Conservative Sorting in Cyanelles

Research project P 14573 Conservative Sorting in Cyanelles Wolfgang LÖFFELHARDT 26.6.2000 The cyanelles of Cyanophora paradoxa and other glaucocystophyceae are the sole plastids that have retained the peptidoglycan wall of their cyanobacterial ancestor. Other parallels between cyanelles (but not the chloroplasts from higher plants) and cyanobacteria are the presence of a periplasmic space and the phycobilisome-bearing thylakoids that do not form tight vesicles after isolation. In the course of the evolution from an endosymbiotic prokaryote to a semiautonomous organelle, gene transfer to the nucleus was paralleled by the development of a protein import apparatus that eventually cooperated with the preexisting prokaryotic protein translocases in the sense of "conservative sorting". Recently it became clear that all known thylakoid transfer and integration mechanisms were prokaryotic achievements. The well established cyanelle in vitro system circumvents the problems encountered with import into cyanobacterial thylakoids. Here we can study the energy requirements for the translocation of lumenal proteins that are not found in plants. The posttranslational thylakoid integration of AtpI takes place only in cyanellvs, since this polytopic integral membrane protein is plastid-encoded in all other known cases. We want to investigate the possible involvement in this process of a signal recognition particle composed of 54 kDa and 43 kDa proteins. Regions with similarity to the SRP-binding motifs of chlorophyll a/b binding proteins are present within the AtpI sequence. Multiple and overlapping transport pathways seem also possible: here we want to exploit that for the Rieske Fe-S protein with respect to the SRP and pH pathways. We want to test our hypothesis that the different composition of the oxygen-evolving complex (OEC) in cyanobacteria and primitive plastids compared to chloroplasts results from a redundancy of subunits in an ancestral cyanobacterium. We want to show that the exchange of certain OEC proteins is accompanied by a shift in the thylakoid transport mechanism. We will try to prove the existence of SecA/E/Y and Tat pre-protein translocases in the thylakoid and inner envelope membranes of cyanelles, in contrast to chloroplasts where these prokaryotic transport apparatus seem to be confined to the thylakoid membranes.

The alga Cyanophora paradoxa constitutes a "missing link" with respect to plastid evolution. Plastids, the organelles harbouring the photosynthetic apparatus in photoautotrophic eukaryotes, comprise chloroplasts of higher plants and green algae, rhodoplasts of red algae, cyanelles of glaucocystophyte algae (the so-called primary plastids, surrounded by two membranes) and a number of secondary plastids that are surrounded by three or four membranes and also show different morphology and pigmentation. The primary plastids are the result of a (single? ) primary endosymbiotic event (cyanobacterium heterotrophic eukaryote), whereas the secondary plastids are thought to originate from multiple (and polyphyletic) secondary endosymbiotic events (eukaryotic alga heterotrophic protist). The cyanelles are the most primitive among the primary plastids in having retained not only the phycobilisome light-harvesting antenna system of the ancestral cyanobacteria (a feature shared with rhodoplasts) but also - unique among eukaryotic organelles - a peptidoglycan layer between the inner and outer envelope membranes. The aim of this project was to compare the intraorganellar protein routing in cyanelles with that in cyanobacteria and in the derived chloroplasts of higher plants ("conservative sorting"). In addition, experiments were done to elucidate the relationship between the protein import apparatus of the different primary plastid types. A single primary endosymbiotic event requires basically similar import mechanisms for cyanelles, rhodoplasts, and chloroplasts. Concerning conservative sorting, we could show functional Sec and Tat pathways for a subset of thylakoid luminal or integral proteins as has been reported for chloroplasts and prokaryotes. Our results render a dual location of the Sec translocase in thylakoid and inner envelope membranes highly likely. Candidate passenger proteins for the two additional pathways (signal recognition particle-dependent and "spontaneous") for thylakoid integration were also found but a definitive proof is still missing. In the cyanelle system, protease protection of proteins within vesicles from phycobilisome-bearing thylakoid membranes could be shown for the first time. Also, the in vitro assembly of phycobilisomes via incorporation after import of a labelled linker polypeptide into this supramolecular structure could be demonstrated. The protein import apparatus of cyanelles appeared to be a prototype that later in evolution was slightly modified: the cyanelle import receptor seems to be specific for phenylalanine at the N-terminus of transit peptides whereas the redundant receptors of chloroplasts don`t show this requirement. This makes sense since cyanelles are expected to most closely resemble the common ancestor of all plastids: our data support the assumption that the kingdom plantae arose from a single primary endosymbiotic event.