The physical and functional borders of transit peptide-like sequences in secondary endosymbionts

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 characterization 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.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.Primary plastids are organelles of endosymbiontic origin [e.g. 1, 2]. In the course of the transition from an (endo-)symbiont to an organelle, most of its genes were either lost or, to a higher degree, transferred into the cell nucleus [e.g. 3, 4, 5]. Hence, most of the plastid proteome is encoded in the nucleus of the host cell, implying that the encoded proteins must be transported post-translationally across the two envelope membranes into the plastid lumen. For accurate trafficking, nearly all nuclear-encoded plastid proteins are equipped with a characteristic N-terminal topogenic signal sequence, the transit peptide [6]. This targeting information is necessary and sufficient for plastid import and interacts with translocons of the outer/inner envelope membrane of chloroplasts [TOC and TIC; recently reviewed in 7]. Interestingly, surveys of transit peptides indicate no strict consensus sequence [8] but some common features such as a positive net charge, elevated levels of hydroxylated amino