Proteinimport in Plastiden von Kieselalgen

Diatoms, like many other algal groups, evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic host cell and the subsequent reduction and specialisation to a “complex” plastid. Comparable to the evolution of primary plastids, targeting mechanisms had to be developed to reimport preproteins into the plastid. Seven putative subunits of the translocons at the inner envelope membrane of chloroplasts (Tic) are encoded in the genome of the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana. Fusion proteins of Tic presequences or full length fusions to GFP show that the investigated Tics are plastid associated. Fusion proteins consisting of bipartite plastid targeting presequences from various algal groups show that they are also functional when heterologously expressed as GFP fusion proteins in the diatom P. tricornutum. Interestingly, also the modified signal peptide of a carbonic anhydrase from Arabidopsis thaliana, which apparently is targeted to A. thaliana plastids via the endoplasmic reticulum, is able to direct GFP into P. tricornutum plastids. This indicates that a conserved transport route is used for protein import into all secondary plastids, and that this route might be related to the signal peptide dependent route to plastids of higher plants. Colocalisation analyses suggest that native and artificial presequences from diatoms lead to an accumulation of GFP in a “blob”-like structure and that this structure is identical in both cases. At the moment, several models for the import of proteins into the “complex” plastids of diatoms are discussed. The models differ in the way they explain transport from the CER into the interenvelope space (ies). In the “pore model” it is proposed that a connection between the CER lumen and the ies might route nucleus encoded proteins across the periplastidic space. With the aid of a self-assembling GFP system we could show that proteins cannot reach the ies from the CER on a direct way. Furthermore from these results it can be concluded that nucleus-encoded plastid proteins from diatoms pass the four plastid envelope membranes via translocators and not via pores or vesicles which are also proposed