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The physical and functional borders of transit peptide-like sequences in secondary endosymbionts
(2010)
Background: Plastids rely on protein supply by their host cells. In plastids surrounded by two membranes (primary plastids) targeting of these proteins is facilitated by an N-terminal targeting signal, the transit peptide. In secondary plastids (surrounded by three or four membranes), transit peptide-like regions are an essential part of a bipartite topogenic signal sequence (BTS), and generally found adjacent to a N-terminally located signal peptide of the plastid pre-proteins. As in primary plastids, for which no wealth of functional information about transit peptide features exists, the transit peptide-like regions used for import into secondary ones show some common features only, which are also poorly characterised. Results: We modified the BTS (in the transit peptide-like region) of the plastid precursor fucoxanthin-chlorophyll a/c binding protein D (FcpD) fused to GFP as model substrate for the characterisation of pre-protein import into the secondary plastids of diatoms. Thereby we show that (i) pre-protein import is highly charge dependent. Positive net charge is necessary for transport across the plastid envelope, but not across the periplastid membrane. Acidic net charge perturbs pre-protein import within the ER. Moreover, we show that (ii) the mature domain of the pre-protein can provide intrinsic transit peptide functions. Conclusions: Our results indicate important characteristics of targeting signals of proteins imported into secondary plastids surrounded by four membranes. In addition, we show a self-targeting mechanism, in which the mature protein domain contributes to the transit peptide function. Thus, this phenomenon lowers the demand for pre-sequences evolved during the course of endosymbiosis.
The plastids of cryptophytes, haptophytes, and heterokontophytes (stramenopiles) (together once known as chromists) are surrounded by four membranes, reflecting the origin of these plastids through secondary endosymbiosis. They share this trait with apicomplexans, which are alveolates, the plastids of which have been suggested to stem from the same secondary symbiotic event and therefore form a phylogenetic clade, the chromalveolates. The chromists are quantitatively the most important eukaryotic contributors to primary production in marine ecosystems. The mechanisms of protein import across their four plastid membranes are still poorly understood. Components of an endoplasmic reticulum-associated degradation (ERAD) machinery in cryptophytes, partially encoded by the reduced genome of the secondary symbiont (the nucleomorph), are implicated in protein transport across the second outermost plastid membrane. Here, we show that the haptophyte Emiliania huxleyi, like cryptophytes, stramenopiles, and apicomplexans, possesses a nuclear-encoded symbiont-specific ERAD machinery (SELMA, symbiont-specific ERAD-like machinery) in addition to the host ERAD system, with targeting signals that are able to direct green fluorescent protein or yellow fluorescent protein to the predicted cellular localization in transformed cells of the stramenopile Phaeodactylum tricornutum. Phylogenies of the duplicated ERAD factors reveal that all SELMA components trace back to a red algal origin. In contrast, the host copies of cryptophytes and haptophytes associate with the green lineage to the exclusion of stramenopiles and alveolates. Although all chromalveolates with four membrane-bound plastids possess the SELMA system, this has apparently not arisen in a single endosymbiotic event. Thus, our data do not support the chromalveolate hypothesis. Key words: Emiliania huxleyi, secondary endosymbiosis, chromalveolate, hypothesis, complex plastid, plastid protein import, algal evolution