact on the import competence of Amphidinium carterae targeting signals in heterologous in vivo systems. Based on results from transfections of Pisum sativum and Phaeodactylum tricornutum, targeting mediated by transit peptides is not merely dependent on net positive charge, N-terminal positive charge, or amino acid content. Therefore, it was concluded that plastid transport into plant and diatom plastids also depended on sequence-specific patterns or motifs that are not present and/or not identical to those in dinoflagellate transit peptides. Based on the partial interchangeability of topogenic signals between an alveolate and a chromist but not between an alveolate and a plant – despite high homologies in mature protein sequences – it was deduced that plastid targeting signals evolve more expediently than the mature protein domains that they intracellularly target.
Analysis of the homologous intracompartmental transport of three Amphidinium carterae plastid proteins showed that differing transport routes exist for plastid proteins. While PsbO and Prk are transported by a Golgi-mediated route to the plastid, RbcL is transported directly from the ER to the plastids. In conclusion, a previously undescribed, possibly protein class-dependent, ER-mediated route seems to exist.|