* Department of Zoology, The Natural History Museum, London, United Kingdom
Department of Biology, University College London, London, United Kingdom
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key Words: heat-shock proteins hydrogenosomes mitochondria anaerobic eukaryotes evolution
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 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.
|
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/carboncoated 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 1530,000x 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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).
|
![]() |
Acknowledgements |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Footnotes |
---|
2 Present address: Dipartimento di Fisiologia e Biochimica Generali, University of Milan, Milan, Italy.
William Martin, Associate Editor
![]() |
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.
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. 149168 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. 133149 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.
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.
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.
Lloyd, D. 1974. The mitochondria of microorganisms. Academic Press, London.
Lucocq, J. 1993. Markers for immunoelectron microscopy. Pp. 279302 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.
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.
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.
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 95:30363041.
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]
|