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1.  Ancestral and Derived Protein Import Pathways in the Mitochondrion of Reclinomonas americana 
Molecular Biology and Evolution  2010;28(5):1581-1591.
The evolution of mitochondria from ancestral bacteria required that new protein transport machinery be established. Recent controversy over the evolution of these new molecular machines hinges on the degree to which ancestral bacterial transporters contributed during the establishment of the new protein import pathway. Reclinomonas americana is a unicellular eukaryote with the most gene-rich mitochondrial genome known, and the large collection of membrane proteins encoded on the mitochondrial genome of R. americana includes a bacterial-type SecY protein transporter. Analysis of expressed sequence tags shows R. americana also has components of a mitochondrial protein translocase or “translocase in the inner mitochondrial membrane complex.” Along with several other membrane proteins encoded on the mitochondrial genome Cox11, an assembly factor for cytochrome c oxidase retains sequence features suggesting that it is assembled by the SecY complex in R. americana. Despite this, protein import studies show that the RaCox11 protein is suited for import into mitochondria and functional complementation if the gene is transferred into the nucleus of yeast. Reclinomonas americana provides direct evidence that bacterial protein transport pathways were retained, alongside the evolving mitochondrial protein import machinery, shedding new light on the process of mitochondrial evolution.
doi:10.1093/molbev/msq305
PMCID: PMC3080133  PMID: 21081480
mitochondria; protein import; Reclinomonas americana; transport pathway; translocon; SecY
4.  Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection 
PLoS Pathogens  2012;8(1):e1002459.
The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms.
Author Summary
Mitochondrial carrier proteins evolved during endosymbiosis to transport substrates across the mitochondrial inner membrane. As such the proteins are associated exclusively with eukaryotic organisms. Despite this, we identified putative mitochondrial carrier proteins in the genomes of different intracellular bacterial pathogens, including Legionella pneumophila, the causative agent of Legionnaire's disease. We named the mitochondrial carrier protein from L. pneumophila LncP and determined that the protein is translocated into host cells during infection by the bacterial Dot/Icm type IV secretion system. From there, LncP accesses the classical mitochondrial import pathway and is incorporated into the mitochondrial inner membrane as an integral membrane protein. Remarkably, LncP crosses five biological membranes to reach its final location. Biochemically, LncP is a unidirectional nucleotide transporter similar to Aac1 in yeast. Although not essential for intracellular replication, the high carriage rate of lncP among isolates of L. pneumophila suggests that the ability of the pathogen to manipulate mitochondrial ATP transport assists survival of the bacteria in an intracellular environment.
doi:10.1371/journal.ppat.1002459
PMCID: PMC3252375  PMID: 22241989
5.  The Protein Import Channel in the Outer Mitosomal Membrane of Giardia intestinalis 
Molecular Biology and Evolution  2009;26(9):1941-1947.
The identification of mitosomes in Giardia generated significant debate on the evolutionary origin of these organelles, whether they were highly reduced mitochondria or the product of a unique endosymbiotic event in an amitochondrial organism. As the protein import pathway is a defining characteristic of mitochondria, we sought to discover a TOM (translocase in the outer mitochondrial membrane) complex in Giardia. A Hidden Markov model search of the Giardia genome identified a Tom40 homologous sequence (GiTom40), where Tom40 is the protein translocation channel of the TOM complex. The GiTom40 protein is located in the membrane of mitosomes in a ∼200-kDa TOM complex. As Tom40 was derived in the development of mitochondria to serve as the protein import channel in the outer membrane, its presence in Giardia evidences the mitochondrial ancestry of mitosomes.
doi:10.1093/molbev/msp117
PMCID: PMC2734158  PMID: 19531743
mitochondria; mitosomes; Giardia intestinalis; protein translocation; evolution
6.  The Essentials of Protein Import in the Degenerate Mitochondrion of Entamoeba histolytica 
PLoS Pathogens  2010;6(3):e1000812.
Several essential biochemical processes are situated in mitochondria. The metabolic transformation of mitochondria in distinct lineages of eukaryotes created proteomes ranging from thousands of proteins to what appear to be a much simpler scenario. In the case of Entamoeba histolytica, tiny mitochondria known as mitosomes have undergone extreme reduction. Only recently a single complete metabolic pathway of sulfate activation has been identified in these organelles. The E. histolytica mitosomes do not produce ATP needed for the sulfate activation pathway and for three molecular chaperones, Cpn60, Cpn10 and mtHsp70. The already characterized ADP/ATP carrier would thus be essential to provide cytosolic ATP for these processes, but how the equilibrium of inorganic phosphate could be maintained was unknown. Finally, how the mitosomal proteins are translocated to the mitosomes had remained unclear. We used a hidden Markov model (HMM) based search of the E. histolytica genome sequence to discover candidate (i) mitosomal phosphate carrier complementing the activity of the ADP/ATP carrier and (ii) membrane-located components of the protein import machinery that includes the outer membrane translocation channel Tom40 and membrane assembly protein Sam50. Using in vitro and in vivo systems we show that E. histolytica contains a minimalist set up of the core import components in order to accommodate a handful of mitosomal proteins. The anaerobic and parasitic lifestyle of E. histolytica has produced one of the simplest known mitochondrial compartments of all eukaryotes. Comparisons with mitochondria of another amoeba, Dictystelium discoideum, emphasize just how dramatic the reduction of the protein import apparatus was after the loss of archetypal mitochondrial functions in the mitosomes of E. histolytica.
Author Summary
All eukaryotic organisms have mitochondria, organelles cordoned by a double membrane, which are descendants of an ancestral bacterial endosymbiont. Nowadays, mitochondria are fully integrated into the context of diverse cellular processes and serve in providing energy, iron-containing prosthetic groups and some of the cellular building blocks like lipids and amino acids. In multi-cellular organisms, mitochondria play an additional vital role in cell signaling pathways and programmed cell death. In some unicellular eukaryotes which inhabit oxygen poor environments, intriguing mitochondrial adaptations have taken place resulting in the creation of specialized compartments known as mitosomes and hydrogenosomes. Several important human pathogens like Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis and microsporidia contain these organelles and in many cases the function and biogenesis of these organelles remain unknown. In this paper, we investigated the protein import pathways into the mitosomes of E. histolytica, which represent one of the simplest mitochondria-related compartment discovered yet. In accordance with the limited organellar proteome, we show that only core components of mitochondria-related protein import machines are present in E. histolytica to serve for the import of a small set of substrate proteins.
doi:10.1371/journal.ppat.1000812
PMCID: PMC2841616  PMID: 20333239
7.  Protein secretion and outer membrane assembly in Alphaproteobacteria 
Fems Microbiology Reviews  2008;32(6):995-1009.
The assembly of β-barrel proteins into membranes is a fundamental process that is essential in Gram-negative bacteria, mitochondria and plastids. Our understanding of the mechanism of β-barrel assembly is progressing from studies carried out in Escherichia coli and Neisseria meningitidis. Comparative sequence analysis suggests that while many components mediating β-barrel protein assembly are conserved in all groups of bacteria with outer membranes, some components are notably absent. The Alphaproteobacteria in particular seem prone to gene loss and show the presence or absence of specific components mediating the assembly of β-barrels: some components of the pathway appear to be missing from whole groups of bacteria (e.g. Skp, YfgL and NlpB), other proteins are conserved but are missing characteristic domains (e.g. SurA). This comparative analysis is also revealing important structural signatures that are vague unless multiple members from a protein family are considered as a group (e.g. tetratricopeptide repeat (TPR) motifs in YfiO, β-propeller signatures in YfgL). Given that the process of the β-barrel assembly is conserved, analysis of outer membrane biogenesis in Alphaproteobacteria, the bacterial group that gave rise to mitochondria, also promises insight into the assembly of β-barrel proteins in eukaryotes.
doi:10.1111/j.1574-6976.2008.00130.x
PMCID: PMC2635482  PMID: 18759741
outer membrane assembly; membrane structure; Omp85; β-barrel proteins; Alphaproteobacteria; mitochondria
8.  Frataxin, a Conserved Mitochondrial Protein, in the Hydrogenosome of Trichomonas vaginalis▿  
Eukaryotic Cell  2007;6(8):1431-1438.
Recent data suggest that frataxin plays a key role in eukaryote cellular iron metabolism, particularly in mitochondrial heme and iron-sulfur (FeS) cluster biosynthesis. We have now identified a frataxin homologue (T. vaginalis frataxin) from the human parasite Trichomonas vaginalis. Instead of mitochondria, this unicellular eukaryote possesses hydrogenosomes, peculiar organelles that produce hydrogen but nevertheless share common ancestry with mitochondria. T. vaginalis frataxin contains conserved residues implicated in iron binding, and in silico, it is predicted to form a typical α-β sandwich motif. The short N-terminal extension of T. vaginalis frataxin resembles presequences that target proteins to hydrogenosomes, a prediction confirmed by the results of overexpression of T. vaginalis frataxin in T. vaginalis. When expressed in the mitochondria of a frataxin-deficient Saccharomyces cerevisiae strain, T. vaginalis frataxin partially restored defects in heme and FeS cluster biosynthesis. Although components of heme synthesis or heme-containing proteins have not been found in T. vaginalis to date, T. vaginalis frataxin was also shown to interact with S. cerevisiae ferrochelatase by using a Biacore assay. The discovery of conserved iron-metabolizing pathways in mitochondria and hydrogenosomes provides additional evidence not only of their common evolutionary history, but also of the fundamental importance of this pathway for eukaryotes.
doi:10.1128/EC.00027-07
PMCID: PMC1951141  PMID: 17573543
9.  Fe-Hydrogenase Maturases in the Hydrogenosomes of Trichomonas vaginalis†  
Eukaryotic Cell  2006;5(3):579-586.
Assembly of active Fe-hydrogenase in the chloroplasts of the green alga Chlamydomonas reinhardtii requires auxiliary maturases, the S-adenosylmethionine-dependent enzymes HydG and HydE and the GTPase HydF. Genes encoding homologous maturases had been found in the genomes of all eubacteria that contain Fe-hydrogenase genes but not yet in any other eukaryote. By means of proteomic analysis, we identified a homologue of HydG in the hydrogenosomes, mitochondrion-related organelles that produce hydrogen under anaerobiosis by the activity of Fe-hydrogenase, in the pathogenic protist Trichomonas vaginalis. Genes encoding two other components of the Hyd system, HydE and HydF, were found in the T. vaginalis genome database. Overexpression of HydG, HydE, and HydF in trichomonads showed that all three proteins are specifically targeted to the hydrogenosomes, the site of Fe-hydrogenase maturation. The results of Neighbor-Net analyses of sequence similarities are consistent with a common eubacterial ancestor of HydG, HydE, and HydF in T. vaginalis and C. reinhardtii, supporting a monophyletic origin of Fe-hydrogenase maturases in the two eukaryotes. Although Fe-hydrogenases exist in only a few eukaryotes, related Narf proteins with different cellular functions are widely distributed. Thus, we propose that the acquisition of Fe-hydrogenases, together with Hyd maturases, occurred once in eukaryotic evolution, followed by the appearance of Narf through gene duplication of the Fe-hydrogenase gene and subsequent loss of the Hyd proteins in eukaryotes in which Fe-hydrogenase function was lost.
doi:10.1128/EC.5.3.579-586.2006
PMCID: PMC1398061  PMID: 16524912

Results 1-9 (9)