Comparative genomic studies suggest that the cyanobacterial endosymbionts of the amoeba Paulinella chromatophora possess an import apparatus for nuclear‐encoded proteins

Plastids evolved from free‐living cyanobacteria through a process of primary endosymbiosis. The most widely accepted hypothesis derives three ancient lineages of primary plastids, i.e. those of glaucophytes, red algae and green plants, from a single cyanobacterial endosymbiosis. This hypothesis was originally predicated on the assumption that transformations of endosymbionts into organelles must be exceptionally rare because of the difficulty in establishing efficient protein trafficking between a host cell and incipient organelle. It turns out, however, that highly integrated endosymbiotic associations are more common than once thought. Among them is the amoeba Paulinella chromatophora, which harbours independently acquired cyanobacterial endosymbionts functioning as plastids. Sequencing of the Paulinella endosymbiont genome revealed an absence of essential genes for protein trafficking, suggesting their residence in the host nucleus and import of protein products back into the endosymbiont. To investigate this hypothesis, we searched the Paulinella endosymbiont genome for homologues of higher plant translocon proteins that form the import apparatus in two‐membrane envelopes of primary plastids. We found homologues of Toc12, Tic21 and Tic32, but genes for other key translocon proteins (e.g. Omp85/Toc75 and Tic20) were missing. We propose that these missing genes were transferred to the Paulinella nucleus and their products are imported and integrated into the endosymbiont envelope membranes, thereby creating an effective protein import apparatus. We further suggest that other bacterial/cyanobacterial endosymbionts found in protists, plants and animals could have evolved efficient protein import systems independently and, therefore, reached the status of true cellular organelles.     

Uniqueness of the mechanism of protein import into the peroxisome matrix: transport of folded, co-factor-bound and oligomeric proteins by shuttling receptors

Based on earlier suggestions that peroxisomes may have arisen from endosymbionts that later lost their DNA, it was expected that protein transport into this organelle would have parallels to systems found in other organelles of endosymbiont origin, such as mitochondria and chloroplasts. This review highlights three features of peroxisomal matrix protein import that make it unique in comparison with these other subcellular compartments - the ability of this organelle to transport folded, co-factor-bound and oligomeric proteins, the dynamics of the import receptors during the matrix protein import cycle and the existence of a peroxisomal quality-control pathway, which insures that the peroxisome membrane is cleared of cargo-free receptors.     

Origin of mitochondria by intracellular enslavement of a photosynthetic purple bacterium

Mitochondria originated by permanent enslavement of purple non-sulphur bacteria. These endosymbionts became organelles through the origin of complex protein-import machinery and insertion into their inner membranes of protein carriers for extracting energy for the host. A chicken-and-egg problem exists: selective advantages for evolving import machinery were absent until inner membrane carriers were present, but this very machinery is now required for carrier insertion. I argue here that this problem was probably circumvented by conversion of the symbiont protein-export machinery into protein-import machinery, in three phases. I suggest that the first carrier entered the periplasmic space via pre-existing beta-barrel proteins in the bacterial outer membrane that later became Tom40, and inserted into the inner membrane probably helped by a pre-existing inner membrane protein, thereby immediately providing the protoeukaryote host with photosynthesate. This would have created a powerful selective advantage for evolving more efficient carrier import by inserting Tom70 receptors. Massive gene transfer to the nucleus inevitably occurred by mutation pressure. Finally, pressure from harmful, non-selected gene transfer to the nucleus probably caused evolution of the presequence mechanism, and photosynthesis was lost.     

Endosymbiosis: bacteria sharing the load

A nested set of bacterial endosymbionts within mealybug cells collectively provides amino acids to their host, but their genomes show that some pathways are distributed between both endosymbionts, while other essential proteins are missing altogether. The possibility that additional functions are shared between partners warrants comparisons with organelles.     

A HYPOTHESIS FOR IMPORT OF THE NUCLEAR‐ENCODED PsaE PROTEIN OF PAULINELLA CHROMATOPHORA (CERCOZOA,RHIZARIA) INTO ITS CYANOBACTERIAL ENDOSYMBIONTS/PLASTIDS VIA THE ENDOMEMBRANE SYSTEM1

The cyanobacterial endosymbionts of Paulinella chromatophora can shed new light on the process of plastid acquisition. Their genome is devoid of many essential genes, suggesting gene transfer to the host nucleus and protein import back into the endosymbionts/plastids. Strong evidence for such gene transfer is provided by the psaE gene, which encodes a PSI component that was efficiently transferred to the Paulinella nucleus. It remains unclear, however, how this protein is imported into the endosymbionts/plastids. We reanalyzed the sequence of Paulinella psaE and identified four potential non‐AUG translation initiation codons upstream of the previously proposed start codon. Interestingly, the longest polypeptide, starting from the first UUG, contains a clearly identifiable signal peptide with very high (90%) predictability. We also found several downstream hairpin structures that could enhance translation initiation from the alternative codon. These results strongly suggest that the PsaE protein is targeted to the outer membrane of Paulinella endosymbionts/plastids via the endomembrane system. On the basis of presence of respective bacterial homologs in the Paulinella endosymbiont/plastid genome, we discuss further trafficking of PsaE through the peptidoglycan wall and the inner envelope membrane. It is possible that other nuclear‐encoded proteins of P. chromatophora also carry signal peptides, but, alternatively, some may be equipped with transit peptides. If this is true, Paulinella endosymbionts/plastids would possess two distinct targeting systems, one cotranslational and the second posttranslational, as has been found in higher plant plastids. Considering the endomembrane system‐mediated import pathway, we also discuss homology of the membranes surrounding Paulinella endosymbionts/plastids.     

Protein trafficking to plastids: one theme, many variations

Plastids are a diverse group of essential organelles in plants that include chloroplasts. The biogenesis and maintenance of these organelles relies on the import of thousands of nucleus-encoded proteins. The complexity of plastid structure has resulted in the evolution of at least four general import pathways that target proteins into and across the double membrane of the plastid envelope. Several of these pathways can be further divided into specialty pathways that mediate and regulate the import of specific classes of proteins. The co-ordination of import by these specialized pathways with changes in gene expression is critical for plastid and plant development. Moreover, protein import is acutely regulated in response to physiological and metabolic changes within the cell. In the present review we summarize the current knowledge of the mechanism of import via these pathways and highlight the regulatory mechanisms that integrate the plastid protein-trafficking pathways with the developmental and metabolic state of the plant.     

N-terminal domain of the dual-targeted pea glutathione reductase signal peptide controls organellar targeting efficiency

Import of nuclear-encoded proteins into mitochondria and chloroplasts is generally organelle specific and its specificity depends on the N-terminal signal peptide. Yet, a group of proteins known as dual-targeted proteins have a targeting peptide capable of leading the mature protein to both organelles. We have investigated the domain structure of the dual-targeted pea glutathione reductase (GR) signal peptide by using N-terminal truncations. A mutant of the GR precursor (pGR) starting with the second methionine residue of the targeting peptide, pGRdelta2-4, directed import into both organelles, negating the possibility that dual import was controlled by the nature of the N terminus. The deletion of the 30 N-terminal residues (pGRdelta2-30) inhibited import efficiency into chloroplasts substantially and almost completely into mitochondria, whereas the removal of only 16 N-terminal amino acid residues (pGRdelta2-16) resulted in the strongly stimulated mitochondrial import without significantly affecting chloroplast import. Furthermore, N-terminal truncations of the signal peptide (pGRdelta2-16 and pGRdelta2-30) greatly stimulated the mitochondrial processing activity measured with the isolated processing peptidase. These results suggest a domain structure for the dual-targeting peptide of pGR and the existence of domains controlling organellar import efficiency therein.     

The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles

Many non-photosynthetic species of protists and metazoans are capable of hosting viable algal endosymbionts or their organelles through adaptations of phagocytic pathways. A form of mixotrophy combining phototrophy and heterotrophy, acquired phototrophy (AcPh) encompasses a suite of endosymbiotic and organelle retention interactions, that range from facultative to obligate. AcPh is a common phenomenon in aquatic ecosystems, with endosymbiotic associations generally more prevalent in nutrient poor environments, and organelle retention typically associated with more productive ones. All AcPhs benefit from enhanced growth due to access to photosynthetic products; however, the degree of metabolic integration and dependency in the host varies widely. AcPh is found in at least four of the major eukaryotic supergroups, and is the driving force in the evolution of secondary and tertiary plastid acquisitions. Mutualistic resource partitioning characterizes most algal endosymbiotic interactions, while organelle retention is a form of predation, characterized by nutrient flow (i.e., growth) in one direction. AcPh involves adaptations to recognize specific prey or endosymbionts and to house organelles or endosymbionts within the endomembrane system but free from digestion. In many cases, hosts depend upon AcPh for the production of essential nutrients, many of which remain obscure. The practice of AcPh has led to multiple independent secondary and tertiary plastid acquisition events among several eukaryote lineages, giving rise to the diverse array of algae found in modern aquatic ecosystems. This article highlights those AcPhs that are model research organisms for both metazoans and protists. Much of the basic biology of AcPhs remains enigmatic, particularly (1) which essential nutrients or factors make certain forms of AcPh obligatory, (2) how hosts regulate and manipulate endosymbionts or sequestered organelles, and (3) what genomic imprint, if any, AcPh leaves on non-photosynthetic host species.     

Peroxisomal matrix protein import: the transient pore model

Peroxisomes import folded, even oligomeric, proteins, which distinguishes the peroxisomal translocation machinery from the well-characterized translocons of other organelles. How proteins are transported across the peroxisomal membrane is unclear. Here, we propose a mechanistic model that conceptually divides the import process into three consecutive steps: the formation of a translocation pore by the import receptor, the ubiquitylation of the import receptors, and pore disassembly/ receptor recycling.     

Peroxisomes: the extended shuttle to the peroxisome matrix

A recent study indicates that protein import into the peroxisomal matrix occurs by a possibly unique mechanism involving the shuttling of cargo receptors into and out of the organelles.     

Elevated levels of stress proteins associated with bacterial symbiosis in Amoeba proteus and soybean root nodule cells

Obligatory bacterial endosymbionts of Amoeba proteus and symbiotic Bradyrhizobium japonicum bacteroids in soybean-root nodules contained large amounts of 67-kDa and 65-kDa proteins, respectively, antigenically related to groEL of E. coli and the 58-kDa heat-shock protein of Tetrahymena. Monoclonal antibodies against the 67-kDa protein recognized groEL analogs from several different organisms. The quantity of the stress protein in symbiotic B. japonicum bacteroids was augmented seven times that in the free-living counterparts. The increase in these proteins in endosymbionts, as determined by immunoblot techniques, indicated that intracellular symbiosis is a stress condition even when the symbiotic relationship is considered to be mutually beneficial. Mitochondria and chloroplasts may also be under a stressed condition like endosymbionts in view of the presence of heat-shock proteins in these cell organelles.     

Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import.

Trichomonads are early-diverging eukaryotes that lack both mitochondria and peroxisomes. They do contain a double membrane-bound organelle, called the hydrogenosome, that metabolizes pyruvate and produces ATP. To address the origin and biological nature of hydrogenosomes, we have established an in vitro protein import assay. Using purified hydrogenosomes and radiolabeled hydrogenosomal precursor ferredoxin (pFd), we demonstrate that protein import requires intact organelles, ATP and N-ethylmaleimide-sensitive cytosolic factors. Protein import is also affected by high concentrations of the protonophore, m-chlorophenylhydrazone (CCCP). Binding and translocation of pFd into hydrogenosomes requires the presence of an eight amino acid N-terminal presequence that is similar to presequences found on all examined hydrogenosomal proteins. Upon import, pFd is processed to a size consistent with cleavage of the presequence. Mutation of a conserved leucine at position 2 in the presequence to a glycine disrupts import of pFd into the organelle. Interestingly, a comparison of hydrogenosomal and mitochondrial protein presequences reveals striking similarities. These data indicate that mechanisms underlying protein targeting and biogenesis of hydrogenosomes and mitochondria are similar, consistent with the notion that these two organelles arose from a common endosymbiont.     

Directed import of macromolecules into mitochondria

Mitochondria are multifunctional organelles of eukaryotic cells that provide the energy for the cells by oxidative phosphorylation, play an important role in the apoptosis and take part in Fe-S clusters formation, fatty acids oxidation and synthesis of some aminoacids. They contain their own genome and are able to transcribe and to translate it. However, the vast majority of the macromolecules which function inside the mitochondria are imported into these organelles from the cytoplasm. The imported macromolecules include proteins and several types of small RNAs. Protein import is a universal process and its mechanism is conserved among all species. This mechanism is now known in detail. RNA import was shown to occur in several groups of eukaryotes, while the pool of imported RNA molecules varies in different organisms. Although the knowledge about the mechanisms of RNA import is less extensive than for the proteins, it becomes clear that these mechanisms are not universal among all the species possessing this pathway. In this review, we summarize the data about the import of macromolecules mentioned above into mitochondria.     

REVERSING MOTHER'S CURSE: SELECTION ON MALE MITOCHONDRIAL FITNESS EFFECTS

Many essential organelles and endosymbionts exhibit a strict matrilineal pattern of inheritance. The absence of paternal transmission of such extranuclear components is thought to preclude a response to selection on their effects on male viability and fertility. We overturn this dogma by showing that two mechanisms, inbreeding and kin selection, allow mitochondria to respond to selection on both male viability and fertility. Even modest levels of inbreeding allow such a response to selection when there are direct fitness effects of mitochondria on male fertility because inbreeding associates male fertility traits with mitochondrial matrilines. Male viability effects of mitochondria are also selectable whenever there are indirect fitness effects of males on the fitness of their sisters. When either of these effects is sufficiently strong, we show that there are conditions that allow the spread of mitochondria with direct effects that are harmful to females, contrary to standard expectation. We discuss the implications of our findings for the evolution of organelles and endosymbionts and genomic conflict.     

The enteropathogenic Escherichia coli (EPEC) Map effector is imported into the mitochondrial matrix by the TOM/Hsp70 system and alters organelle morphology

Enteropathogenic Escherichia coli (EPEC) is a human intestinal pathogen and a major cause of diarrhoea, particularly among infants in developing countries. EPEC target the Map and EspF multifunctional effector proteins to host mitochondria - organelles that play crucial roles in regulating cellular processes such as programmed cell death (apoptosis). While both molecules interfere with the organelles ability to maintain a membrane potential, EspF plays the predominant role and is responsible for triggering cell death. To learn more about the Map-mitochondria interaction, we studied Map localization to mitochondria with purified mitochondria (from mammalian and yeast cells) and within intact yeast. This revealed that (i) Map targeting is dependent on the predicted N-terminal mitochondrial targeting sequence, (ii) the N-terminal 44 residues are sufficient to target proteins to mitochondria and (iii) Map import involves the mitochondrial outer membrane translocase (Tom22 and Tom40), the mitochondrial membrane potential, and the matrix chaperone, mtHsp70. These results are consistent with Map import into the mitochondria matrix via the classical import mechanism. As all known, Map-associated phenotypes in mammalian cells are independent of mitochondrial targeting, this may indicate that import serves as a mechanism to remove Map from the cytoplasm thereby regulating cytoplasmic function. Intriguingly, Map, but not EspF, alters mitochondrial morphology with deletion analysis revealing important roles for residues 101-152. Changes in mitochondrial morphology have been linked to alterations in the ability of these organelles to regulate cellular processes providing a possible additional role for Map import into mitochondria.     

Surviving the passage: Non-canonical stromal targeting of an Arabidopsis mitogen-activated protein kinase kinase

In plants, mitogen-activated protein kinases (MAPK) have been implicated in signalling associated with many processes, including cellular differentiation, organ development, cell death and stress/hormone signalling. While MAPK cascades are known to act in the cytosol and the nucleus, sequence analysis of the Arabidopsis MAPK cascade proteins predicts the presence of import signals that would target some of them to other organelles. In vitro uptake experiments confirm the predicted import of an oxidant-responsive MAPKK, AtMKK4, into the chloroplast. Unexpectedly, the imported MKK4 protein was not processed through stromal peptidase-dependent cleavage of the N-terminal signal peptide, thus leaving the pre-protein intact. Nevertheless, the N-terminal region was shown to be essential both for the import process and for the ability of MKK4 to activate its cognate MAPK targets in vivo. MKK4 import also occurred irrespective of the activation status of the kinase. The import of this primarily cytosolic oxidant-stimulated AtMKK4 into the chloroplasts, organelles with high redox fluxes, suggests that one of the functions of MKK4 might be to help coordinate intercompartment responses to cellular redox imbalances.Key words: cell death, chloroplast, compartmentation, MAPK, MAPK kinase, MPK6, MPK3, signal transduction, stroma, transit peptide     

Organelle evolution: what's in a name?

Plastids are organelles derived from cyanobacterial endosymbionts and the evolutionary process that gave rise to them is well understood. Or is it? The complete genome sequence of a recently evolved photosynthetic body in Paulinella chromatophora is cause for reflection on the distinction between 'endosymbiont' and 'organelle', and how the boundaries between these terms can blur.     

The intracellular cyanobacteria of Paulinella chromatophora: endosymbionts or organelles?

Endosymbiotic relationships are common across the tree of life and have had profound impacts on cellular evolution and diversity. Recent molecular investigations of the amoeba Paulinella chromatophora have raised a timely and important question: should obligatory intracellular cyanobacteria in Paulinella be considered new organelles, or do plastids and mitochondria hold a unique stature in the history of endosymbiotic events? We argue that drawing a sharp distinction between these two organelles and all other endosymbionts is not supported by accumulating data, neither is it a productive framework for investigating organelle evolution.     

A novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts

Most chloroplast and mitochondrial precursor proteins are targeted specifically to either chloroplasts or mitochondria. However, there is a group of proteins that are dual targeted to both organelles. We have developed a novel in vitro system for simultaneous import of precursor proteins into mitochondria and chloroplasts (dual import system). The mitochondrial precursor of alternative oxidase, AOX was specifically targeted only to mitochondria. The chloroplastic precursor of small subunit of pea ribulose bisphosphate carboxylase/oxygenase, Rubisco, was mistargeted to pea mitochondria in a single import system, but was imported only into chloroplasts in the dual import system. The dual targeted glutathione reductase GR precursor was targeted to both mitochondria and chloroplasts in both systems. The GR pre-sequence could support import of the mature Rubisco protein into mitochondria and chloroplasts in the single import system but only into chloroplasts in the dual import system. Although the GR pre-sequence could support import of the mature portion of the mitochondrial FAd subunit of the ATP synthase into mitochondria and chloroplasts, mature AOX protein was only imported into mitochondria under the control of the GR pre-sequence in both systems. These results show that the novel dual import system is superior to the single import system as it abolishes mistargeting of chloroplast precursors into pea mitochondria observed in a single organelle import system. The results clearly show that although the GR pre-sequence has dual targeting ability, this ability is dependent on the nature of the mature protein.     

Possible import routes of proteins into the cyanobacterial endosymbionts/plastids of <Emphasis Type="Italic">Paulinella chromatophora</Emphasis>

The rhizarian amoeba Paulinella chromatophora harbors two photosynthetically active and deeply integrated cyanobacterial endosymbionts acquired ~60 million years ago. Recent genomic analyses of P. chromatophora have revealed the loss of many essential genes from the endosymbiont’s genome, and have identified more than 30 genes that have been transferred to the host cell’s nucleus through endosymbiotic gene transfer (EGT). This indicates that, similar to classical primary plastids, Paulinella endosymbionts have evolved a transport system to import their nuclear-encoded proteins. To deduce how these proteins are transported, we searched for potential targeting signals in genes for 10 EGT-derived proteins. Our analyses indicate that five proteins carry potential signal peptides, implying they are targeted via the host endomembrane system. One sequence encodes a mitochondrial-like transit peptide, which suggests an import pathway involving a channel protein residing in the outer membrane of the endosymbiont. No N-terminal targeting signals were identified in the four other genes, but their encoded proteins could utilize non-classical targeting signals contained internally or in C-terminal regions. Several amino acids more often found in the Paulinella EGT-derived proteins than in their ancestral set (proteins still encoded in the endosymbiont genome) could constitute such signals. Characteristic features of the EGT-derived proteins are low molecular weight and nearly neutral charge, which both could be adaptations to enhance passage through the peptidoglycan wall present in the intermembrane space of the endosymbiont’s envelope. Our results suggest that Paulinella endosymbionts/plastids have evolved several different import routes, as has been shown in classical primary plastids.