Cyanelles are the peculiar plastids of glaucocystophyte algae that retained a peptidoglycan wall from the ancestral cyanobacterial endosymbiont. All cyanobacteria and most algae possess an inorganic carbon-concentrating mechanism (CCM) that involves a microcompartment--carboxysomes in prokaryotes and pyrenoids in eukaryotes--harboring the bulk of cellular (plastidic) Rubisco. In the case of the living fossil, Cyanophora paradoxa, the existence of a CCM was a matter of debate. Microarray data revealing 142 CO(2)-responsive genes (induced or repressed through a shift from high to low CO(2) conditions), gas exchange measurements and measurements of photosynthetic affinity provided strong support for a CCM. We favor a recent hypothesis that glaucocystophyte cyanelles as the closest cousins to cyanobacteria among plastids contain 'eukaryotic carboxysomes': bicarbonate enrichment within cyanelles should be considerably higher than in chloroplasts with their pyrenoid-based CCM. Thus, the stress-bearing function of the peptidoglycan layer, the other unique heritage, would be indispensable. An isolation method for cyanelle 'carboxysomes' was developed and the protein components other than Rubisco analyzed by MS. Rubisco activase was identified and corroborated by western blotting. The well-established cyanelle in vitro import system allows to use them as 'honorary cyanobacteria': assembly processes of supramolecular structures as phycobilisomes and carboxysomes thus can be studied after import of nucleus-encoded precursor proteins and subsequent fractionation. Even minor components can easily be tracked and a surprisingly dynamic view is obtained. Labeled pre-activase was imported into isolated cyanelles and 30% of the mature protein was found to be incorporated into the carboxysome fraction. A final decision between carboxysome or pyrenoid must await the identification of cyanelle carbonic anhydrase and, especially, the demonstration of shell proteins.

ABSTRACT

Abstract:The cyanelles of the glaucocystophyte Cyanophora paradoxa combine two prokaryotic features not found in other phototrophic eukaryotes: a peptidoglycan wall and a putative carboxysome. Both of them would be indispensable when a inorganic carbon concentrating mechanism involving high accumulation of bicarbonate in the cyanelle stroma is assumed. Two approaches were used. (i) An expressed sequence tag library was generated allowing access to interesting genes and microarray technology. Hybridization of the microarrays to RNA from cells grown at high and low CO 2 yielded 97 genes that were upregulated under CO 2 stress whereas 87 genes were found to be downregulated.(ii) Cyanelle central bodies were isolated and protein components other than Rubisco were investigated by mass spectrometry. So far, mass spectrometric analysis of putative carboxysomal proteins yielded only sequences with no match in the databases. Rubisco activase could be shown via in vitro import and Western blotting to be copackaged with Rubisco in isolated purified central bodies. While our data support the presence of an inorganic carbon concentrating mechanism in cyanelles, they do not allow us to distinguish the microcompartment as a carboxysome or pyrenoid.Key words: Cyanophora paradoxa, cyanelles, carboxysome, Rubisco activase, carbon-concentrating mechanism, microarrays.Résumé : Les cyanelles de la glaucocystophyte, Cyaonophora paradoxa, combinent deux caractéristiques procaryotiques qu'on ne retrouve pas chez d'autres eucaryotes phototrophes; une paroi de peptidoglucans et un présumé carboxysome. Ces deux caractéristiques seraient indispensables, si on assume qu'un mécanisme de concentration du CO 2 est impliqué dans l'accumulation du bicarbonate dans le stroma des cyanelles. Les auteurs ont utilisé deux approches. (i) Ils ont généré une librairie des séquences génomiques exprimées qui permet d'avoir accès à des gènes intéressants et à la technologie des puce à ADN. L'hybridation de puces à ADN avec l'ARN de cellules cultivées en présence de faibles ou de fortes teneurs en CO 2 , montre que 97 gènes ont été activés, alors que 87 ont été réprimés. (ii) Ils ont isolé les corps centraux des cyanelles et ils ont examiné les constituants protéiques, autres que la Rubisco, par spectroscopie de masse. Jusqu'ici, les analyses par spectroscopie de masse des protéines des carboxysomes ne conduit qu'à des séquen-ces sans correspondance dans les banques de données. On a pu montrer que l'activase de la Rubisco, via importations in vitro et les transferts Western, est attachée à la Rubisco, dans des corps centraux isolés et purifiés. Bien que les données supportent la présence d'un mécanisme de concentration du CO 2 dans les cyanelles, elles ne permettent pas de distinguer le microcompartiment comme un carboxysome ou comme un pyrénoïde.