Proteomics reveals plastid- and periplastid-targeted proteins in the chlorarachniophyte alga Bigelowiella natans

Abstract

Chlorarachniophytes are unicellular marine algae with plastids (chloroplasts) of secondary endosymbiotic origin. Chlorarachniophyte cells retain the remnant nucleus (nucleomorph) and cytoplasm (periplastidial compartment, PPC) of the green algal endosymbiont from which their plastid was derived. To characterize the diversity of nucleus-encoded proteins targeted to the chlorarachniophyte plastid, nucleomorph, and PPC, we isolated plastid-nucleomorph complexes from the model chlorarachniophyte Bigelowiella natans and subjected them to high-pressure liquid chromatography-tandem mass spectrometry. Our proteomic analysis, the first of its kind for a nucleomorph-bearing alga, resulted in the identification of 324 proteins with 95% confidence. Approximately 50% of these proteins have predicted bipartite leader sequences at their amino termini. Nucleus-encoded proteins make up >90% of the proteins identified. With respect to biological function, plastid-localized light-harvesting proteins were well represented, as were proteins involved in chlorophyll biosynthesis. Phylogenetic analyses revealed that many, but by no means all, of the proteins identified in our proteomic screen are of apparent green algal ancestry, consistent with the inferred evolutionary origin of the plastid and nucleomorph in chlorarachniophytes.

Fig. 1.—

The chlorarachniophyte alga Bigelowiella natans. (A) Transmission electron micrograph showing ultrastructural features of B. natans. Asterisks (*) highlight the PPC, that is, the residual cytosol of the engulfed algal cell. (B) Simplified diagram showing main subcellular compartments and the protein synthesis/trafficking in chlorarachniophytes relevant to this study (highlighted by cartoon ribosomes and white arrows, respectively). The “?” indicates uncertainty with respect to protein trafficking from the ER to the outermost plastid membrane (see text). The numbers of protein genes present in the plastid, nucleomorph, and host nucleus genomes are taken from Rogers et al. (2007), Gilson et al. (2006), and Curtis et al. (2012), respectively. Details surrounding the mitochondrion have been omitted for simplicity. PL, plastid; N, nucleus; NM, nucleomorph; MT, mitochondrion; PY, pyrenoid; C, pyrenoid cap; FR, flagellar root; CB, cyanobacterium derived.

Fig. 2.—

Bigelowiella natans subcellular fractionation and protein isolation. (A) Three-step sucrose density gradient of French press-disrupted B. natans cells. Three distinct bands are apparent. (B) Fluorescence microscopy of material present in “fraction 3.” The image is an overlay of two different channels showing DAPI-stained nuclei (blue) and chlorophyll autofluorescence from the plastids under a rhodamine filter (red). (C) Fluorescence microscopy showing material in fraction 1, which was enriched in plastid–nucleomorph complexes and largely devoid of host cell nuclei. Faint blue spots corresponding to nucleomorphs (NM) can be seen in close association with plastids (PL). A large blue spot corresponding to a nucleus (N) can also be seen. (D) Transmission electron micrograph of isolated B. natans plastids obtained from fraction 1 of the sucrose gradients after subcellular fractionation. (E) TEM of an isolated plastid–nucleomorph complex from B. natans. (F) One-dimensional SDS-PAGE of B. natans proteins isolated from fraction 1. Fourteen discrete bands were excised from the gel, as shown, for proteomic analysis (refer to text for further details).

Fig. 3.—

Maximum likelihood phylogenetic trees of proteins (A) cytochrome C6 (50246) and (B) Hcf136 (236790). The Bigelowiella natans cytochrome C6 protein shows a strong grouping (>75% bootstrap support) with green algae, whereas the tree of Hcf136 suggests a red algal origin.