Fungal Hydrogenosomes Contain Mitochondrial Heat-Shock Proteins

Mark van der Giezen*,1, Graeme M. Birdsey{dagger}, David S. Horner*,2, John Lucocq{ddagger}, Patricia L. Dyal*, Marlene Benchimol§, Christopher J. Danpure{dagger} and T. Martin Embley*,

* Department of Zoology, The Natural History Museum, London, United Kingdom
{dagger} Department of Biology, University College London, London, United Kingdom
{ddagger} School of Life Sciences, University of Dundee, Dundee, United Kingdom
§ Universidade Santa Ursula, Rio de Janeiro, Brazil

Correspondence: E-mail: tme{at}nhm.ac.uk.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
At least three groups of anaerobic eukaryotes lack mitochondria and instead contain hydrogenosomes, peculiar organelles that make energy and excrete hydrogen. Published data indicate that ciliate and trichomonad hydrogenosomes share common ancestry with mitochondria, but the evolutionary origins of fungal hydrogenosomes have been controversial. We have now isolated full-length genes for heat shock proteins 60 and 70 from the anaerobic fungus Neocallimastix patriciarum, which phylogenetic analyses reveal share common ancestry with mitochondrial orthologues. In aerobic organisms these proteins function in mitochondrial import and protein folding. Homologous antibodies demonstrated the localization of both proteins to fungal hydrogenosomes. Moreover, both sequences contain amino-terminal extensions that in heterologous targeting experiments were shown to be necessary and sufficient to locate both proteins and green fluorescent protein to the mitochondria of mammalian cells. This finding, that fungal hydrogenosomes use mitochondrial targeting signals to import two proteins of mitochondrial ancestry that play key roles in aerobic mitochondria, provides further strong evidence that the fungal organelle is also of mitochondrial ancestry. The extraordinary capacity of eukaryotes to repeatedly evolve hydrogen-producing organelles apparently reflects a general ability to modify the biochemistry of the mitochondrial compartment.

Key Words: heat-shock proteins • hydrogenosomes • mitochondria • anaerobic eukaryotes • evolution


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Anaerobic habitats are common in nature and contain large numbers of eukaryotes that lack mitochondria (Fenchel and Finlay 1995). Some of these eukaryotes contain organelles called hydrogenosomes, which make a small amount of ATP and excrete hydrogen gas (Yarlett et al. 1986; Müller 1993). Hydrogenosome-containing species do not form a single phylogenetic group, so it is apparent that hydrogenosomes have been invented repeatedly during eukaryotic evolution. A key question is whether different eukaryotes use the same fundamental organelle to host the hydrogen-generating biochemistry or whether different cell compartments can fulfill this function. For anaerobic ciliates and the human parasite Trichomonas the balance of data support the hypothesis that their hydrogenosomes share common ancestry with mitochondria (Embley, Horner, and Hirt 1997; Akhmanova et al. 1998; Dyall and Johnson 2000; Embley et al. 2003). The origins of fungal hydrogenosomes are more controversial and potentially exceptional.

Electron micrographs for fungal hydrogenosomes have been interpreted to support either a single boundary membrane like peroxisomes (Marvin-Sikkema et al. 1993b; Hackstein and Vogels 1997; Akhmanova et al. 1998; Hackstein et al. 2001) or a double membrane as seen in mitochondria (Benchimol, Durand, and Almeida 1997; van der Giezen et al. 1997b). The debate is complicated further by the lack of an associated organelle genome (van der Giezen et al. 1997b), which would provide the most incisive evidence regarding the identity of fungal hydrogenosomes. Protein import into fungal hydrogenosomes has also been reported to have features of both mitochondrial (van der Giezen et al. 1998) and peroxisomal (Marvin-Sikkema et al. 1993a; Hackstein et al. 1998) import systems. The strongest evidence for a common origin with mitochondria is the recent demonstration that fungal hydrogenosomes and yeast mitochondria use the same pathway for ADP/ATP exchange (van der Giezen et al. 2002; Voncken et al. 2002a). Thus, not only is the hydrogenosomal ADP/ATP carrier protein correctly imported into yeast mitochondria, a process which requires conservation of internal targeting signals that can be recognized by the yeast protein import machinery (Pfanner and Geissler 2001), but the protein also complements a yeast mutant deficient in ATP import and restores mitochondrial function (van der Giezen et al. 2002).

In the present study we have investigated whether the genome of Neocallimastix patriciarum contained genes coding for the mitochondrial heat shock proteins 60 and 70 (Hsp60 and Hsp70). In aerobic eukaryotes, these proteins, the genes for which are of alpha-proteobacterial ancestry consistent with their arrival with the mitochondrial endosymbiont (e.g., Boorstein, Ziegelhoffer, and Craig 1994; Viale and Arakaki 1994), carry out key functions in protein import and folding within mitochondria. Mitochondrial Hsp70 is the central component of an ATP-dependent molecular motor that drives import of pre-proteins into the mitochondrial matrix (Matouschek, Pfanner, and Voos 2000). The chaperonin Hsp60 assists in folding the imported proteins into their correct active form (Hartl 1996). Both proteins are imported into the mitochondria of aerobic eukaryotes using a characteristic amino-terminal presequence-dependent pathway, which is different from the pathway used for mitochondrial membrane proteins including the ADP/ATP carrier (Pfanner and Geissler 2001). Published data suggest that this import pathway is present in Neocallimastix frontalis L2 and is used to import hydrogenosomal malic enzyme and the ß-subunit of succinyl-CoA synthetase (Brondijk et al. 1996; van der Giezen et al. 1997a, 1998). Moreover, there are fragments of a mitochondrial-type Hsp60 gene already published for Neocallimastix frontalis L2, although the cellular localization of the protein was not investigated (Voncken et al. 2002a). Here we show that Neocallimastix patriciarum contains orthologues of mitochondrial Hsp60 and mitochondrial Hsp70, that the proteins are located within hydrogenosomes, and that both proteins contain N-terminal presequences capable of sorting either chaperone or green fluorescent protein (GFP) into mammalian mitochondria.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Fungal and Bacterial Strains and Growth Conditions
Neocallimastix patriciarum strain CX was grown anaerobically at 39°C in semidefined medium (Hobson 1969) supplemented with 20 mM cellobiose. Escherichia coli DH5{alpha} (Bethesda Reserach Laboratory) was grown at 37°C in LB medium and supplemented with ampicillin (100 µg/ml) or kanamycin (50 µg/ml). Growth medium for Escherichia coli XL1-Blue MRF' (Stratagene) was supplemented with MgSO4 (10 mM) and maltose (0.2 %) for bacteriophage {lambda}-experiments.

General DNA Techniques and PCR Amplification Experiments
Standard recombinant DNA techniques were used for nucleic acid preparation and analysis (Sambrook, Fritsch, and Maniatis 1989). Fungal DNA was isolated as described previously (Brookman et al. 2001) and published degenerate oligonucleotide polymerase chain reaction (PCR) primers (Hirt et al. 1997) were used to amplify a large fragment of the Neocallimastix Hsp70. Published Hsp60 primers (Horner et al. 1996) and specific internal primers were used in a nested PCR to amplify part of the Neocallimastix Hsp60 gene. The new primers were: G1Neo, 5'-ggw gay ggw acy acy acy gcy acy gt-3' and G2Neo, 5'-tcs ccr aas ccs ggr gcy ttr acr-3', and 1150F Neo, 5'-ggy ggy cgt tay ggt-3' and 1370R Neo, 5'-rcc dar rcc cca raa-3'. The resulting fragments were cloned into pGEM-T-Easy (Promega) and sequenced to confirm their identity.

Construction of Expression Constructs Harboring Hsp60::6(His) and Hsp70::6(His) and Generation of Homologous Antibodies
The PCR fragments were subcloned for protein expression into the NcoI-site and XhoI-site of pET-30b(+) (Novagen). The primers used to amplify Hsp60 from pGEM-T-Easy were as follows: sense, 5'-aga cca tgg agt tga act tag aaa gag gtg-3'; anti-sense: 5'-tct ctc gag tta ccc gaa ccc cgg agc ttt aac-3'; for Hsp70 the primers were sense, 5'-aga cca tgg cga ttc aag caa cta agg atg c-3' and anti-sense, 5'-tct ctc gag tta aca gtt ctt aac aga ctt ac-3' (restriction sites are indicated in italics). Affinity purification of the histidine-tagged partial chaperone fragments was performed according to the manufacturer's instructions (Novagen). Rabbit polyclonal antisera against the purified partial Hsp60 and Hsp70 were raised by Cymbus Biotechnology (UK).

Identification and Isolation of Full-Length Clones from a cDNA Library
The complete N. patriciarum Hsp60 and Hsp70 genes were isolated by screening a {lambda}ZAP II cDNA library (Xue et al. 1992), using the PCR products as homologous probes. Positive plaques were isolated and recombinant pBluescript SK(-) plasmids were excised according to the manufacturer's instructions (Stratagene) and sequenced. The cDNA sequences were deposited in DDBJ/EMBL/GenBank under the following accession numbers: N. patriciarum Hsp60 cDNA, AY033884; N. patriciarum Hsp70 cDNA, AF419853.

Phylogenetic Analysis
The conceptually translated N. patriciarum Hsp60 and Hsp70 sequences were aligned using ClustalW (Thompson, Higgins, and Gibson 1994) to reference sequences from GenBank. The alignments were manually refined, and unambiguously aligned regions were used for phylogenetic analysis, leaving two data sets of 23 taxa, one with 362 amino acid positions (Hsp60) and another with 370 amino acid positions (Hsp70). Likelihood searches were performed in a Baysian framework under the JTT-f substitution model accommodating site rate variation (fraction of invariable sites plus four variable gamma rates) using the program MrBayes (Huelsenbeck 2000). In all, 200,001 search generations were performed with trees sampled every 100 generations. The first 200 trees recovered in the likelihood search were not used in the calculation of the consensus tree, as the likelihood model had not yet stabilized. Maximum likelihood branch lengths were estimated for the Baysian consensus topologies using the program Puzzle v4 (Strimmer and von Haeseler 1996). Bootstrap analyses were performed using the custom software MrBOOT (Peter Foster, Natural History Museum [NHM], London), which automates MrBayes analyses of resampled datasets generated by the custom software P4 (Peter Foster, NHM). For bootstrap replicate analyses, 20,001 search generations were sampled every 50 generations, and "burn-ins" of 100 trees were used.

Cell Fractionation, Protein Electrophoresis, and Immunoblotting
Neocallimastix cell fractionation by mechanical disruption and differential centrifugation was performed as described by Marvin-Sikkema et al. (1993b), followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting using a semi-dry electroblotter. Blots were stained using homologous antiserum (1:10,000) against Neocallimastix Hsp60 and Hsp70, followed by secondary anti-rabbit IgG antibodies conjugated with horseradish peroxidase and visualized by chemiluminescence.

In Vitro Translation Experiments
Coupled in vitro translation experiments were performed with a rabbit reticulocyte translation system (STP3 system; Novagen) using either the full-length Hsp60 or Hsp70. The proteins produced were analyzed by SDS-PAGE and immunoblotted as described above.

Construction of Expression Constructs for COS-1 Cell Transfection
To investigate the presence of mitochondrial targeting sequences in Neocallimastix Hsp60 and Hsp70, various expression constructs were made using custom PCR primers (table 1). All constructs were confirmed by sequence analyses prior to transfection.


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Table 1 Description of the Various Chaperone Constructs Used in This Study and Their Distribution in Transfected COS Cells.

 
Cell Culture, Transfection, and Immunofluorescence Microscopy for Heterologous Targeting in Mammalian COS Cell Lines
Mammalian COS-1 cells were transfected with the different plasmid constructs using Superfect (Qiagen) and the manufacturer's protocol. Fixation, immunofluorescence, mitochondrial labeling, and confocal immunofluorescence microscopy were performed as described previously (Leiper, Oatey, and Danpure 1996; Oatey, Lumb, and Danpure 1996). Hsp60 and Hsp70 were visualized with the homologous polyclonal antisera described in this paper and with fluorescein isothiocyanate-conjugated goat anti-rabbit immunoglobulin (IgG; Sigma). Mammalian mitochondria were visualized using the autofluorescent vital stain MitoTracker (Molecular Probes). The fluorescence of fluorescein isothiocyanate and GFP was observed with excitation and emission wavelengths of 488 nm and 522 nm, respectively. For MitoTracker, excitation and emission wavelengths of 568 and 585 nm were used. A BioRad MRC1024 laser-scanning confocal fluorescence microscope was used.

Quantitative Immunoelectron Microscopy
Neocallimastix cells were fixed in 4% paraformaldehyde in 0.2 M Pipes pH 7.2 (Pipes buffer) at room temperature for at least 30 minutes and then stored in this fixative at 4°C. Pellets were prepared by centrifugation at for 20 min and 0.5-mm sized blocks were cryoprotected in 2.1 M sucrose in phosphate-buffered saline (PBS), mounted on iron panel pins, and frozen in liquid nitrogen. Cryosections were cut with a diamond knife on a Leica Ultracryomicrotome, mounted on pioloform/carbon–coated electron micrograph (EM) support grids. Grids were immunogold-labeled according to the following protocol: The grids were placed on drops of 0.5% fish skin gelatin in PBS (PBS/gelatin; Sigma), then on drops of 0.1 M ammonium chloride in PBS, each for 5 min. The grids were then incubated using rabbit antisera raised against Neocallimastix Hsp60 or Hsp70 diluted 1/20 and 1/40, respectively, in PBS/gelatin, washed in PBS, and then on protein A Gold (8 nm particle size; prepared as described in Lucocq [1993]). After final washes in PBS and distilled water, the sections were embedded and contrasted in methyl cellulose/uranyl acetate.

Labeling was quantified using stereological techniques to estimate the area of each compartment including hydrogenosomes, cytosol, and nucleus according to the method of Lucocq (1994). Pictures were recorded at systematically placed locations with a random start at 15–30,000x magnification on phosphoimaging plates and scanned in a plate reader (DITABIS AG, Birkenfeld, Germany). Images were displayed in Adobe Photoshop 5.5 and overlaid with an electronically generated square lattice grid with spacing of 0.5 µm. Area of compartments was estimated from in which P is the sum of points situated over the compartment (point hits) and a is the area associated with each point (in this case 0.25 µm2). For hydrogenosomes, a total of 210 point hits and 706 gold particles were counted. For cytosol, totals of 469 point hits and 83 gold particles were recorded; and for nucleus, 46 point hits and 8 gold particles were counted.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Sequence Features of Hsp60 and Hsp70 Genes from Neocallimastix patriciarum
The GC-content of both the Hsp60 and Hsp70 coding regions is 37%, with the noncoding regions ranging from 7% to 16%. These values are comparable to other Neocallimastix coding sequences (see van der Giezen et al. 1997a). The fungal genes share strong sequence similarity to chaperone sequences from aerobic mitochondria-containing eukaryotes, and they contain all of the key features of Hsp60 and Hsp70 (fig. 1). The Neocallimastix Hsp60 and Hsp70 genes encode proteins of 600 and 657 amino acids with calculated masses of 64.3 and 71.3 kDa, respectively. Computer prediction software MITOPROT (Claros and Vincens 1996), PSORT (Nakai and Horton 1999), and iPSORT (Bannai et al. 2002) identified plausible amino-terminal mitochondrial-like presequences on both proteins (fig. 2). To investigate whether these putative targeting signals were processed, the mobility of Hsp60 and Hsp70 produced from full-length genes in a coupled in vitro reticulocyte translation system (Novogen) was compared to the mobility of proteins in a Neocallimastix cell-free extract, detected using the homologous antisera. For Hsp60 the size of the (mature) protein in the cell-free extract was 61 kDa, about 3 kDa smaller than the protein produced by in vitro translation (64 kDa), in agreement with the predicted size of the leader peptide. In contrast, no size difference was observed for Hsp70, both bands being 70 kDa, despite the in silico prediction of a targeting signal.



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FIG. 1. Schematic representation of the typical features of the Neocallimastix patriciarum hydrogenosomal chaperones. Top: Comparison of the N. patriciarum and Saccharomyces cerevisiae Hsp60 proteins. Shown are residues that are conserved in most mitochondrial isoforms and implicated in substrate binding in Escherichia coli (bold) (Horner and Embley 2001); residues that are conserved in most mitochondrial isoforms and implicated in Hsp60/Hsp10 equatorial interactions (bold) (Horner and Embley 2001); and Prosite motif PS00296, typical for Hsp60. Bottom: Comparison of the N. patriciarum hydrogenosomal Hsp70 and S. cerevisiae mitochondrial Hsp70 proteins. Shown are: residue numbers defining the approximate domain borders, known structural features, and functions of domains; and the three Prosite motifs PS00297 (Hsp70 1), PS00329 (Hsp70 2), and PS01036 (Hsp70 3)

 


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FIG. 2. Analyses of the amino-terminal region of the Neocallimastix patriciarum hydrogenosomal chaperones. A, Alignment of amino-termini of eukaryotic Hsp60 chaperonins from N. patriciarum, S. cerevisiae, Homo sapiens, Rattus norvegicus, Cucurbita maxima (winter squash), Zea mays, Entamoeba histolytica, and Trichomonas vaginalis and bacterial homologues from Rickettsia tsutsugamushi, Desulfovibrio vulgaris, and Haemophilus influenzae. The 43 N-terminal amino acids of N. patriciarum Hsp60 used in the GFP targeting experiment are underlined. Predicted or confirmed cleavage sites are indicated by a dash. Subcellular localization is indicated on the right. B, Alignment of amino-termini of eukaryotic Hsp70 chaperones from N. patriciarum, S. cerevisiae, H. sapiens, Cricetulus griseus (Chinese hamster), Pisum sativa (pea), Solanum tuberosum (potato), E. histolytica, and T. vaginalis and bacterial homologues from Rickettsia prowazekii, E. coli, and H. influenzae. The 38 N-terminal amino acids of N. patriciarum Hsp70 used in the GFP targeting experiment are underlined. Predicted or confirmed cleavage sites are indicated by a dash. Subcellular localization is indicated on the right

 
Phylogenetic Analysis of the Neocallimastix Heat-Shock Proteins
Bayesian analysis of both Hsp60 and Hsp70 coding regions yielded similar tree topologies with both N. patriciarum genes placed within the radiation defined by mitochondrial isoforms (fig. 3). Both the Hsp60 and Hsp70 of N. patriciarum were recovered with high bootstrap support (100% and 97%) as the basal branches of the fungi. This placement is consistent with phylogenies derived from short subunit (SSU) ribosomal RNA (van der Auwera and De Wachter 1996), which suggests that chytrids like Neocallimastix are the earliest diverging fungal lineage.



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FIG. 3. Phylogenetic analysis of Neocallimastix hydrogenosomal chaperone protein sequences using similar taxonomic sampling for Hsp60 and Hsp70. Unrooted maximum likelihood phylogenetic tree of 23 Hsp60 and Hsp70 protein sequences. The Neocallimastix sequences are recovered as part of a monophyletic group otherwise defined by mitochondrial chaperones of aerobic fungi. Numbers at the nodes represent bootstrap values

 
Localization Studies of the Neocallimastix Molecular Chaperones
Immunoblotting of N. patriciarum cellular fractions showed the presence of a cross-reacting protein with the Neocallimastix polyclonal anti-Hsp60 serum in the cell-free extract and in the hydrogenosomal fraction, but not in the cytosolic fraction (fig. 4A). The apparent molecular mass of 61 kDa is 3 kDa smaller than the calculated molecular mass of the predicted gene product (64.3 kDa). When the antiserum raised against the Neocallimastix Hsp70 was used, a similar distribution pattern was observed, but a faint band was also visible in the cytosolic fraction (fig. 4B). The cross-reacting band in each case was 70 kDa, being only slightly smaller than the expected molecular mass of the translated cDNA sequence (71.3 kDa). Quantitative immunoelectron microscopy revealed that in Neocallimastix both Hsp60 (fig. 5) and Hsp70 (fig. 6) are present (fig. 7) in double-membrane-bounded hydrogenosomes.



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FIG. 4. Localization of the Neocallimastix patriciarum chaperones. Western blot of N. patriciarum cellular fractions (15 µg) probed with antiserum raised against Neocallimastix Hsp60 (A), Hsp70 (B), and the hydrogenosomal marker the ß-subunit of succinyl-CoA synthetase (Brondijk et al. 1996; Benchimol, Durand, and Almeida 1997) (C). Lane 1: cell-free extract; lane 2: cytosolic fraction; and lane 3: hydrogenosomal fraction

 


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FIG. 5. Immunocytochemical localization of the N. patriciarum Hsp60. Gold particles are located in the hydrogenosomal matrix. Arrows indicate the double membrane surrounding the fungal hydrogenosomes. A, Overview of two hydrogenosomes. B, Detail of closely apposed hydrogenosomal double membrane. Scale bar: 200 nm

 


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FIG. 6. Immunocytochemical localization of Hsp70 in hydrogenosomes of N. patriciarum. The gold particles are located in the hydrogenosomal matrix. Note the double membrane (thin arrows). A, overview showing a hydrogenosome with typical concentric membranous structures (short arrows) as observed previously (see text). B and C, detail of closely apposed double membrane. Scale bar: 200 nm (A, B) or 50 nm (C)

 


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FIG. 7. Quantitative analysis of gold label distribution in N. patriciarum cells when incubated with antiserum raised against Hsp60 (gray bars) or Hsp70 (black bars). Both Hsp60 and Hsp70 are specifically localized in the fungal hydrogenosomes. Data are from a representative experiment and obtained as described in Materials and Methods (error bars are SEM calculated for ratio estimates according to Cochran [1977]; Hsp60 ; Hsp70 )

 
COS Cell Targeting
Because both Hsp60 and Hsp70 contained predicted mitochondrial targeting motifs, we investigated whether these targeting motifs could function in mammalian COS-1 cells containing both mitochondria and peroxisomes. Both proteins were specifically targeted to mitochondria, as shown by co-localization with the mitochondrial stain MitoTracker (fig. 8). To investigate whether the putative presequences were sufficient by themselves for mitochondrial import, the amino-terminal amino acids corresponding to the predicted targeting signals (N-terminal 43 amino acids for Hsp60; N-terminal 38 amino acids for Hsp70) were each fused in-frame with GFP. When COS-1 cells were transfected with these constructs, the GFP reporter protein was localized to mitochondria (fig. 9). The N-terminal amino acids of Hsp60 seem to be less efficient in targeting GFP to mitochondria than those of Hsp70, as some nuclear label could be seen in the former, as is the case in the control.



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FIG. 8. Intracellular distribution of N. patriciarum chaperones in transfected COS cells. COS cells were transfected with either pHsp60 (A and B) or pHsp70 (C and D). Cells were double-labeled for either Hsp60 (A) or Hsp70 (C) and the mitochondrial marker MitoTracker (B and D). Both chaperones co-localized with MitoTracker. Bar, 10 µm

 


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FIG. 9. Intracellular distribution of N. patriciarum chaperone fusion proteins in transfected COS cells. COS cells were transfected with GFP vector (A, B), pHsp60-43-GFP (C, D), or pHsp70-38-GFP (E, F). Cells were double-labeled for GFP autofluorescence (A, C, E) and MitoTracker (B, D, F). GFP vector without a targeting signal was diffusely distributed with no mitochondrial localization (A, B). Both pHsp60-43-GFP and pHsp70-38-GFP fusion proteins co-localized with the mitochondrial marker MitoTracker (C–F). Bar, 10 µm

 

    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
Shortly after hydrogenosomes were first discovered in Tritrichomonas foetus (Lindmark and Müller 1973) it was suggested that they were derived from an endosymbiontic Clostridium (Whatley, John, and Whatley 1979). The reasoning behind this suggestion was that Tritrichomonas hydrogenosomes made hydrogen via a pathway otherwise found only in anaerobic bacteria. It was subsequently shown that Trichomonas hydrogenosomes contained Hsp60 (and Hsp70) that clustered with mitochondrial orthologues rather than clostridial GroEL, that they imported proteins the same way that mitochondria do, and that they contained a member of the mitochondrial carrier protein family of unknown function (reviewed in Dyall and Johnson 2000; Embley et al. 2003). At least four distinct groups of ciliates, surrounded by aerobic mitochondria-containing species, are anaerobic and contain hydrogenosomes (Embley et al. 1995). In some cases the hydrogenosomes strongly resemble mitochondria in aerobic relatives (Finlay and Fenchel 1989; Fenchel and Finlay 1995), and at least one has been reported to contain a mitochondrial genome (Akhmanova et al. 1998). Thus, for ciliates and trichomonads, the available data are consistent with their hydrogenosomes sharing a common ancestry with mitochondria. The key enzyme, hydrogenase, for which there is no evidence for an alpha-proteobacterial ancestry, appears to have been acquired by eukaryotes early in their evolution and has subsequently been targeted to different cell compartments in different eukaryotes, including the cytosol, hydrogenosomes, and plastids (Horner, Foster, and Embley 2000; Davidson et al. 2002; Horner et al. 2002; Voncken et al. 2002b).

It has been argued repeatedly that fungal hydrogenosomes are different from those of other eukaryotes (Marvin-Sikkema et al. 1992, 1993a; Hackstein and Vogels 1997; Hackstein et al. 1998, 1999). The inference drawn from these arguments is that the differences are more fundamental than can be explained by the variation produced by descent with modification in separate lineages of a common progenitor organelle. The debate has largely been based on conflicting interpretations of ultrastructural "peculiarities" of fungal hydrogenosomes, including their lack of cristae or tubuli and the presence of membranous internal structures, and on whether fungal hydrogenosomes are surrounded by one boundary membrane, like peroxisomes, or two, like mitochondria (Hackstein et al. 2001). It is already apparent that the mitochondria of Saccharomyces cerevisiae cells grown under anoxic conditions have poorly organized inner membranes (Lloyd 1974) and that the total area of the inner membrane is related to the capacity for oxidative phosphorylation (Scheffler 1999), which hydrogenosomes lack (Müller 1993). The presence of concentric membranous structures in fungal hydrogenosomes (fig. 6A) is noteworthy, because these are not commonly reported for mitochondria (Hackstein et al. 2001). However, internal membrane structures of similar appearance and unknown function have been observed in the hydrogenosomes of Tritrichomonas foetus (Benchimol, Almeida, and De Souza 1996). Lastly, in our experiments we observed two closely apposed unit membranes surrounding fungal hydrogenosomes (fig. 6C), in agreement with the findings of van der Giezen et al. (1997b) and Benchimol, Durand, and Almeida (1997), and again like those shown around Tritrichomonas hydrogenosomes (Benchimol, Almeida, and De Souza 1996).

In the present study, antibodies homologous to the Neocallimastix Hsp60 and Hsp70 proteins localized overwhelmingly to fungal hydrogenosomes. Fungal hydrogenosomes therefore contain two key proteins of the mitochondrial protein import and folding pathway (Pfanner and Geissler 2001). We found none of the currently recognized fungal peroxisomal targeting signals (Rachubinski and Subramani 1995) on either of the Neocallimastix proteins. Indeed, none of the published fungal hydrogenosomal proteins contain such signals (Brondijk et al. 1996; van der Giezen et al. 1997a, 2002; Davidson et al. 2002; Voncken et al. 2002a, 2002b). The claim that fungal hydrogenosomes contain a hydrogenase with a consensus peroxisomal targeting signal in the form of an SKL motif (Marvin-Sikkema et al. 1993a), has not been confirmed by subsequent work (Davidson et al. 2002; Voncken et al. 2002b). Neocallimastix hydrogenosomes appear to contain an iron-only hydrogenase, the gene for which encodes a putative N-terminal mitochondrial targeting signal but no SKL motif.

Fungal hydrogenosomes lack an associated genome (van der Giezen et al. 1997b), so any proteins they contain must be synthesized in the cytosol and then correctly targeted and imported. In mitochondria there are two main protein import pathways (Pfanner and Geissler 2001). Some proteins that are destined for the inner mitochondrial membrane, for example the ADP/ATP carrier protein, carry within the mature protein poorly characterized internal targeting signals that are necessary for import (Sirrenberg et al. 1996). There is now evidence that fungal hydrogenosomes also use this import pathway, although the import machinery itself has not been isolated (van der Giezen et al. 2002; Voncken et al. 2002a). In heterologous transfection experiments, the ADP/ATP transporter from fungal hydrogenosomes is correctly processed into yeast inner mitochondrial membranes where it functions to transport ATP (van der Giezen et al. 2002). The second main mitochondrial import pathway processes mitochondrial proteins that are synthesized in the cytosol as preproteins carrying a positively charged targeting sequence at their amino-terminus (von Heijne, Steppuhn, and Herrmann 1989). The targeting sequence allows the preprotein to dock at the translocator of the outer mitochondrial membrane (TOM) complex, before being transported through the translocase of the inner membrane (TIM) complex (Schatz and Dobberstein 1996; Neupert 1997; Pfanner 1998). Import is dependent on an electrochemical gradient across the inner mitochondrial membrane and on the action of a mitochondrial Hsp70 in the matrix. During import, the presequence is cleaved off by the mitochondrial processing peptidase. Once inside the mitochondrial matrix, the newly imported proteins are transferred to the mitochondrial Hsp60 for folding into their native state (Hartl 1996; Bukau and Horwich 1998).

The hydrogenosomal Hsp60 and Hsp70 sequences reported here contain positively charged N-terminal extensions relative to bacterial sequences, which are enriched in alanine, arginine, and serine. Both of these extensions were recognized as potential mitochondrial targeting sequences by computer prediction software. They also resemble the presequences found on other fungal hydrogenosomal proteins in this respect (fig. 10), including the one on Neocallimastix hydrogenosomal malic enzyme, which is known to be processed (van der Giezen et al. 1997a). The computer-predicted presequences on Hsp60 and Hsp70 are sufficient to sort both proteins, or a green fluorescent reporter protein, into mammalian mitochondria. There was no evidence for a peroxisomal localization for either protein. Our data are in agreement with previous work demonstrating that hydrogenosomal malic enzyme was correctly processed to mitochondria, rather than peroxisomes, in the yeast Hansenula (van der Giezen et al. 1998). The retention of functional transit peptides on all three proteins strongly suggests that fungal hydrogenosomes possess the same presequence-dependent import machinery as is found in mitochondria. Comparison of the size of the in silico and experimentally translated products of the Hsp60 gene, with the protein detected in Neocallimastix cell extracts, is consistent with cleavage of a transit peptide from the protein. In the case of Hsp70, our data were more ambiguous over cleavage; we detected no size difference in our in vitro coupled assays and a smaller than predicted size difference between the in silico translated protein and the one detected in Neocallimastix extracts. Further work on the processing of Neocallimastix hydrogenosomal Hsp70 is required to understand the reasons for this discrepancy. Our attempts to sequence the N-terminus of the processed Hsp70 have so far failed, possibly because the protein appears to be blocked at the N-terminus (data not shown).



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FIG. 10. Alignment of amino-termini of the hydrogenosomal proteins from Neocallimastix. The arrow indicates the confirmed cleavage site of the malic enzyme targeting signal (van der Giezen et al. 1997a). The boxed arginine residues in bold indicate amino acids predicted to be involved in correct processing of the presequence

 
The demonstration that fungal hydrogenosomes contain Hsp60 and Hsp70 proteins that share common ancestry with mitochondrial orthologues is most simply explained by the hypothesis that fungal hydrogenosomes, like those of ciliates and trichomonads, share common ancestry with mitochondria. The topology of the phylogenetic trees, placing both proteins at the base of the fungal radiation, is entirely consistent with vertical inheritance of the genes for these proteins from a common ancestor they shared with aerobic mitochondria-containing fungi. The strong inference from this and previously published work (van der Giezen et al. 1997a, 2002; Voncken et al. 2002a), is that fungal hydrogenosomes use mitochondrial import pathways, sophisticated multicomponent complexes that are unlikely to have evolved twice, to import their proteins. In recent years it has become apparent that the biochemistry of mitochondria is more variable than revealed by studies of aerobic model organisms (van Hellemond, Opperdoes, and Tielens 1997; Embley and Martin 1998; Tielens et al. 2002). Hydrogenosomes provide some of the most striking examples of this apparent ease with which eukaryotes are able to change the metabolism of the mitochondrial compartment.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 Literature Cited
 
We thank Gang Ping Xue (CSIRO Plant Industry, Australia) for the N. patriciarum cDNA library. This project was supported in part by a Leverhulme Trust grant (F/696A) to T.M.E. and in part by an EMBO long-term fellowship (ALTF 520-1997) to M.v.d.G. D.S.H. was funded by the Natural History Museum. J.M.L. was supported by a Research Leave Fellowship from the Wellcome Trust (059767/Z/99/Z) and by Tenovus Scotland. Technical help was provided by Calum Thomson James of the Centre for High Resolution Imaging and Processing (CHIPS) at Dundee University of Dundee.


    Footnotes
 
1 Present address: School of Biological Sciences, Royal Holloway, University of London, Surrey, UK. Back

2 Present address: Dipartimento di Fisiologia e Biochimica Generali, University of Milan, Milan, Italy. Back

William Martin, Associate Editor Back


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 Abstract
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 Literature Cited
 

    Akhmanova, A., F. Voncken, A. T. van Alen, H. A. van Hoek, B. Boxma, G. Vogels, M. Veenhuis, and J. H. Hackstein. 1998. A hydrogenosome with a genome. Nature 396:527-528.[CrossRef][ISI][Medline]

    Bannai, H., Y. Tamada, O. Maruyama, K. Nakai, and S. Miyano. 2002. Extensive feature detection of N-terminal protein sorting signals. Bioinformatics 18:298-305.[Abstract/Free Full Text]

    Benchimol, M., J. C. A. Almeida, and W. De Souza. 1996. Further studies on the organization of the hydrogenosome in Tritrichomonas foetus. Tissue Cell 28:287-299.[ISI][Medline]

    Benchimol, M., R. Durand, and J. C. A. Almeida. 1997. A double membrane surrounds the hydrogenosomes of the anaerobic fungus Neocallimastix frontalis. FEMS Microbiol. Lett. 154:277-282.[CrossRef][ISI][Medline]

    Boorstein, W. R., T. Ziegelhoffer, and E. A. Craig. 1994. Molecular evolution of the HSP70 multigene family. J. Mol. Evol. 38:1-17.[ISI][Medline]

    Brondijk, T. H. C., R. Durand, M. van der Giezen, J. C. Gottschal, R. A. Prins, and M. Fèvre. 1996. scsB, a cDNA encoding the hydrogenosomal protein ß-succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis. Mol. Gen. Genet. 253:315-323.[CrossRef][ISI][Medline]

    Brookman, J. L., G. Mennim, A. P. Trinci, M. K. Theodorou, and D. S. Tuckwell. 2001. Identification and characterization of anaerobic gut fungi using molecular methodologies based on ribosomal ITS1 and 185 rRNA. Microbiology 146:393-403.[ISI]

    Bukau, B., and A. L. Horwich. 1998. The Hsp70 and Hsp60 chaperone machines. Cell 92:351-366.[ISI][Medline]

    Claros, M. G., and P. Vincens. 1996. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241:779-786.[Abstract]

    Cochran, A. J. 1977. Immunologic mechanisms in malignant melanoma of the skin. Trans. Ophthalmol. Soc. U.K. 97:385-388.[ISI][Medline]

    Davidson, E. A., M. van der Giezen, D. S. Horner, T. M. Embley, and C. J. Howe. 2002. An [Fe] hydrogenase from the anaerobic hydrogenosome-containing fungus Neocallimastix frontalis L2. Gene 296:45-52.[CrossRef][ISI][Medline]

    Dyall, S. D., and P. J. Johnson. 2000. Origins of hydrogenosomes and mitochondria: evolution and organelle biogenesis. Curr. Opin. Microbiol. 3:404-411.[CrossRef][ISI][Medline]

    Embley, T. M., B. J. Finlay, P. L. Dyal, R. P. Hirt, M. Wilkinson, and A. G. Williams. 1995. Multiple origins of anaerobic ciliates with hydrogenosomes within the radiation of aerobic ciliates. Proc. R. Soc. Lond. Ser. B. Biol. Sci. 262:87-93.[ISI][Medline]

    Embley, T. M., D. S. Horner, and R. P. Hirt. 1997. Anaerobic eukaryote evolution: hydrogenosomes as biochemically modified mitochondria? Trends Ecol. Evol. 12:437-441.[CrossRef][ISI]

    Embley, T. M., and W. Martin. 1998. A hydrogen-producing mitochondrion. Nature 396:517-519.[CrossRef][ISI][Medline]

    Embley, T. M., M. van der Giezen, D. S. Horner, P. L. Dyal, and P. Foster. 2003. Mitochondria and hydrogenosomes are two forms of the same fundamental organelle. Phil. Trans. R. Soc. Lond. 358:191-204.[CrossRef][ISI][Medline]

    Fenchel, T., and B. J. Finlay. 1995. Ecology and evolution in anoxic worlds. Oxford University Press, Oxford.

    Finlay, B. J., and T. Fenchel. 1989. Hydrogenosomes in some anaerobic protozoa resemble mitochondria. FEMS Microbiol. Lett. 65:311-314.[CrossRef][ISI]

    Hackstein, J. H., A. Akhmanova, B. Boxma, H. R. Harhangi, and F. G. Voncken. 1999. Hydrogenosomes: eukaryotic adaptations to anaerobic environments. Trends Microbiol. 7:441-447.[CrossRef][ISI][Medline]

    Hackstein, J. H. P., A. Akhmanova, F. Voncken, A. H. A. M. van Hoek, T. van Alen, B. Boxma, S. Y. Moon-van der Staay, G. van der Staay, J. Leunissen, M. Huynen, J. Rosenberg, and M. Veenhuis. 2001. Hydrogenosomes: convergent adaptations of mitochondria to anaerobic environments. Zoology 104:290-302.[ISI]

    Hackstein, J. H. P., and G. D. Vogels. 1997. Endosymbiotic interactions in anaerobic protozoa. Antonie Leeuwenhoek Int. J. Gen. Mol. Microbiol. 71:151-158.

    Hackstein, J. H. P., F. G. J. Voncken, G. D. Vogels, J. Rosenberg, and U. Mackenstedt. 1998. Hydrogenosomes and plastid-like organelles in amoeboflagellates, chytrids, and apicomplexan parasites. Pp. 149–168 in G. H. Coombs, K. Vickerman, M. A. Sleigh, and A. Warren, eds. Evolutionary relationships among protozoa. Chapman & Hall, London.

    Hartl, F. U. 1996. Molecular chaperones in cellular protein folding. Nature 381:571-580.[CrossRef][ISI][Medline]

    Hirt, R. P., B. Healy, C. R. Vossbrinck, E. U. Canning, and T. M. Embley. 1997. A mitochondrial Hsp70 orthologue in Vairimorpha necatrix: molecular evidence that microsporidia once contained mitochondria. Curr. Biol. 7:995-998.[ISI][Medline]

    Hobson, P. N. 1969. Rumen bacteria. Pp. 133–149 in J. R. Norris and D. W. Ribbons, eds. Methods in microbiology. Academic Press, London.

    Horner, D. S., and T. M. Embley. 2001. Chaperonin 60 phylogeny provides further evidence for secondary loss of mitochondria among putative early branching eukaryotes. Mol. Biol. Evol. 18:1970-1975.[Free Full Text]

    Horner, D. S., P. G. Foster, and T. M. Embley. 2000. Iron hydrogenases and the evolution of anaerobic eukaryotes. Mol. Biol. Evol. 17:1695-1709.[Abstract/Free Full Text]

    Horner, D. S., B. Heil, T. Happe, and T. M. Embley. 2002. Iron hydrogenases, ancient enzymes in modern eukaryotes. Trends Biochem. Sci. 27:148-153.[CrossRef][ISI][Medline]

    Horner, D. S., R. P. Hirt, S. Kilvington, D. Lloyd, and T. M. Embley. 1996. Molecular data suggest an early acquisition of the mitochondrion endosymbiont. Proc. R. Soc. Lond. Ser. B. Biol. Sci. 263:1053-1059.[ISI][Medline]

    Huelsenbeck, J. P. 2000. MrBayes. Department of Biology, University of Rochester.

    Leiper, J., P. Oatey, and C. Danpure. 1996. Inhibition of alanine: glyoxylate aminotransferase 1 dimerization is a prerequisite for its peroxisome-to-mitochondrion mistargeting in primary hyperoxaluria type 1. J. Cell Biol. 135:939-951.[Abstract]

    Lindmark, D. G., and M. Müller. 1973. Hydrogenosome, a cytoplasmic organelle of the anaerobic flagellate Tritrichomonas foetus, and its role in pyruvate metabolism. J. Biol. Chem. 248:7724-7728.[Abstract/Free Full Text]

    Lloyd, D. 1974. The mitochondria of microorganisms. Academic Press, London.

    Lucocq, J. 1993. Markers for immunoelectron microscopy. Pp. 279–302 in G. Griffiths, ed. Fine structure immunocytochemistry. Springer-Verlag, Berlin.

    Lucocq, J. 1994. Quantitation of gold labelling and antigens in immunolabelled ultrathin sections. J. Anat. 184:1-13.[ISI][Medline]

    Marvin-Sikkema, F. D., M. N. Kraak, M. Veenhuis, J. C. Gottschal, and R. A. Prins. 1993a. The hydrogenosomal enzyme hydrogenase from the anaerobic fungus Neocallimastix sp. L2 is recognized by antibodies, directed against the C-terminal microbody protein targetting signal SKL. Eur. J. Cell Biol. 61:86-91.[ISI][Medline]

    Marvin-Sikkema, F. D., G. A. Lahpor, M. N. Kraak, J. C. Gottschal, and R. A. Prins. 1992. Characterization of an anaerobic fungus from llama faeces. J. Gen. Microbiol. 138:2235-2241.[ISI][Medline]

    Marvin-Sikkema, F. D., T. M. Pedro Gomes, J. Grivet, J. C. Gottschal, and R. A. Prins. 1993b. Characterization of hydrogenosomes and their role in glucose metabolism of Neocallimastix sp. L2. Arch. Microbiol. 160:388-396.[ISI][Medline]

    Matouschek, A., N. Pfanner, and W. Voos. 2000. Protein unfolding by mitochondria. The Hsp70 import motor. EMBO Rep. 1:404-10.[Abstract/Free Full Text]

    Müller, M. 1993. The hydrogenosome. J. Gen. Microbiol. 139:2879-2889.[ISI][Medline]

    Nakai, K., and P. Horton. 1999. PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem. Sci. 24:34-36.[CrossRef][ISI][Medline]

    Neupert, W. 1997. Protein import into mitochondria. Annu. Rev. Biochem. 66:863-917.[CrossRef][ISI][Medline]

    Oatey, P. B., M. J. Lumb, and C. J. Danpure. 1996. Molecular basis of the variable mitochondrial and peroxisomal localisation of alanine-glyoxylate aminotransferase. Eur. J. Biochem. 241:374-385.[Abstract]

    Pfanner, N. 1998. Mitochondrial import: crossing the aqueous intermembrane space. Curr. Biol. 8:R262-R265.[ISI][Medline]

    Pfanner, N., and A. Geissler. 2001. Versatility of the mitochondrial protein import machinery. Nat. Rev. Mol. Cell. Biol. 2:339-349.[CrossRef][ISI][Medline]

    Rachubinski, R. A., and S. Subramani. 1995. How proteins penetrate peroxisomes. Cell 83:525-528.[ISI][Medline]

    Sambrook, J., E. Fritsch, and T. Maniatis. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

    Schatz, G., and B. Dobberstein. 1996. Common principles of protein translocation across membranes. Science 271:1519-1526.[Abstract]

    Scheffler, I. E. 1999. Mitochondria. Wiley Liss, New York.

    Sirrenberg, C., M. F. Bauer, B. Guiard, W. Neupert, and M. Brunner. 1996. Import of carrier proteins into the mitochondrial inner membrane mediated by Tim22. Nature 384:582-585.[CrossRef][ISI][Medline]

    Strimmer, K., and A. von Haeseler. 1996. Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol. Biol. Evol. 13:964-969.[Free Full Text]

    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. ClustalW: improving the sensitivity of progressive multiple alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673-4680.[Abstract]

    Tielens, A. G. M., C. Rotte, J. J. van Hellemond, and W. Martin. 2002. Mitochondria as we don't know them. Trends Biochem. Sci. 27:564-572.[CrossRef][ISI][Medline]

    van der Auwera, G., and R. De Wachter. 1996. Large-subunit rRNA sequence of the chytridiomycete Blastocladiella emersonii, and implications for the evolution of zoosporic fungi. J. Mol. Evol. 43:476-483.[ISI][Medline]

    van der Giezen, M., J. A. K. W. Kiel, K. A. Sjollema, and R. A. Prins. 1998. The hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis is targeted to mitochondria of the methylotrophic yeast Hansenula polymorpha. Curr. Genet. 33:131-135.[CrossRef][ISI][Medline]

    van der Giezen, M., K. B. Rechinger, I. Svendsen, R. Durand, R. P. Hirt, M. Fèvre, T. M. Embley, and R. A. Prins. 1997a. A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: evidence for the hypothesis that hydrogenosomes are modified mitochondria. Mol. Microbiol. 23:11-21.[ISI][Medline]

    van der Giezen, M., K. A. Sjollema, R. R. E. Artz, W. Alkema, and R. A. Prins. 1997b. Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome. FEBS Lett. 408:147-150.[CrossRef][ISI][Medline]

    van der Giezen, M., D. J. Slotboom, D. S. Horner, P. L. Dyal, M. Harding, G.-P. Xue, T. M. Embley, and E. R. S. Kunji. 2002. Conserved properties of hydrogenosomal and mitochondrial ADP/ATP carriers: a common origin for both organelles. EMBO J. 21:572-579.[Abstract/Free Full Text]

    van Hellemond, J. J., F. R. Opperdoes, and A. G. M. Tielens. 1997. Trypanosomatidae produce acetate via a mitochondrial acetate: succinate CoA-transferase. Proc. Natl. Acad. Sci. USA 95:3036–3041.

    Viale, A. M., and A. K. Arakaki. 1994. The chaperone connection to the origins of the eukaryotic organelles. FEBS Lett. 341:146-151.[CrossRef][ISI][Medline]

    von Heijne, G., J. Steppuhn, and R. G. Herrmann. 1989. Domain structure of mitochondrial and chloroplast targeting peptides. Eur. J. Biochem. 180:535-545.[Abstract]

    Voncken, F., B. Boxma, J. Tjaden, A. Akhmanova, M. Huynen, F. Verbeek, A. G. Tielens, I. Haferkamp, H. E. Neuhaus, G. Vogels, M. Veenhuis, and J. H. Hackstein. 2002a. Multiple origins of hydrogenosomes: functional and phylogenetic evidence from the ADP/ATP carrier of the anaerobic chytrid Neocallimastix sp. Mol. Microbiol. 44:1441-1454.[CrossRef][ISI][Medline]

    Voncken, F. G., B. Boxma, A. H. van Hoek, A. S. Akhmanova, G. D. Vogels, M. Huynen, M. Veenhuis, and J. H. Hackstein. 2002b. A hydrogenosomal [Fe]-hydrogenase from the anaerobic chytrid Neocallimastix sp. L2. Gene 284:103-112.[CrossRef][ISI][Medline]

    Whatley, J. M., P. John, and F. R. Whatley. 1979. From extracellular to intracellular: the establishment of mitochondria and chloroplasts. Proc. R. Soc. Lond. Ser. B. 204:165-187.[ISI][Medline]

    Xue, G. P., C. G. Orpin, K. S. Gobius, J. H. Aylward, and G. D. Simpson. 1992. Cloning and expression of multiple cellulase cDNAs from the anaerobic rumen fungus Neocallimastix patriciarum in Escherichia coli. J. Gen. Microbiol. 138:1413-1420.[ISI][Medline]

    Yarlett, N., C. G. Orpin, E. A. Munn, and C. Greenwood. 1986. Hydrogenosomes in the rumen fungus Neocallimastix patriciarum. Biochem. J. 236:729-739.[ISI][Medline]

Accepted for publication February 5, 2003.





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