|Fig.1 Cyanophora paradoxa|
Cyanophora paradoxa , a photoautotrophic protist harboring a special type of plastids named cyanelles, has been the object of our studies for more than 20 years. This peculiar system per se constitutes a proof for plastid evolution according to the endosymbiotic theory: the cyanelles combine a plastid-like genome with an unusual gene complement with a peptidoglycan-containing "organelle wall" that is unique among eukaryotes (Fig. 1). In studying the interesting features of this organism we use approaches and methods from biochemistry, cell biology, and molecular biology.
In the past years we have been able to characterize 15 cyanelle genes that are nucleus-encoded in higher plants [1,2,11]. Among those are the genes for ferredoxin, for six ribosomal proteins, for enzymes involved in the biosynthesis of low MW compounds (NADP+, chlorophyll, and isoprenoids) and for SecY, a component of the bacterial pre-protein translocase. The latter gene can complement a secY mutation in E. coli that leads to a thermosensitive phenotype. The Sec transport system, once postulated by the "Conservative Sorting" hypothesis, is now established for the thylakoid membranes of chloroplasts. We want to demonstrate by means of specific antibodies its localization on the cyanelle inner envelope membrane, too. Dual translocons, as found in cyanobacteria, might be another primitive feature of cyanelles (in addition to the peptidoglycan wall) that later on was abandoned in chloroplasts (Fig. 2).
Significant contributions by the labs of Don Bryant and Hans Bohnert led to the completion of the nucleotide sequence of the 135,599 bp cyanelle genome. A surplus of more than 50 genes specifying chaperonins, transcription factors, biosynthetic enzymes, ribosomal proteins etc. became apparent. On comparison of the organization of the plastid genome of C. paradoxa with the counterparts from algae and higher plants we and others arrived at the conclusion that all plastid types are derived from a common precursor, the "protoplastid", that resulted from a singular primary endosymbiotic event. This means that the successful endosymbiosis between a mitochondriate eukaryotic cell and a cyanobacterium (involving extensive gene transfer to the host cell nucleus and the development of a protein import apparatus on the endosymbiont envelope) happened only once. Then, a sister group relationship should exist between glaucocystophytes, rhodophytes and chlorophytes (i.e. green algae and higher plants). Recently, support for this concept came from phylogenetic analyses of nuclear genes (where we contributed the sequences of b-1-tubulin and elongation factor 1a) as well as mitochondrial genes.
The import of nucleus-encoded cyanelle polypeptides across the two envelope membranes and the peptidoglycan wall is a major topic of our present research. 13 transit peptides of C. paradoxa are now known, from 11 different precursor proteins. They are analogous to chloroplast stroma-targeting peptides with respect to amino acid composition and hydropathy plots. Pre-ferredoxin-NADP+-reductase (FNR), pre-cytochrome c6, and pre-transketolase from C. paradoxa could be imported in vitro into isolated cyanelles as well as into isolated pea chloroplasts . This points to an analogous import apparatus for cyanelles and chloroplasts and is in accordance with the postulated origin of all plastids from a semiautonomous endosymbiont that had retained the wall of its cyanobacterial ancestor. Isolation of cyanelle envelope membranes and probing of protein blots with antisera directed against identified components of the import apparatus of higher plant chloroplasts yielded signals only in the case of two cyanobacterial homologs to Toc75 (the putative outer membrane channel protein) and Tic55 (an inner membrane protein of unknown function). Thus the cyanelle Toc/Tic translocon has to be viewed as kind of a prototype that underwent modifications in the course of chloroplast evolution. A highly conserved phenylalanine in the third (sometimes fourth) position at the amino terminus of C. paradoxa transit peptides seems to be crucial for recognition of precursors by the cyanelle protein import apparatus: replacement through other amino acids severely impeded or abolished envelope translocation.
In the case of the pre-cytochrome c6 a leader sequence for translocation across the thylakoid membrane follows immediately after the transit sequence . This precursor was used for cyanelle/chloroplast and thylakoid import experiments: cytochrome c6 appeared to be transported to the thylakoid lumen via the SecA/Y pathway, an example for "conservative sorting". Sec-dependence of thylakoid import could also be demonstrated for a cyanelle-encoded protein of the thylakoid lumen: cytochrome c550 . We are also interested if the other mechanisms for protein transport across and integration into the thylakoid membrane that exist in chloroplasts, i.e. the DpH dependent Tat pathway, the posttranslational signal recognition particle (SRP) pathway, and the spontaneous (unassisted) pathway are operative in a primitive plastid. Our candidates are Rieske Fe-S protein (Tat pathway) and the nucleus-encoded ATP synthase subunit AtpI (SRP pathway), respectively. In the pea chloroplast system we investigated the energy requirements for thylakoid integration of the Sec translocase core component SecE (from Arabidopsis) and found support for the operation of the spontaneous pathway .
Cyanelle inorganic pyrophosphatase appeared to represent the rare case where a host cell gene product was directed to the organelle after the endosymbiotic event . These data which are based upon N-terminal sequencing and mass spectrometry of proteins still have to be complemented by gene sequences.
As a side product of this line of work, ATP citrate lyase (an enzyme providing acetyl coenzyme A for fatty acid biosynthesis) reported for long to be a chloroplast protein was localized to the cytoplasm in C. paradoxa and, likely, also in higher plants .
Previously, we identified seven penicillin-binding proteins in the cyanelle envelope, performing the periplasmic steps of the cyanelle wall biogenesis and a number of stromal enzymes responsible for providing the soluble precursor, UDP-N-acetylmuramylpentapeptide . During investigations on the fine structure of C. paradoxa cyanelle peptidoglycan which can be immunodecorated using antisera directed against the cell wall of E. coli (Fig.1), N-acetylputrescine was identified as a substituent at the D-glutamyl moiety of many peptide side chains. 29 different muropeptide monomers, dimers, trimers, and tetramers were characterized using HPLC and MALDI mass spectrometry. Substitution with N-acetylputrescine was also detected in cyanelle peptidoglycan from two other glaucocystophyte algae but not in a cyanobacterial peptidoglycan. We postulate that this modification renders the organelle wall more permeable for the >2000 precursor polypeptides that continuously have to be imported. Recently, the membrane-bound steps of cyanelle peptidoglycan biosynthesis have been investigated: they are organized in analogy to the pathway in E. coli. The amidation with putrescine was shown to occur at the level of lipid II . Surprisingly, the cyanelle genome does not encode any of the more than 30 enzymes involved in peptidoglycan biosynthesis (with the possible exception of ftsW). The respective nuclear genes have been searched for using several different strategies but thus far without success.
Cyanelles contain a central body (Fig.1) which we postulate, due to preliminary evidence, as the only example of an eukaryotic carboxysome. Cyanobacteria and microalgae possess inducible (by low CO2 concentrations) transporters for CO2 and HCO3- leading to accumulation of inorganic carbon (Ci) inside the cell (chloroplast). Carbonic anhydrase (CA) associated to a Rubisco-containing microcompartment (a carboxysome in cyanobacteria, a pyrenoid in algal plastids) creates an optimal environment for CO2 fixation. We plan to characterize as many components (transporters, CAs, carboxysomal shell proteins, etc.) of the CCM of C. paradoxa as possible. A microarray approach will also be involved, in order to find genes the expression of which is increased upon shift from 2% CO2 to ambient (0.035%) concentration. For that purpose, cDNA libraries were established from cells grown at high CO2 as well as from cells adapting to low CO2 for 2, 6, and 24 h. Thus far, 5000 expressed sequence tags have been produced, comprising about 2200 unique genes. Some of them appeared to be very valuable for our various lines of research.
The plastids of euglenoids are surrounded by three membranes and are thought to be the result of a secondary endosymbiosis involving a green algal invader. Thus protein sorting to these plastids is a complex process involving ER and Golgi apparatus . We could show that precursors to thylakoid lumen proteins as pre-cytochrome c6 and pre-OE33 (from the oxygen evolving complex of photosystem II) possess tripartite presequences (signal peptide-transit peptide-thylakoid transfer peptide) with three hydrophobic domains . The expression of nucleus-encoded chloroplast proteins was studied in wild type Euglena and bleached mutants obtained by treatment with xenobiotics [7,10].
Wolfgang Löffelhardt, Assoc. Professor, tit.ao. Professor, Ph. D.
phone +43-1-4277-512811, fax +43-1-4277-9528,
e-mail: Wolfgang Löffelhardt