Investigations on functional aspects of secondary endocytobiosis using the diatom Phaeodactylum tricornutum as a model organism Dissertation Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften

Diatoms and a variety of other algae evolved by secondary endocytobiosis, i.e. by uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent transformation of the endosymbiont into an organelle. Due to this explicit evolutionary history diatoms possess plastids bound by four membranes. Nucleus encoded plastid preproteins in diatoms have N-terminal bipartite presequences consisting of a signal and a transit peptide domain, respectively, to enter the plastids. However, only little information is available about the mechanism how the actual import reaction occurs. Here, the possible evolution of such protein transport systems is discussed and targeting of polypeptides within diatoms characterized. DNA constructs using green fluorescent protein (GFP) fused to targeting sequences have been generated to investigate on in vivo expression in the diatom Phaeodactylum tricornutum. Correct in vivo targeting into various organelles and membranes has been demonstrated. We also isolated and characterized a tightly regulated, inducible promotor from Phaeodactylum. Experimental data is provided for the minimal requirements of a diatom plastid targeting presequence. It is demonstrated i) that the transit peptide of at least one plastid preprotein is largely removable without affecting plastid import, ii) that the signal peptides of plastid preproteins and of ER targeted preproteins in diatoms are functionally equivalent and iii) that a conserved amino acid sequence motif at the signal peptide cleavage site is present in diatoms and a variety of other secondary algae. As revealed by site directed mutagenesis, a single phenylalanine within this motif is crucial for protein transport into the diatom plastids in vivo. Additional results also show that a vesicular transport step within the envelope membranes may be involved in the import of plastid preproteins and strongly support the hypothesis of a multi-step plastid targeting process in chromophytic algae. Comparing cDNAs and respective genomic DNA fragments encoding diatom plastid preproteins, single spliceosomal introns in the regions encoding the presequences were found indicating that the bipartite targeting sequences in these secondary algae might have been acquired by recombination events via introns.