According to the actual view plastids evolved from free-living cyanobacteria that were engulfed by phagotrophic eukaryotic cells. During this process called primary endo-cytobiosis, the endosymbionts were reduced to cell organelles, resulting in plastids surrounded by two membranes. Primary plastids are characteristic for three eukary-otic lineages: glaucophytes, red algae, and green algea, the latter including the land plants [122, 5].
Diatoms and other groups of algae originated from a secondary endocytobiotic event, which means the uptake of a eukaryotic alga possessing a primary plastid into another eukaryotic host cell followed by the reduction of the endosymbiotic alga to an organelle (secondary plastid). Such processes occurred at least twice in evolution, involving a green or a red alga as endosymbiont [25, 26]. Secondary endocytobiosis dramatically increased algal diversity and led to the evolution of cryptophytes, eugleno-phytes, haptoeugleno-phytes, heterokontoeugleno-phytes, dinophytes and chlorarachniophytes but also in the apicomplexa. Due to this explicit evolutionary history diatoms, which belong to the heterokontophytes, possess plastids bound by four membranes [24, 25, 107, 27].
The outermost of these envelope membranes is studded with ribosomes and appears to be continuous with the host endoplasmic reticulum (ER) and is therefore called chloroplast ER (CER; [47, 48]) membrane. The second membrane most likely repre-sents the plasma membrane of the red algal endosymbiont, whereas the two innermost membranes are homologous to the plastid envelope of the red alga’s primary plastid [49]. Additional envelope membranes represent more barriers for the transport of substrates between the cytosol and the plastids stroma, including nucleus-encoded plastid proteins.
As outlined in chapter 5 there are three plausible models for proteins to be trans-ported across the different envelope membranes: the “pore model”, the “vesicular shuttle model” and the “translocator model” (Figure 6.1) [47, 89, 52]. Common to all these models is a co-translational transport across the CER membrane and translo-cation across the innermost envelope membrane via a Tic (translocon at the inner envelope membrane of chloroplasts) complex. The models differ in the way they explain the passage of the proteins across the second and the third outermost mem-brane. The “pore model” suggests import of nucleus-encoded plastid proteins via a pore through the pps [52]. The “vesicular shuttle model” postulates vesicular trans-port across the pps between these membranes [47]. Finally, the “translocator model”
cators and then are imported into the plastid across the remaining two membranes via a Toc/Tic system (Toc/Tic: translocon at the outer/inner envelope membrane of chloroplasts) similarly to land plant plastids.
CER lumen SPFTP mature protein
SP TP mature protein
CER lumen
SP FTP mature protein
TP mature protein
Figure 6.1.: Schematic illustration of possible import pathways in P. tricornutum plastids. (A) In the “vesicular shuttle model”, preproteins are transported co-translationally into the chloroplast endoplasmic reticulum (CER) lumen via a N-terminal bipartite presequence, then cross the periplastidic space (pps) via vesicles and are released into the interenvelope space (ies). Final import occurs via Tic translocator. (B)In the “translocator model”, proteins are transferred into the pps by an additional translocator (ERAD-like) in the second outermost membrane followed by transport into the plastid stroma via two additional translocators similar to the Toc and Tic in higher plants; (C)In the “pore model”, proteins cross the pps via a pore. Fluorescence images represent different localisations of GFP withinP. tricornutum plastid; red: Chlorophyll autofluorescence, green: GFP fluorescence; Sp: signal peptide; Tp: transit peptide; scissors: peptidase; Fluorescence images: by Sascha Vugrinec; EM image:
by Ansgar Gruber
For successful protein trafficking into plastids, nucleus-encoded polypeptides have to be equipped with additional import information. While in higher plants a transit peptide is sufficient for protein targeting across two chloroplast envelope membranes, diatom preproteins need an additional signal peptide (at the N-terminus of the pre-proteins) for protein transport across the first two envelope membranes into the pps.
For the import into the plastid stroma, they need an additional conserved sequence motif at the signal peptide cleavage site (ASAFAP).
Investigations on FCP-preproteins of the diatom P. tricornutum showed that
pre-proteins containing a bipartite leader sequence were successfully imported into canine microsomes, indicating that transport through ER membranes might be the initial import step into diatom plastids [92].
The “vesicular shuttle model” proposed by Gibbs [47], is based on the presence of vesicles and a periplastidic reticulum within the pps observed as by electron mi-croscopy. Experimental support for this model is the finding that protein transport intoP. tricornutum plastids is blocked by an antibiotic that inhibits Golgi-mediated trafficking (Brefeldin A) [115, 82]. Furthermore it was found that the membranes con-tent of the pps proliferates upon inhibition of plastid protein biosynthesis, while the extension of the periplastidic reticulum decreases when cytoplasmic protein biosynthe-sis is blocked [138]. However, proteins that might be involved in vesicular transport within the pps in diatoms were not detected up to now, although they should be easily distinguishable from their cytosolic counterparts by the presence of a signal peptide.
In the case of the “pore model” there is no experimental support available. By using self-assembling GFP we show in this work, that there is no connection between the pps and the ies (chapter 5). The main experimental support for the “translocator model” is the recognition of transit peptide-like domains from bipartite chromalveo-late presequences consisting of a signal and transit peptide by the Toc complex in heterologousin vitro import experiments [92, 29].
Another suggestion for translocator mediated protein import was proposed by Som-mer et al. [142]. They identified genes for components of the ER-associated degra-dation machinery (ERAD) on the nucleomorph (small, reduced eukaryotic nuclei) genome of the cryptophyteG. theta. The respective genes are also duplicated in the genomes of the diatoms P. tricornutum and T. pseudonana but also in Plasmodium falciparum. An ERAD related machinery involved in the protein import, out of the ER and into the periplastidic space was therefore suggested [142, 62]. However, there is no functional proof yet that this ERAD-like machinery is involved in plastid protein targeting.
To date, a number of potentially pps located proteins have been identified in the diatomsP. tricornutum and T. pseudonana and the cryptophyteG. theta. Especially the identification of a thioredoxin indicates, that enzymatic processes (regulated by thioredoxin) might take place within the periplastidic compartment [54]. It is likely that the pps has a physiological function in diatoms, because of the identification of a number of pps targeted proteins. Different structures have been observed, as the periplastidic reticulum [48], or in the case of G. theta a nucleomorph envelope [36].
reasons and therefore need to be degraded [52]. Clear experimental evidence for an ERAD-like dependent protein transport across the second membrane (counted from outside) is missing so far.
Findings in this work (chapter 5 and 4), but also the necessity of a transit peptide as an import signal may imply that nucleus-encoded plastid proteins cross the third and fourth envelope membrane also through translocons similar to Toc/Tic components from higher plants. However, analyses of the genomes of the diatoms T. pseudonana andP. tricornutum did not yield putative elements of the Toc apparatus (chapter 2) [150, 107]. It was proposed that a component derived from the cyanobacterial outer membrane protein Omp85, which is related to the Toc75 protein in higher plants, could act as a phenylalanine specific receptor and membrane channel across the third outermost membrane [160, 20].
As mentioned before, final transport across the innermost envelope membrane is mediated by a Tic complex. In higher plants seven or eight Tic components have been proposed to form the Tic complex, while in the diatoms P. tricornutum and T. pseudonana seven proteins might be involved in protein translocation (chapter 2).
In higher plants the stromal membrane chaperones GroEL and Hsp93 (ClpC) interact with the Tic complex to receive the imported proteins and fold them, after cleavage of the transit peptide, to their mature conformation [153]. We were able to identify the ClpC gene within the genome of P. tricornutum and T. pseudonana, suggesting that it is possible that inP. tricornutum the last step is similar to higher plants (chapter 2).