How protein targeting to primary plastids via the endomembrane system could have evolved? A new hypothesis based on phylogenetic studies

Abstract

Background: It is commonly assumed that a heterotrophic ancestor of the supergroup Archaeplastida/Plantae engulfed a cyanobacterium that was transformed into a primary plastid; however, it is still unclear how nuclear-encoded proteins initially were imported into the new organelle. Most proteins targeted to primary plastids carry a transit peptide and are transported post-translationally using Toc and Tic translocons. There are, however, several proteins with N-terminal signal peptides that are directed to higher plant plastids in vesicles derived from the endomembrane system (ES). The existence of these proteins inspired a hypothesis that all nuclear-encoded, plastid-targeted proteins initially carried signal peptides and were targeted to the ancestral primary plastid via the host ES.

Results: We present the first phylogenetic analyses of Arabidopsis thaliana α-carbonic anhydrase (CAH1), Oryza sativa nucleotide pyrophosphatase/phosphodiesterase (NPP1), and two O. sativa α-amylases (αAmy3, αAmy7), proteins that are directed to higher plant primary plastids via the ES. We also investigated protein disulfide isomerase (RB60) from the green alga Chlamydomonas reinhardtii because of its peculiar dual post- and co-translational targeting to both the plastid and ES. Our analyses show that these proteins all are of eukaryotic rather than cyanobacterial origin, and that their non-plastid homologs are equipped with signal peptides responsible for co-translational import into the host ES. Our results indicate that vesicular trafficking of proteins to primary plastids evolved long after the cyanobacterial endosymbiosis (possibly only in higher plants) to permit their glycosylation and/or transport to more than one cellular compartment.

Conclusions: The proteins we analyzed are not relics of ES-mediated protein targeting to the ancestral primary plastid. Available data indicate that Toc- and Tic-based translocation dominated protein import into primary plastids from the beginning. Only a handful of host proteins, which already were targeted through the ES, later were adapted to reach the plastid via the vesicular trafficking. They represent a derived class of higher plant plastid-targeted proteins with an unusual evolutionary history.

Figure 1

Three evolutionary scenarios for the origin of endomembrane system-mediated protein targeting to higher plant plastids. The phagotrophic ancestor of the kingdom Archaeplastida, including glaucophytes, red algae, and green plants, regularly fed on cyanobacteria, from which genes migrated to the host nucleus via endosymbiotic gene transfer (A). When these endosymbionts evolved into primary plastids, they made use of both cyanobacteria- and host-derived genes present in the host nucleus. According to the ‘relic’ hypothesis for endomembrane system (ES)-mediated plastid protein targeting [33], all such proteins were targeted to the new primary plastid via the endoplasmic reticulum and/or Golgi apparatus (B1). In a later evolutionary stage, this co-translational pathway was replaced by a post-translational route involving Toc and Tic translocons for most plastid-targeted proteins (C). The hypothesis implies that proteins currently imported into higher plant plastids via the ES, such as αAmy3, αAmy7, CAH1, NPP1, are relics of ancestral ES-mediated protein trafficking to the primary plastid. Two alternative scenarios (B2 and B3) conflict with the ‘relic’ hypothesis; they postulate that the Toc and Tic translocons evolved very early in the primary endosymbiosis. In one (B2), a limited subset of host-derived proteins, previously targeted via the ES to different compartments within the host cell, exploited their pre-existing signal peptides to reach the primary plastid. Alternatively (B3), host-derived proteins carrying signal peptides were directed to primary plastids much later, well after the initial primary endosymbiosis, and possibly only in some higher plant lineages (C). Thickness of the colored arrows is proportional to the presumed or known commonality of a given pathway: ES (pink) or Toc-Tic translocons (orange). Stacked thylakoids only evolved in the green primary plastid lineage.

Figure 2

The Bayesian tree for α-amylases obtained in PhyloBayes under the LG + Γ(5) model. Sequence in which more than 50% algorithms recognized SP are indicated in bold. Numbers at nodes, in the presented order, correspond respectively to posterior probabilities estimated in PhyloBayes for LG + Γ(5) model (PP-LG) and CAT + Γ(5) model (PP-CAT), as well as support values resulting from bootstrap analysis in PhyMl (B-Ph) and TreeFinder (B-TF). Values of the posterior probabilities and bootstrap percentages lower than 0.5 and 50% were omitted or indicated by a dash “-“. All bacterial sequences, apart from cyanobacterial ones, are in white background.

Figure 3

Part of the Bayesian tree from Figure 2for α-amylases of green algae and land plants obtained in PhyloBayes under the LG + Γ(5) model. Sequences experimentally proved to be imported to plastids are in red font. Sequence in which more than 50% algorithms recognized signal peptide are indicated in bold and those which possess plastid transit peptide or mitochondrial transit peptide are signed respectively (pTP) and (mTP). Proteins that were shown to be located in the cell wall by mass spectrometry are indicated by (W). Other explanations as in Figure 2.

Figure 4

The Bayesian tree for purple acid phosphatases obtained in PhyloBayes under the LG + Γ(5) model. Sequences experimentally proved to be imported to plastids are in red font. Proteins that were shown to be located in the cell wall by mass spectrometry are indicated by (W), and those that were experimentally proved to reside in endomembrane system by (E). Other explanations as in Figure 2.

Figure 5

The Bayesian tree for protein disulfide isomerases obtained in PhyloBayes under the LG + Γ(5) model. Sequences experimentally proved to be imported to plastids are in red font. Other explanations as in Figure 2.