Survival of digestion may occur by preventing lysosome fusion to the food vacuole, resistance to the hydrolytic enzymes of lysosomes, or escaping from the digestive vacuole into the host cytosol Matz and Kjelleberg, ; Kodama et al. Retention of viable captured cells is an obvious key step for the establishment of permanent endosymbiosis Kodama and Fujishima, , , but another crucial event is the establishment of a steady flux of metabolites between the photosynthetic endosymbiont and the host cytoplasm Weber et al.
The stable exchange of compounds must have been critical as well for the evolution of metabolic interdependencies e. What molecular mechanisms may have facilitated the flux of metabolites during early stages of organelle evolution? Key subcellular events and proteins involved in the evolution of primary photosynthetic organelles. Successfully transferred genes green double strands should have been expressed and translated in the host nucleocytoplasm system.
Cytoplasm-translated proteins Pro of non-cyanobacterial origin red ribbons and cyanobacterial provenance green ribbons were imported into the cyanobacterial compartment via the ancestral protein import machinery PIM. B The recruitment of cytoplasm-translated membrane transporters of host e.
OEP represents diverse host-derived pore proteins e. C The evolution of the core plastid protein import system relied on the activity of the cyanobacterial proteins Toc75 and Tic These two pore-forming proteins were present originally in the plastid ancestor genome but the encoding genes were likely transferred early during endosymbiosis to the host genome Tic20 are still plastid-encoded in some algal lineages.
The ancestral organelle import system was able to translocate both cyanobacterial green ribbons and non-cyanobacterial red ribbons proteins translated in the host cytoplasm. D Other transferred cyanobacterial genes with putative key roles on plastid evolution were those encoding subunits of the Photosytems I psaE, psaI, psaK and II psbO, psbP, and psbU with important roles for the photosynthetic activity under stress conditions.
The cytochrome b6f complex b6f is indicated. E Initial host control over the endosymbiont division likely relied on transferred cyanobacterial genes encoding cytokinesis-associated proteins, such as FtsZ, which is a tubulin-like protein fundamental to assemble the contractile Z-ring. The protein FtsZ is nuclear-encoded in most eukaryotes bearing plastids. Red and green rounded rectangles represent non-cyanobacterial and cyanobacterial-derived proteins, respectively.
Sharing Resources with the Photosynthetic Partner Photosynthetic organelles of plants and algae are bound by at least two membranes e. The membranes surrounding recently captured photosynthetic cells are significant physical barriers for the flux of metabolites between the potential endosymbiont and the host cytoplasm.
The recruitment of membrane proteins able to translocate carbon compounds produced by the cyanobacterial photosynthesis was a critical step for the metabolic integration of the host and the endosymbionts, allowing the interconnection of the metabolite pools of both cells Weber et al.
Interestingly, alternative solutions may have evolved to take advantage of the endosymbiont photosynthates and different membrane transporters have been recruited to translocate metabolites from the photosynthetic compartment. For example, the glaucophyte Cyanophora paradoxa lacks plastid antiporters of the NST family, and UhpC-type transporters Uhp: uptake of hexose phosphate are possibly responsible for the carbohydrate extraction Price et al.
Other plastid-localized transporters that regulate the export and import of diverse metabolites, such as 2-oxoglutarate, glucoseP, riboseP, pyruvate, and tetrahydrofolate, evolved from proteins encoded in the host genome.
The evidence suggests that a basic set of metabolite membrane transporters has been fundamental to highjack photosynthetic endosymbionts and initiate the metabolic integration Tyra et al. Besides the translocation of photosynthates into the host cytoplasm, the metabolic integration must require also the flux of minerals, vitamins and nutrients into the endosymbiont.
A central event to facilitate the recruitment of host proteins for organelle functions is the relocation i. What do we know about the ancestral mechanisms of protein import underlining the evolution of primary photosynthetic organelles?
Regardless of the increased molecular complexity i. A plausible scenario is that a gene encoding a protein of the Omp85 family the ancestral Toc75 was transferred from the cyanobacterial genome into the host nucleus. Subsequently, the Omplike protein was translated in the host cytoplasm and relocated via the host endomembrane system e. The evolution of the protein import function of the TOC pore involved an opposite topological orientation of Toc75 with respect to the orientation of the original Omplike protein in the cyanobacterial OEM Steiner et al.
Organelle import systems have evolved and been remodeled several times during eukaryote history e. The chromatophores of Paulinella are able to import proteins translated in the cytoplasm, probably using both elements of the host endomembrane system and pore-forming proteins localized in the endosymbiont envelope Mackiewicz et al. Overall, phylogenetic and functional data suggest that in principle the import of unfolded proteins into an endosymbiotic compartment can be achieved with a relatively simple set of pore-forming proteins and the participation of the host endomembrane system.
The Plastid Proteome is a Phylogenetic Mosaic Plastid genomes contain, at the most, protein-coding genes Wang et al. Remarkably, only between and of the plastid-localized proteins are of cyanobacterial provenance Price et al. Minimizing the Genetic Program to Run a Photosynthetic Endosymbiont An inevitable consequence of the endosymbiotic life style is the reduction of the genome of the captured cells Selosse et al.
Besides the losses of genetic material, the transference of genes to the host nucleus i. The transference of endosymbiont genetic material seems to be stochastic, relatively frequent and correlated with the amount of endosymbiotic genomic copies available Stegemann and Bock, However, successful EGT implies not only the relocation of the genetic material to the nuclear genome, but the acquisition of elements necessary for expression e.
However, the magnitude and overall impact of EGT during early stages of primary plastid evolution is not entirely understood. A pivotal issue in plastid evolution is the identification of those transferred cyanobacterial genes that were critical for the success of the early endosymbiosis. Most of the genomic evidence to answer that question in plants and algae with primary plastids has been diluted after more than 1 billion years of evolution Yoon et al.
However, comparative genomics studies including information from Archaeplastida lineages and P. The idea that photosynthesis was the major driving force of plastid evolution correlates with the relatively high number of plastid genes related with the photosynthetic function and carbon fixation.
The genome-size reduction was accompanied by movement of genes from the endosymbiont to the host nuclear genome, a process known as endosymbiotic gene transfer EGT. The genome and transcriptome analyses identified more than 30 such EGT-derived genes in the Paulinella nuclear genome Nakayama and Ishida ; Reyes-Prieto et al.
The debate about the organellar status of Paulinella photosynthetic endosymbionts was finally settled by the recent studies of Nowack and Grossman The authors showed that the endosymbionts import nucleus-encoded and EGT-derived photosynthetic proteins, such as PsaE, PsaK1, and PsaK2, and thus should be called true cell organelles.
They also demonstrated that one of these proteins, PsaE, is targeted via the endomembrane system to these organelles but, unexpectedly, not by means of a classic signal peptide, which is commonly used by proteins imported to this system. The co-translational import of PsaE clearly contrasts with the post-translational transit peptide- and Toc-Tic-based mechanism that is characteristic of the Archaeplastida primary plastids Fig. The latter consists of many specialized protein subunits that function as transit peptide receptors Toc34, Toc64, and Toc , protein-conducting channels Toc75, Tic20, Tic21, and Tic , regulatory elements Tic55, Tic62, and Tic32 , scaffold proteins Tic , Toc-Tic translocons connecting subunits Toc12, Tic22 , chaperones Hsp70, Hsp93 , and co-chaperones Tic There are two independent molecular motors pulling imported proteins into the stroma: one consists of Tic, Tic40, and Hsp93, whereas the second engages Hsp70, possibly GrpE, and some unknown J-domain protein J?
After the release of imported proteins into the intermembrane space IMS , their further trafficking through the peptidoglycan wall could be facilitated by molecular chaperones, such as DegP, FkpA, PpiA, and Hsp The final import step, translocation across the inner membrane, may proceed via the Tic21 channel homolog with the help of Ticlike protein, a calcium- and redox-sensing regulatory subunit. The imported proteins are hypothesized to be finally pulled into the organelle matrix by a molecular motor composed of Hsp93, Hsp70, Hsp40, and GrpE Although the findings of Nowack and Grossman are invaluable contributions to the subject of Paulinella endosymbiosis, and thus endosymbiosis in general, there are still two critical issues unsolved: i the nature of targeting signals in proteins imported into Paulinella photosynthetic organelles and ii mechanisms of further protein transport through the subsequent organelle envelope barriers, including the peptidoglycan wall and the inner plastid membrane.
Therefore, we have decided to raise these important questions here. We also discuss implications of the new findings for understanding of the classic primary plastid endosymbiosis, especially early stages in the evolution of photosynthetic organelles derived from cyanobacterial endosymbionts.
Thus, although both PsaE homologs are imported into the same cellular compartment via the endomembrane system, they appear to have evolved distinct targeting mechanisms in the two Paulinella strains.
Notably, their mature sequences differ by one indel and 21 substitutions per only 69 amino acid residues Fig. Eight of these substitutions are non-conserved according to Blosum62 substitution matrix and lead to changes in biochemical properties of amino acid residues.
Consequently, these substitutions could reflect the evolution of an internal PsaE targeting signal in the Paulinella CCAC strain investigated by Nowack and Grossman Eight non-conserved substitutions according to Blosum62 matrix in red , which change biochemical properties of residues, may constitute some internal PsaE targeting signal in the CCAC strain Although targeting mechanisms for the Paulinella PsaK proteins were not determined by Nowack and Grossman , it is possible that they are also imported into Paulinella photosynthetic organelles via the endomembrane system.
According to these authors, the C-terminal hydrophobic domain of Paulinella PsaK proteins could function as an internal targeting signal; however, it seems more likely that their N-terminal hydrophobic domains represent signal peptides Mackiewicz et al.
Compared to their cyanobacterial homologs, these domains have lost several hydrophilic residues and acquired some hydrophobic ones, thereby gaining features of signal peptides Mackiewicz et al. In support of this, experimental studies showed that similar changes in mitochondrial and plastid membrane proteins caused their targeting to the endomembrane system Lee et al. These data suggest that not only PsaE but also other Paulinella endosymbiont-derived proteins encoded by the host nuclear genome can utilize vesicular trafficking to reach the photosynthetic organelles.
Unfortunately, in contrast to the glaucophyte muroplast Pfanzagl et al. Nevertheless, adaptations required for this translocation appear to be the low molecular weight and nearly neutral charge. These features are common to Paulinella EGT-derived proteins and fit well with the properties characteristic of proteins known to cross dense and negatively charged peptidoglycan walls Mackiewicz et al. In support of this, some studies identified Hsp70 in the intermembrane space of primary plastids indicating its role in protein translocation across this space Schnell et al.
Interestingly, Tic21 molecular weight and number of transmembrane domains are characteristic of other protein-conducting channels, e. Tic20 — the classic Tic translocon Chen et al. Although plant Tic21 was claimed to be an iron transporter Duy et al. It is probable that the Paulinella Tic21 homolog fulfills much the same functions in its photosynthetic organelles because the genetic complementation of Arabidopsis tic21 knockout mutant with its cyanobacterial Synechocystis ortholog turned out to be successful Lv et al.
This protein, together with Tic55 and Tic62, is responsible for regulation of protein import by redox-sensing and calcium-signaling in the plastids of green plants or chloroplasts Li and Chiu ; Shi and Theg ; Stengel et al. All these three proteins would then constitute a redox-sensing complex in the Paulinella organelle inner membrane, but it cannot be excluded that Tic32 plays the regulatory function alone.
There are two such Tic-associated motor complexes that act in parallel Fig. In the first motor, Tic, Hsp93 the Hsptype chaperone, and Tic40 co-chaperone cooperate with each other Kovacheva et al. The Tic40 is anchored in the inner membrane with its N-terminal transmembrane region Chou et al. The ATP hydrolysis generates energy necessary for conformational changes of Hsp93 that enable pulling of proteins into the stroma.
The second, recently discovered, import motor involves stromal Hsp70 chaperone as the driving force for protein import and was identified in both the moss Physcomitrella patens Shi and Theg and the higher plant Arabidopsis thaliana Su and Li Because Hsp70 requires proteins with J domain and nucleotide exchange factors e. GrpE for effective functioning Kampinga and Craig , such partners are expected in the Hspbased molecular motor.
In agreement with that, it was found that one of the stromal co-chaperones, CGE Chloroplast GrpE homolog , has an essential role in plastid protein import Shi and Theg Although the involvement of J-domain proteins as a molecular motor component has not yet been confirmed, their role cannot be excluded on the basis of dnaJ-J8 mutants, which still at low level could express truncated J proteins masking expected defects in protein import Chiu et al.
Their absence from the Paulinella organelle genome should be expected because the plant tic and tic40 genes are of eukaryotic origin Shi and Theg The preliminary analyses by Nowack et al. Consequently, Paulinella photosynthetic organelles are probably devoid of a TicHspTicbased molecular motor typical of higher plant plastids.
Such organization of the Paulinella Hsp40 suggests that it could indeed represent a component of the Hspbased molecular motor. The GrpE-like protein, encoded by the Paulinella organelle genome, could function as a nucleotide exchange factor for HspAdditionally, there is a large photosynthesis bp only in KR01, suggesting future complete loss. For definition, some of the proteins that choreograph the plastid division are nuclear encoded and were likely selected from the host repertoire to control organellar functions. The stable exchange P1 aqa revision checklist for essays compounds must have been critical as well for the photosynthesis of metabolic interdependencies e. It has to be explored if the sulA gene has been transferred into the Paulinella nuclear genome and if the encoded protein has any particular role regulating the chromatophore division Nowack et al.
This category encompassed a variety of functional categories, implying that relaxed selection is occurring at the genome level. Even though this process has slowed down, many chromatophore genes remain under relaxed selection when compared to homologs in free-living cyanobacterial relatives. This result suggests that AT-enrichment is an ongoing process in chromatophore genomes. After inactivation, DNA removal occurs because mutational bias favors deletions over insertions in bacterial genomes
Organelle import systems have evolved and been remodeled several times during eukaryote history e. I want to thank Oscar Juarez Illinois Institute of Technology for critical reading of the manuscript and valuable comments.
Lipopolysaccharides are large molecules present in the outer membrane of Gram-negative bacteria The recruitment of membrane proteins able to translocate carbon compounds produced by the cyanobacterial photosynthesis was a critical step for the metabolic integration of the host and the endosymbionts, allowing the interconnection of the metabolite pools of both cells Weber et al. These lineages are expected to provide invaluable information because comparing the genome of free-living cyanobacterial relatives to that of the endosymbionts offers fundamental insights into organellogenesis.
Their absence from the Paulinella organelle genome should be expected because the plant tic and tic40 genes are of eukaryotic origin Shi and Theg The evolution of a stable endosymbiotic relationship between eukaryotic cells and photosynthetic cyanobacteria involved series of cellular and molecular processes that are not entirely understood. Plastids evolved once, a long time ago Photosynthetic eukaryotes are a tremendously diverse collection of organisms, from bacterium-sized unicells and giant kelp in the oceans to the plants and trees that inhabit dry land. Therefore, although Paulinella photosynthetic organelles provide exciting new insights into mechanisms of organellogenesis, it is difficult to use them as a model for the evolution of early stages of protein targeting to classic primary plastids. A total of 22, positions
The observation of relaxed selection on chromatophore genes when compared to free-living alpha-cyanobacteria may, therefore, be explained by the homogeneous intracellular environment. In the more advanced stages of genome degradation, most of the unnecessary information has been completely lost, resulting in highly reduced genomes with a scarcity of pseudogenes. Full size table Figure 1 a Gene synteny comparison of the chromatophore genomes from Paulinella longichromatophora, P.
Other plastid-localized transporters that regulate the export and import of diverse metabolites, such as 2-oxoglutarate, glucoseP, riboseP, pyruvate, and tetrahydrofolate, evolved from proteins encoded in the host genome. Adaptive codon bias is correlated with gene expression level 49 , 50 , 51 , thus Synechococcus genes remaining in the chromatophore may have higher expression.
Even though lost genes in the endosymbiont can be counteracted by the host, some interesting trends are apparent within the retained inventory. Subsequently, the Omplike protein was translated in the host cytoplasm and relocated via the host endomembrane system e. A widely accepted scenario suggests that those endosymbiotic cyanobacteria evolved into the photosynthetic organelles i. Multiple rounds of eukaryote-eukaryote endosymbioses have resulted in a tangled web of plastid-bearing lineages [ 5 ]. The observation of relaxed selection on chromatophore genes when compared to free-living alpha-cyanobacteria may, therefore, be explained by the homogeneous intracellular environment.
In the case of M. This is emphasized by the fact that, in contrast to Paulinella, no photosynthetic proteins are imported into primary plastids via the endomembrane system. The stable exchange of compounds must have been critical as well for the evolution of metabolic interdependencies e. The ancestral organelle import system was able to translocate both cyanobacterial green ribbons and non-cyanobacterial red ribbons proteins translated in the host cytoplasm. Even though this process has slowed down, many chromatophore genes remain under relaxed selection when compared to homologs in free-living cyanobacterial relatives. Lost KEGG pathways from cyanobacteria to chromatophore, and different pathways between chromatophore and Archaeplastida plastids are listed.
This testate filose amoeba is surrounded by the cell wall called theca T , which is composed of silica scales. Among these, only 22 OGFs had intensified selection, whereas the remainder OGFs showed relaxed selection in chromatophore genes. When comparing the chromatophore genomes of P. However, comparative genomics studies including information from Archaeplastida lineages and P.