BACKGROUND
the plastids of glaucophytes, rhodophytes, green algae and higher plants are surrounded by an envelope consisting of two membranes.
these "primary" plastids are thought to have originated from a single primary endosymbiotic event .
cyanelles  are the peptidoglycan-armored plastids of glaucocystophyte algae which represent the first diverging phototrophic eukaryotes, on the earliest branch after initial endosymbiosis.
cyanelles can be envisaged as the closest cousins to free-living cyanobacteria among plastids  <cit> .
the unique murein layer serving as "organelle wall" is modified through amidation of the free c- <dig> carboxylic group of the d-isoglutamyl moiety with n-acetylputrescine  <cit> , which is unusual in the eubacterial kingdom and reduces the negative net charge of the murein layer that might interfere with protein import.
cyanelle  protein import machineries in plastids should be considered as prototypes of translocation systems that later underwent substantial modifications.
these primitive translocons strictly require phenylalanine in the n-terminal domain of the transit peptide, even when they fulfil their function in a secondary plastid derived from a red alga  <cit> .
a key event for transition of the "primordial plastids" to the chloroplasts of green algae and higher plants is the gain of  receptors in the toc complex conferring less stringent and overlapping specificities finally leading to dispensability of the once crucial phenylalanine  <cit> .
recent reports confirmed the dependence of protein translocation across the outer membrane of cyanelles or the third outermost membrane of plastids derived from secondary endosymbiosis through a red alga, on the presence of a phenylalanine within the transit sequence  <cit> .
in the absence of import experiments with red algae, the similarity of the n-terminal phenylalanine motif of porphyra yezoensis plastid precursor proteins  <cit>  to their counterparts from c. paradoxa is striking.
however, the translocation of precursor proteins across the outer membrane of higher plant and green algal chloroplasts does not show such dependence  <cit> .
thereby, the phenylalanine represents a key for the understanding of the development of translocation machinery and their receptors.
yet, one question has not been explored satisfactorily, namely, what the phenylalanine is needed for.
current models favour an interpretation where this amino acid interacts with the ancestral translocon built up by endosymbiont-derived toc <dig>  <cit> .
however, other models on the evolutionary development and mechanism of translocation focused on an initial specific interaction between the precursor protein and the organellar membrane .
we reconsidered both possibilities, since phenylalanine is an aromatic amino acid known to exhibit pronounced hydrophobic properties  <cit> , and it is known to insert more deeply into the membrane than other less hydrophobic amino acids like alanine  <cit> .
furthermore, phenylalanine is one important component in "aromatic belts" demarcating the hydrophobic surface immersed in the lipid bilayer  <cit> .
it is noteworthy that all bacterial outer membrane protein structures analyzed so far show an interaction between their ultimate amino- and carboxyterminal domains.
in some cases this contact is closing the β-barrel structure completely  or in addition forming a hydrophobic interaction between a phenylalanine in the n-terminal domain and the c-terminal phenylalanine .
these observations would favour a role of phenylalanine in membrane association of the cytosolic precursor as first contact with the cyanelle.
the importance of aromatic amino acids in protein-protein interactions or substrate recognition is manifold documented.
for example, aromatic residues are found in the substrate-binding site of the aaa+ chaperone clpb that are located at the central pore of the first aaa domain.
these aromatic residues may act as a molecular clamp by binding and releasing substrates in a nucleotide-dependent manner  <cit> .
translocation of exposed segments enriched in aromatic residues  of a protein aggregate by clpb would lead to the continuous extraction of unfolded polypeptides from an aggregate.
therefore hydrophobic interactions between aromatic residues are a common principle of protein-protein cooperation.
furthermore, phenylalanine is reported to occupy prominent positions in the sequences of proteins targeted to the bacterial outer membrane  <cit> , such as porins  and type iv pilins .
recently it has been shown that the penultimate residue of bacterial outer membrane proteins is involved in the species specificity of omp <dig> recognition  <cit> .
interestingly, cyanobacterial outer membrane proteins show the c-terminal consensus sequence fxf  <cit> .
thereby, it is tempting to speculate that a phenylalanine  residue is important for the interaction of precursor proteins with the toc translocon of primitive plastids.
in here we describe the influence of the phenylalanine on the interaction with lipids and lipid surfaces represented by liposomes and proteoliposomes.
the obtained specificity for proteoliposomes is subsequently discussed in the context of the two "initial receptor models" and the evolutionary development of the chloroplast translocon.
RESULTS
the phenylalanine defines lipid specificities of pfnr
we first analyzed the association of the precursor of the ferredoxin:nadp+-oxidoreductase  from cyanophora to lipids found in the membrane of chloroplasts isolated from higher plants .
even though the lipid content of the outer membrane of cyanelles is not yet known, it is envisioned that loss of the lipopolysaccharides paralleled the endosymbiotic process  and that they were replaced by phosphatidylcholine and galactolipids .
hence, the composition of the outer membrane of e.g.
pea chloroplasts serves as a model for the lipid composition of the outer membrane of cyanelles.
we used the well established lipid dot blot  analysis  <cit>  to determine the interaction between precursor and specific lipids.
the precursor protein interacts with phosphatidylcholine  .
interestingly, pfnr did not show an interaction with other neutral lipids , but with the two charged lipids sulfoquinovosyldiacylglycerol  and phosphatidylglycerol , though to a reduced extent when compared to the interaction with pc .
we further controlled whether the lipids are capable of rejecting pfnr by mixing  <dig> or  <dig> mol% of the lipids to pc .
however, at the lower concentration of the added lipid the association of pfnr is comparable to its binding to pc .
to explore the influence of the phenylalanine on this interaction, we used the two types of mutants previously generated  <cit> .
in a first set, the phenylalanine was replaced either by a tyrosine or by glycine.
in a second set, the phenylalanine itself or with the two neighbouring amino acids were removed.
when binding of the mutants to lipids was analyzed we did obtain the highest affinity for pc as seen before for pfnr.
however, some changes in lipid binding can be found.
the most conservative mutant f→y parallels the feature of wild type in terms of not interacting with mono – or digalactosyldiaclglycerol.
.
it still binds to sqdg, but not to pg, suggesting that its interaction to sqdg is not driven by electrostatic interactions.
in contrast, the exchange of the phenylalanine by glycine introduces marked alterations  as this mutant shows no binding to pg but to all galactolipids.
the two deletion mutants reveal a reduced but still recognizable binding to pc and sqdg, but not to the other lipids .
hence, we obtained a chain of alterations of lipid specificities or binding capacities, which somewhat, but not fully parallel the import behaviour observed  <cit> .
the aromatic properties of the phenylalanine define the lipid specificity
to further support our findings we have focused on the comparison between wild type and the two mutants with amino acid exchange.
these two mutants have differential import rates  <cit>  and an altered lipid binding behaviour .
we analyzed the binding of the three proteins to pc, dgdg and pg as pg was found to be specifically recognized by the wild type precursor and dgdg specifically by the mutant with phenylalanine to glycine exchange .
we were indeed able to confirm the highest binding capacity for all proteins to pc  and the enhanced capacity for this association by the f→g mutant.
based on these experiments we were able to determine an apparent dissociation constant.
to directly explore differential features the ratio between the apparent dissociation constants of the mutant and wild type for each lipid, was calculated.
by this it becomes obvious that the conservative exchange from phenylalanine to tyrosine somewhat reduces the association with phospholipids, but does not effect the affinity for galactolipids .
in turn, the exchange of phenylalanine to glycine enhances the association to galactolipids or pc considerably, whereas the association to pg is somewhat reduced .
however, this reduction is not a result of the charge as pfnr f→g binds do sqdg better than wild type .
therefore, the exchange of phenylalanine to another aromatic amino acid only slightly affects binding by reducing the affinity to phospholipids but not altering the affinity for galactolipids, whereas removal of the aromatic amino acid at this position significantly enhances affinity for galactolipids without significant alterations of the affinity for phospholipids.
binding of pfnr to liposomes
so far we have determined the association of the proteins to immobilized lipids.
to further support our findings and to establish a system to reconstitute the toc <dig> like proteins, we have analyzed the association of the precursor proteins to liposomes .
we were able to reproduce the findings with immobilized lipids in terms of the enhanced affinity of pfnr f→g for the liposome surface composed of pc or pc and dgdg, the loss of interaction with pg or dgdg containing surfaces by the f→y replacement and the reduced affinity of the mutant with deleted phenylalanine .
however, we also obtained a difference when compared to the fat western analysis, as pfnr wt binds significantly better to liposomes containing  <dig> mol% pg than to liposomes composed of pc only .
we subsequently analyzed the binding in more detail by incubation of radioactive precursor proteins with different concentrations of liposomes .
the specificity of the interaction was controlled by analyzing the precipitation of the precursor in the absence of liposomes .
quantification of these experimental data revealed a four to five fold lower apparent dissociation constant  for the binding of pfnr to pc/pg liposomes than to pc or pc/dgdg liposomes .
for pfnr f→g we again obtained the highest efficiency for pc liposomes revealing an apparent dissociation constant, which is two times lower than the binding of the mutant to pc/dgdg liposomes or the wild type protein to pc.
the mutant shows the lowest binding capacity for pc/pg liposomes.
here, the apparent dissociation constant is  <dig> times higher then for pc liposomes.
summarizing, we document that the alteration of the phenylalanine modifies the lipid binding behavior of the precursor protein to lipids immobilized to a surface or present in liposomes.
in both cases, deletion of the phenylalanine results in a reduction of the association .
the exchange of the phenylalanine to tyrosine somewhat changes the specificity for the lipids, but does not significantly change the affinity to phosphatidylcholine .
in contrast, exchange of phenylalanine to glycine on one hand enhances the association to phosphatidylcholine and changes the specificity of the protein from preferred interaction with phosphatidylglycerol to preferred interaction with galactolipids .
this discrepancy can not be explained by a difference of the hydrophobicity within the mutants.
considering the first seven amino acids, the wild type protein shows a mean hydrophobicity of  <dig>  according to the eisenberg scale , and the f → y and f → g mutants a hydrophobicity of  <dig>  and  <dig> , respectively.
binding of cyanelle pfnr to omp <dig> from anabaena or toc <dig> from pea
our results for the lipid binding do no parallel the previous observations while importing the respective constructs into pea chloroplasts or cyanelles  <cit> .
we therefore determined the association of pfnr with two omp85-like proteins from pea  and anabaena sp.
pcc <dig>  because the latter protein encoded by alr <dig> is thought to be related to the ancestral translocon  <cit> .
pfnr binds to both proteins  with comparable efficiency to the precursor of the small subunit of ribulose- <dig> -bisphosphate carboxylase/oxygenase  from pea.
however, no interaction to the matrix was obtained in the absence of a coating protein  or using the mutant lacking the amino acids alanine, phenylalanine and valine .
hence, the precursor protein is recognized by the two omp <dig> family proteins, and the n-terminal portion of the transit peptide is responsible for this interaction.
the results are also in line with the protein translocation into pea chloroplasts, where the deletion of the three amino acids causes a significant reduction.
to further explore this interaction we studied it in a membrane environment.
the omp85-like protein from anabaena sp.
pcc <dig> was reconstituted into liposomes composed of phosphatidylcholine.
the binding of the wild type precursor to liposomes is significantly enhanced in the presence of the omp85-like protein .
the apparent dissociation constant determined was about  <dig> fold reduced .
the exchange of phenylalanine to tyrosine somewhat reduced the affinity for the proteoliposomes  resulting in a  <dig> fold reduction of the apparent dissociation constant , which is half of that found for wild type pfnr.
this observation parallels the decrease in in vitro import efficiency into cyanelles of this mutant when compared to wild type.
the mutant carrying a glycine did not show an enhanced binding to proteoliposomes .
interestingly, this precursor was also not imported into isolated cyanelles.
in line, the binding to liposomes of the two deletion mutants, which are incompetent for import into cyanelles  <cit> , was insignificantly enhanced when using proteoliposomes , because the increase was in the error range of the experiment.
therefore, binding of the two deletion mutants and the mutant with a phenylalanine exchange to glycine did not show a significant dependence on the presence of the omp <dig> homologue in the liposome which parallels the loss of import competence  <cit> .
we can therefore postulate that the dependence of protein translocation into cyanelles on the presence of n-terminal phenylalanine is caused by omp <dig> protein, most likely via its receptor domain.
discussion
previous reports implicated a monogalactosyldiacylglycerol dependent interaction of the transit peptide of tobacco pssu with liposomes  <cit>  and a preference of the transit peptide of ferredoxin from silene pratensis for  <dig> -dioleoyl-sn-glycero-3-phosphoglycerol and sulfoquinovosyl-diacylglycerol  <cit> .
these findings are in contrast to our observations for the ferredoxin:nadp+-oxidoreductase  from cyanophora, which binds strongest to phophatidylcholine .
the exchange of phenylalanine to glycine within the cyanelle precursor causes the most significant alteration of lipid binding, especially with respect to the affinity and lipid preference.
for this mutant we obtained almost no binding to lipid mixtures with phosphatidylglycerol, but an enhanced interaction with liposomes containing galactosyldiacylglycerol .
hence, our results suggest that exchange of the phenylalanine by a non aromatic amino acid renders the physiochemical properties of the transit peptide from cyanophora closer to those found for transit peptides of organisms with chloroplasts sensu stricto  <cit> .
however, our analysis revealed a picture of lipid binding significantly different to that observed during import of precursor proteins into cyanelles  <cit> .
hence, we have to conclude that lipid association alone does not account for the observed phenylalanine dependence of protein translocation into cyanelles.
in turn, the efficiency of precursor binding to proteoliposomes clearly parallels the import efficiencies of the mutant precursors.
interestingly, pstoc <dig>  a member of the omp <dig> family, discriminates between c. paradoxa pfnr with and without phenylalanine.
however, the binding of pssu from pea also documents that this does not mean a dependence on phenylalanine per se  but rather on so far not understood physicochemical features of the transit peptide.
nevertheless, this results points toward the presence of an omp <dig> like protein in cyanelles functioning as receptor recognizing the phenylalanine present in the transit sequence.
in line, omp <dig> proteins are found to be involved in protein translocation across membranes or integration of outer membrane proteins of bacteria and endosymbiont-derived organelles .
furthermore, in the diatom phaeodactylum tricornutum, where translocation across the third outermost membrane is phenylalanine-dependent as well  <cit> , a member of the omp <dig> family can be identified by sequence similarity.
this omp85-like protein possesses a bipartite plastid targeting signal , displays a potra domain  and ends with the cyanobacterial c-terminal consensus .
thereby, the c-terminus differs from that found in toc <dig> proteins of green algae and higher plants , but is similar to that found in an omp <dig> homologue in the red alga cyanidioschyzon merolae .
the two proteins from p. tricornutum and c. merolae do not display the n-terminal polyglycine stretch typical for the toc <dig> proteins  <cit> , but contain an lrggg motif in the n-terminal region expected to be soluble.
additionally, the two identified sequences contain motifs  <dig> and  <dig> identified in the omp <dig> family .
hence, these observations together with preliminary data from heterologous western blots  support the existence of an omp <dig> like protein in the outer membrane of cyanelles, which serves as receptor for the incoming precursor.
CONCLUSIONS
summarizing, the following model for toc translocon evolution can be assumed from the presented data.
primordial transit sequences evolved with a phenylalanine for two reasons; at first this amino acid attenuates the interaction with the cyanelle surface containing mono- and digalactosyldiacylglycerol.
interestingly, previous studies determined that diacylglycerol was needed to interfere with spontaneous insertion of proteins into liposomes using an e. coli in vitro system  <cit> .
in the absence of diacylglycerol spontaneous integration into phospholipids was observed even for multi-spanning membrane proteins.
it was concluded that diacylglycerol seals the cytoplasmic membrane of e. coli against spontaneous insertion of hydrophobic proteins.
hence, the low affinity of pfnr for galactosyldiacylglycerol containing membranes might be essential to warrant the recognition by proteins present in the outer membrane.
one such protein is omp <dig> involved in outer membrane protein assembly in bacteria and predestined to interact with sequences composed like transit peptides  <cit> .
in a primitive translocon, this receptor pore likely is the only candidate to interact with precursor proteins whereas in the chloroplast system a whole set of receptors is available for binding and able to overrule precursor-lipid affinities.
secondly, the phenylalanine reflects a match to properties of bacterial omp <dig> proteins, which are thought to recognize omps via their c-termini enriched in aromatic amino acids .
in the course of evolution and paralleled by development of a more sophisticated translocation apparatus including cytosolic guidance complexes and regulatory receptor components like toc <dig> and toc <dig> , the importance of the n-terminal phenylalanine might have been gradually lost in the "green line", which allowed a more flexible and versatile  regulation of the import process.
future research, especially on the translocon of cyanelles, will have to corroborate this model.

 the plastids of glaucophytes, rhodophytes, green algae and higher plants are surrounded by an envelope consisting of two membranes. these "primary" plastids are thought to have originated from a single primary endosymbiotic event. cyanelles are the peptidoglycan-armored plastids of glaucocystophyte algae which represent the first diverging phototrophic eukaryotes, on the earliest branch after initial endosymbiosis. cyanelles can be envisaged as the closest cousins to free-living cyanobacteria among plastids. the unique murein layer serving as "organelle wall" is modified through amidation of the free c- carboxylic group of the d-isoglutamyl moiety with n-acetylputrescine, which is unusual in the eubacterial kingdom and reduces the negative net charge of the murein layer that might interfere with protein import. cyanelle protein import machineries in plastids should be considered as prototypes of translocation systems that later underwent substantial modifications. these primitive translocons strictly require phenylalanine in the n-terminal domain of the transit peptide, even when they fulfil their function in a secondary plastid derived from a red alga. a key event for transition of the "primordial plastids" to the chloroplasts of green algae and higher plants is the gain of receptors in the toc complex conferring less stringent and overlapping specificities finally leading to dispensability of the once crucial phenylalanine. recent reports confirmed the dependence of protein translocation across the outer membrane of cyanelles or the third outermost membrane of plastids derived from secondary endosymbiosis through a red alga, on the presence of a phenylalanine within the transit sequence. in the absence of import experiments with red algae, the similarity of the n-terminal phenylalanine motif of porphyra yezoensis plastid precursor proteins to their counterparts from c. paradoxa is striking. however, the translocation of precursor proteins across the outer membrane of higher plant and green algal chloroplasts does not show such dependence. thereby, the phenylalanine represents a key for the understanding of the development of translocation machinery and their receptors. yet, one question has not been explored satisfactorily, namely, what the phenylalanine is needed for. current models favour an interpretation where this amino acid interacts with the ancestral translocon built up by endosymbiont-derived toc. however, other models on the evolutionary development and mechanism of translocation focused on an initial specific interaction between the precursor protein and the organellar membrane. we reconsidered both possibilities, since phenylalanine is an aromatic amino acid known to exhibit pronounced hydrophobic properties, and it is known to insert more deeply into the membrane than other less hydrophobic amino acids like alanine. furthermore, phenylalanine is one important component in "aromatic belts" demarcating the hydrophobic surface immersed in the lipid bilayer. it is noteworthy that all bacterial outer membrane protein structures analyzed so far show an interaction between their ultimate amino- and carboxyterminal domains. in some cases this contact is closing the β-barrel structure completely or in addition forming a hydrophobic interaction between a phenylalanine in the n-terminal domain and the c-terminal phenylalanine. these observations would favour a role of phenylalanine in membrane association of the cytosolic precursor as first contact with the cyanelle. the importance of aromatic amino acids in protein-protein interactions or substrate recognition is manifold documented. for example, aromatic residues are found in the substrate-binding site of the aaa+ chaperone clpb that are located at the central pore of the first aaa domain. these aromatic residues may act as a molecular clamp by binding and releasing substrates in a nucleotide-dependent manner. translocation of exposed segments enriched in aromatic residues of a protein aggregate by clpb would lead to the continuous extraction of unfolded polypeptides from an aggregate. therefore hydrophobic interactions between aromatic residues are a common principle of protein-protein cooperation. furthermore, phenylalanine is reported to occupy prominent positions in the sequences of proteins targeted to the bacterial outer membrane, such as porins and type iv pilins. recently it has been shown that the penultimate residue of bacterial outer membrane proteins is involved in the species specificity of omp recognition. interestingly, cyanobacterial outer membrane proteins show the c-terminal consensus sequence fxf. thereby, it is tempting to speculate that a phenylalanine residue is important for the interaction of precursor proteins with the toc translocon of primitive plastids. in here we describe the influence of the phenylalanine on the interaction with lipids and lipid surfaces represented by liposomes and proteoliposomes. the obtained specificity for proteoliposomes is subsequently discussed in the context of the two "initial receptor models" and the evolutionary development of the chloroplast translocon.  the phenylalanine defines lipid specificities of pfnr we first analyzed the association of the precursor of the ferredoxin:nadp+-oxidoreductase from cyanophora to lipids found in the membrane of chloroplasts isolated from higher plants. even though the lipid content of the outer membrane of cyanelles is not yet known, it is envisioned that loss of the lipopolysaccharides paralleled the endosymbiotic process and that they were replaced by phosphatidylcholine and galactolipids. hence, the composition of the outer membrane of e.g. pea chloroplasts serves as a model for the lipid composition of the outer membrane of cyanelles. we used the well established lipid dot blot analysis to determine the interaction between precursor and specific lipids. the precursor protein interacts with phosphatidylcholine. interestingly, pfnr did not show an interaction with other neutral lipids, but with the two charged lipids sulfoquinovosyldiacylglycerol and phosphatidylglycerol, though to a reduced extent when compared to the interaction with pc. we further controlled whether the lipids are capable of rejecting pfnr by mixing or mol% of the lipids to pc. however, at the lower concentration of the added lipid the association of pfnr is comparable to its binding to pc. to explore the influence of the phenylalanine on this interaction, we used the two types of mutants previously generated. in a first set, the phenylalanine was replaced either by a tyrosine or by glycine. in a second set, the phenylalanine itself or with the two neighbouring amino acids were removed. when binding of the mutants to lipids was analyzed we did obtain the highest affinity for pc as seen before for pfnr. however, some changes in lipid binding can be found. the most conservative mutant f→y parallels the feature of wild type in terms of not interacting with mono – or digalactosyldiaclglycerol.. it still binds to sqdg, but not to pg, suggesting that its interaction to sqdg is not driven by electrostatic interactions. in contrast, the exchange of the phenylalanine by glycine introduces marked alterations as this mutant shows no binding to pg but to all galactolipids. the two deletion mutants reveal a reduced but still recognizable binding to pc and sqdg, but not to the other lipids. hence, we obtained a chain of alterations of lipid specificities or binding capacities, which somewhat, but not fully parallel the import behaviour observed. the aromatic properties of the phenylalanine define the lipid specificity to further support our findings we have focused on the comparison between wild type and the two mutants with amino acid exchange. these two mutants have differential import rates and an altered lipid binding behaviour. we analyzed the binding of the three proteins to pc, dgdg and pg as pg was found to be specifically recognized by the wild type precursor and dgdg specifically by the mutant with phenylalanine to glycine exchange. we were indeed able to confirm the highest binding capacity for all proteins to pc and the enhanced capacity for this association by the f→g mutant. based on these experiments we were able to determine an apparent dissociation constant. to directly explore differential features the ratio between the apparent dissociation constants of the mutant and wild type for each lipid, was calculated. by this it becomes obvious that the conservative exchange from phenylalanine to tyrosine somewhat reduces the association with phospholipids, but does not effect the affinity for galactolipids. in turn, the exchange of phenylalanine to glycine enhances the association to galactolipids or pc considerably, whereas the association to pg is somewhat reduced. however, this reduction is not a result of the charge as pfnr f→g binds do sqdg better than wild type. therefore, the exchange of phenylalanine to another aromatic amino acid only slightly affects binding by reducing the affinity to phospholipids but not altering the affinity for galactolipids, whereas removal of the aromatic amino acid at this position significantly enhances affinity for galactolipids without significant alterations of the affinity for phospholipids. binding of pfnr to liposomes so far we have determined the association of the proteins to immobilized lipids. to further support our findings and to establish a system to reconstitute the toc like proteins, we have analyzed the association of the precursor proteins to liposomes. we were able to reproduce the findings with immobilized lipids in terms of the enhanced affinity of pfnr f→g for the liposome surface composed of pc or pc and dgdg, the loss of interaction with pg or dgdg containing surfaces by the f→y replacement and the reduced affinity of the mutant with deleted phenylalanine. however, we also obtained a difference when compared to the fat western analysis, as pfnr wt binds significantly better to liposomes containing mol% pg than to liposomes composed of pc only. we subsequently analyzed the binding in more detail by incubation of radioactive precursor proteins with different concentrations of liposomes. the specificity of the interaction was controlled by analyzing the precipitation of the precursor in the absence of liposomes. quantification of these experimental data revealed a four to five fold lower apparent dissociation constant for the binding of pfnr to pc/pg liposomes than to pc or pc/dgdg liposomes. for pfnr f→g we again obtained the highest efficiency for pc liposomes revealing an apparent dissociation constant, which is two times lower than the binding of the mutant to pc/dgdg liposomes or the wild type protein to pc. the mutant shows the lowest binding capacity for pc/pg liposomes. here, the apparent dissociation constant is times higher then for pc liposomes. summarizing, we document that the alteration of the phenylalanine modifies the lipid binding behavior of the precursor protein to lipids immobilized to a surface or present in liposomes. in both cases, deletion of the phenylalanine results in a reduction of the association. the exchange of the phenylalanine to tyrosine somewhat changes the specificity for the lipids, but does not significantly change the affinity to phosphatidylcholine. in contrast, exchange of phenylalanine to glycine on one hand enhances the association to phosphatidylcholine and changes the specificity of the protein from preferred interaction with phosphatidylglycerol to preferred interaction with galactolipids. this discrepancy can not be explained by a difference of the hydrophobicity within the mutants. considering the first seven amino acids, the wild type protein shows a mean hydrophobicity of according to the eisenberg scale, and the f → y and f → g mutants a hydrophobicity of and, respectively. binding of cyanelle pfnr to omp from anabaena or toc from pea our results for the lipid binding do no parallel the previous observations while importing the respective constructs into pea chloroplasts or cyanelles. we therefore determined the association of pfnr with two omp85-like proteins from pea and anabaena sp. pcc because the latter protein encoded by alr is thought to be related to the ancestral translocon. pfnr binds to both proteins with comparable efficiency to the precursor of the small subunit of ribulose- -bisphosphate carboxylase/oxygenase from pea. however, no interaction to the matrix was obtained in the absence of a coating protein or using the mutant lacking the amino acids alanine, phenylalanine and valine. hence, the precursor protein is recognized by the two omp family proteins, and the n-terminal portion of the transit peptide is responsible for this interaction. the results are also in line with the protein translocation into pea chloroplasts, where the deletion of the three amino acids causes a significant reduction. to further explore this interaction we studied it in a membrane environment. the omp85-like protein from anabaena sp. pcc was reconstituted into liposomes composed of phosphatidylcholine. the binding of the wild type precursor to liposomes is significantly enhanced in the presence of the omp85-like protein. the apparent dissociation constant determined was about fold reduced. the exchange of phenylalanine to tyrosine somewhat reduced the affinity for the proteoliposomes resulting in a fold reduction of the apparent dissociation constant, which is half of that found for wild type pfnr. this observation parallels the decrease in in vitro import efficiency into cyanelles of this mutant when compared to wild type. the mutant carrying a glycine did not show an enhanced binding to proteoliposomes. interestingly, this precursor was also not imported into isolated cyanelles. in line, the binding to liposomes of the two deletion mutants, which are incompetent for import into cyanelles, was insignificantly enhanced when using proteoliposomes, because the increase was in the error range of the experiment. therefore, binding of the two deletion mutants and the mutant with a phenylalanine exchange to glycine did not show a significant dependence on the presence of the omp homologue in the liposome which parallels the loss of import competence. we can therefore postulate that the dependence of protein translocation into cyanelles on the presence of n-terminal phenylalanine is caused by omp protein, most likely via its receptor domain. discussion previous reports implicated a monogalactosyldiacylglycerol dependent interaction of the transit peptide of tobacco pssu with liposomes and a preference of the transit peptide of ferredoxin from silene pratensis for -dioleoyl-sn-glycero-3-phosphoglycerol and sulfoquinovosyl-diacylglycerol. these findings are in contrast to our observations for the ferredoxin:nadp+-oxidoreductase from cyanophora, which binds strongest to phophatidylcholine. the exchange of phenylalanine to glycine within the cyanelle precursor causes the most significant alteration of lipid binding, especially with respect to the affinity and lipid preference. for this mutant we obtained almost no binding to lipid mixtures with phosphatidylglycerol, but an enhanced interaction with liposomes containing galactosyldiacylglycerol. hence, our results suggest that exchange of the phenylalanine by a non aromatic amino acid renders the physiochemical properties of the transit peptide from cyanophora closer to those found for transit peptides of organisms with chloroplasts sensu stricto. however, our analysis revealed a picture of lipid binding significantly different to that observed during import of precursor proteins into cyanelles. hence, we have to conclude that lipid association alone does not account for the observed phenylalanine dependence of protein translocation into cyanelles. in turn, the efficiency of precursor binding to proteoliposomes clearly parallels the import efficiencies of the mutant precursors. interestingly, pstoc a member of the omp family, discriminates between c. paradoxa pfnr with and without phenylalanine. however, the binding of pssu from pea also documents that this does not mean a dependence on phenylalanine per se but rather on so far not understood physicochemical features of the transit peptide. nevertheless, this results points toward the presence of an omp like protein in cyanelles functioning as receptor recognizing the phenylalanine present in the transit sequence. in line, omp proteins are found to be involved in protein translocation across membranes or integration of outer membrane proteins of bacteria and endosymbiont-derived organelles. furthermore, in the diatom phaeodactylum tricornutum, where translocation across the third outermost membrane is phenylalanine-dependent as well, a member of the omp family can be identified by sequence similarity. this omp85-like protein possesses a bipartite plastid targeting signal, displays a potra domain and ends with the cyanobacterial c-terminal consensus. thereby, the c-terminus differs from that found in toc proteins of green algae and higher plants, but is similar to that found in an omp homologue in the red alga cyanidioschyzon merolae. the two proteins from p. tricornutum and c. merolae do not display the n-terminal polyglycine stretch typical for the toc proteins, but contain an lrggg motif in the n-terminal region expected to be soluble. additionally, the two identified sequences contain motifs and identified in the omp family. hence, these observations together with preliminary data from heterologous western blots support the existence of an omp like protein in the outer membrane of cyanelles, which serves as receptor for the incoming precursor.  summarizing, the following model for toc translocon evolution can be assumed from the presented data. primordial transit sequences evolved with a phenylalanine for two reasons; at first this amino acid attenuates the interaction with the cyanelle surface containing mono- and digalactosyldiacylglycerol. interestingly, previous studies determined that diacylglycerol was needed to interfere with spontaneous insertion of proteins into liposomes using an e. coli in vitro system. in the absence of diacylglycerol spontaneous integration into phospholipids was observed even for multi-spanning membrane proteins. it was concluded that diacylglycerol seals the cytoplasmic membrane of e. coli against spontaneous insertion of hydrophobic proteins. hence, the low affinity of pfnr for galactosyldiacylglycerol containing membranes might be essential to warrant the recognition by proteins present in the outer membrane. one such protein is omp involved in outer membrane protein assembly in bacteria and predestined to interact with sequences composed like transit peptides. in a primitive translocon, this receptor pore likely is the only candidate to interact with precursor proteins whereas in the chloroplast system a whole set of receptors is available for binding and able to overrule precursor-lipid affinities. secondly, the phenylalanine reflects a match to properties of bacterial omp proteins, which are thought to recognize omps via their c-termini enriched in aromatic amino acids. in the course of evolution and paralleled by development of a more sophisticated translocation apparatus including cytosolic guidance complexes and regulatory receptor components like toc and toc, the importance of the n-terminal phenylalanine might have been gradually lost in the "green line", which allowed a more flexible and versatile regulation of the import process. future research, especially on the translocon of cyanelles, will have to corroborate this model. 
here, bacterial outer membrane proteins of the omp family have recently been discussed as evolutionary seeds for the development of translocation systems. the phenylalanine in the n-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. however, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by omp this finding is placed into the context of the evolutionary development of the plastid translocon. the phenylalanine has subsequently been lost in the transit sequence, but can be found at the c-terminal position of the translocating pore. to further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. this process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. at present, most information on protein translocation is obtained by the analysis of land plants.
 today it is widely accepted that plastids are of cyanobacterial origin. during their evolutionary integration into the metabolic and regulatory networks of the host cell the engulfed cyanobacteria lost their independency. this process was paralleled by a massive gene transfer from symbiont to the host nucleus challenging the development of a retrograde protein translocation system to ensure plastid functionality. such a system includes specific targeting signals of the proteins needed for the function of the plastid and membrane-bound machineries performing the transfer of these proteins across the envelope membranes. at present, most information on protein translocation is obtained by the analysis of land plants. however, the analysis of protein import into the primitive plastids of glaucocystophyte algae, revealed distinct features placing this system as a tool to understand the evolutionary development of translocation systems. here, bacterial outer membrane proteins of the omp family have recently been discussed as evolutionary seeds for the development of translocation systems.  to further explore the initial mode of protein translocation, the observed phenylalanine dependence for protein translocation into glaucophyte plastids was pursued in detail. we document that indeed the phenylalanine has an impact on both, lipid binding and binding to proteoliposomes hosting an omp homologue. comparison to established import experiments, however, unveiled a major importance of the phenylalanine for recognition by omp this finding is placed into the context of the evolutionary development of the plastid translocon.  the phenylalanine in the n-terminal domain signs as a prerequisite for protein translocation across the outer membrane assisted by a "primitive" translocon. this amino acid appears to be optimized for specifically targeting the omp protein without enforcing aggregation on the membrane surface. the phenylalanine has subsequently been lost in the transit sequence, but can be found at the c-terminal position of the translocating pore. thereby, the current hypothesis of omp being the prokaryotic contribution to the ancestral toc translocon can be supported. 