B09: Exploring the symbiotic interface of amoebal host and its nascent photosynthetic organelle in the amoeba Paulinella chromatophora (Rhizaria, Cercozoa)

Eukaryotes co-opted photosynthetic carbon fixation from prokaryotes by engulfing a cyanobacterium and stably integrating it as a photosynthetic organelle (plastid) in a process known as primary endosymbiosis. The sheer complexity of interactions between a plastid and the surrounding cell that started to evolve over 1 billion years ago, make it challenging to reconstruct intermediate steps in organelle evolution by studying extant plastids. Recently, the cercozoan amoeba Paulinella chromatophora was identified as a much sought-after intermediate stage in the evolution of a photosynthetic organelle. P. chromatophora harbors nascent photosynthetic organelles termed ‘chromatophores’ which evolved from a cyanobacterium much more recently than the plastids of plants and algae. The chromatophores localize to the cytoplasm of P. chromatophora and are surrounded by two envelope membranes. These membranes that form the interface between host and cyanobacterial symbiont -and therewith mediate communication between the two partners- play a critical role during organellogenesis. Metabolic reconstructions from recently obtained genomic and transcriptomic data imply an intricate metabolic entanglement of host and chromatophore and allowed us to make specific predictions about many metabolites that seem to be exchanged between host and chromatophore. Since membrane transport systems encoded on the chromatophore genome are extremely limited, insertion of nuclear-encoded solute transporters into the chromatophore envelope membranes (CrEMs) that control metabolic fluxes between the two symbiotic partners have to be postulated. In our sequencing data we identified >400 nuclear-encoded transporter candidate genes. For some predicted transporters amino acid sequence homology searches suggest specific transport functions; however, for most only general functions are predicted.

In addition to transporter based inter-organellar metabolite exchange, in plants, recently it was shown that exchange of non-polar substrates between plastid and ER can proceed without the involvement of specific transport proteins by a membrane hemifusion mechanism. Proteins of the DUF3411 family are suspected to be involved in mediating this inter-organellar contact (see Project B11, Weber). Interestingly, blast searches of our new P. chromatophora transcriptome dataset revealed a DUF3411 homologue in P. chromatophora (PcDUF3411). This gene is typically restricted to photosynthetic eukaryotes with canonical plastids, and our phylogenetic analyses of PcDUF3411 suggest that it was obtained by horizontal gene transfer from a green alga.

The main aims of this project will be (i) to identify solute transporters that localize to the CrEMs; (ii) to characterize their phylogenetic origin and substrate specificity; (iii) and to test the hypothesis that the P. chromatophora DUF3411 homologue is involved in mediating interactions between CrEMs and the endomembrane system. For this purpose, we will infer CrEM transporter candidates from our list of in silico predicted transporter genes. This approach will be complemented through a targeted yeast mutant complementation screening. Substrate specificities for candidate transporters identified by any of these two approaches will be characterized in a heterologous system. Using antibodies raised against specific transport proteins, their localization within the P. chromatophora cell will be determined. Furthermore, we will test if PcDUF3411 can complement plant mutants that are lacking specific DUF3411 proteins in collaboration with the group of Andreas Weber. And finally, subcellular localization of PcDUF3411 in P. chromatophora will be tested and interaction partners identified using specific antibodies raised against PcDUF3411.

This research will be critical for a better understanding of how integration of chromatophore physiology into the biological networks of the host cell is accomplished, and -in more general- will provide insights into mechanisms that eukaryotic cells employ to take control over a bacterial endosymbiont and that might be important early events in the evolution of an organelle.

Figure 1: (A) Light microscopic image of P. chromatophora. (B) Transmission electron microscopic image of a cross-sectioned chroma-tophore. Cb: carboxysome; Ch: chromatophore; E: envelope membrane; P: pseudopodium; S: silica scales forming the cell wall or theka; T: thylakoids.

Project leader:
Dr. Eva Nowack, undefined email, Institute of Microbiology,
                         undefinedEmmy Noether Group “Microbial Symbiosis and Organelle Evolution"

Researcher: Linda Oberleitner, undefined email

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