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Implications of Protein Import on the Origin of Hydrogenosomes
Protist, Vol. 149, 303-311, December 1998 © Gustav Fischer Verlag
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Implications of Protein Import on the Origin of Hydrogenosomes Introduction Hydrogenosomes were first described 25 years ago in trichomonads, parasitic protists belonging to the phylum Parabasalia (Lindmark and Muller 1973). These double membrane-bounded redox organelles metabolize glycolytic pyruvate, generate ATP by substrate level phosphorylation and concomitantly evolve vast amounts of hydrogen due to the action of their marker enzyme hydrogenase (Muller 1993). Organelles with similar characteristics have since also been described in the amoeboflagellate Psalteriomonas lanterna, in rumen-dwelling and free living ciliates as well as in rumen-dwelling chytrid fungi (see Johnson et al. 1995; Muller 1993). These protists comprise a broad range of phylogenetically distant eukaryotes; they do, however, share two characteristics: they all live in anaerobic habitats and they all lack mitochondria. The origin of hydrogenosomes has been a matter of debate ever since their discovery. At a time when both morphological and biochemical data on fungal hydrogenosomes was very limited, Cavalier-Smith (1987) proposed that these organelles evolved from microbodies, such as peroxisomes. Later, data which was interpreted to support this hypothesis, was reported based on studies of the rumendwelling fungus Neocallimastix frontalis. These fungal hydrogenosomes were reported to most likely be bounded by only a single membrane, a microscopic property of peroxisomes that distinguishes these organelles from double-membrane bounded mitochondria. In addition, immunological crossreactivity of a fungal hydrogenosomal protein with antibodies against the three amino acid motif, Ser-Lys-Leu, that comprises a peroxisomal targeting signal (type 1) was reported (for details see Muller 1993). However, the presence of a hydrogenosomal protein with an epitope against such a simple sequence could occur by random chance and thus this observation alone cannot be interpreted to imply similar target-
ing mechanisms for fungal hydrogenosomes and peroxisomes. In fact, recent molecular analyses of two fungal hydrogenosomal proteins failed to reveal the existence of a C-terminal peroxisomal targeting sequence and re-examination of the Ultrastructure has clearly demonstrated the existence of two membranes surrounding fungal hydrogenosomes (Benchimol et al. 1997; Brondijk et al. 1996; van der Giezen et al. 1997a; van der Giezen et al. 1997b). Moreover, ciliate and trichomonad hydrogenosomes have also been shown to be surrounded by double membranes (Benchimol and De Souza 1983; Finlay and Fenchel 1989; Paul et al. 1990). Thus, at present, it appears that all examined hydrogenosomes are bounded by a double-membrane, contrary to that observed for microbody-derived organelles. The existence of two surrounding membranes as well as biochemical evidence indicating that trichomonad hydrogenosomes are anaerobic equivalents of mitochondria prompted two hypotheses to explain the origin of this organelle. Both hypotheses proposed an endosymbiotic origin. One took into account the biochemical differences between mitochondria and hydrogenosomes, whereas the other focussed on their similiarities. With regard to their differences, mitochondria lack hydrogenase, the marker enzmye of the hydrogenosome, and pyruvate:ferredoxin oxidoreductase, the hydrogenosomal counterpart to the mitochondrial pyruvate dehydrogenase complex. Furthermore, hydrogenosomes lack cytochromes, a respiratory chain, FoF1 -ATPase activity and DNA (see Johnson et al. 1995; Muller 1993) all of which are present in mitochondria. Based on these differences, one hypothesis proposed that hydrogenosomes arose via an endosymbiosis of an anaerobic bacterium with a eukaryotic host (Muller 1980; Whatley et al. 1979). In this scenario, hydrogenosomes and mitochondria would have evolved entirely independent of each other and
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their similarities would be accounted for by convergent evolution. The second hypothesis proposed that hydrogenosomes arose via the conversion of an established mitochondrion, based on the similiarities of these two organelles and their mutually exclusive appearance (Cavalier-Smith 1987). This hypothesis has since been supported by molecular analyses of a number of hydrogenosomal proteins which showed close homology to their mitochondrial counterparts, such as ferredoxin and succinyl-CoA synthetase (summarized in Johnson et al. 1995). According to this hypothesis, the biochemical differences between hydrogenosomes and mitochondria result from the anaerobic versus aerobic lifestyles of the respective hosts. This is not difficult to envisage as the majority of components that are found in mitochondria but that are absent in hydrogenosomes playa role in respiration, a property that is lacking in the anaerobic environment of the hydrogenosome. Interestingly, it is even possible to account for the lack of DNA in hydrogenosomes based on a lack of respiration. The proteins encoded in the mitochondrial DNA of most contemporary organisms are inner membrane proteins of the respiratory chain (Neupert 1997). As noted previously, since hydrogenosomes do not possess an active respiratory chain in their inner membranes, the maintenance of a mitochondrion-like genome in hydrogenosomes would be unnecessary (Johnson et al. 1993). In the late 80s and early 90s, phylogenetic analyses using rRNA sequences revealed that the lineage that gave rise to trichomonads diverged early from the main line of eukaryotic descent, prior to the advent of mitochondria-containing eukaryotes (Baroin et al. 1988; Sogin 1991). This phylogenetic position for trichomonads is in contrast to other hydrogenosome-containing organisms, such as ciliates and fungi, which evolved much later and belong to the crown group of eukaryotes. Based on the early divergence of the trichomonad lineage, the common origin hypothesis for mitochondria and hydrogenosomes was refined to state that hydrogenosomes and mitochondria evolved from a common progenitor organelle (Johnson et al. 1993) as opposed to the popular notion that mitochondria gave rise to hydrogenosomes. This refined hypothesis implies that the progenitor organelle was present in eukaryotes before the advent of true mitochondria or hydrogenosomes. This review is intended to summarize our current knowledge on the synthesis and targeting of hydrogenosomal proteins and to discuss what these data imply regarding the origin of hydrogenosomes. As almost nothing is known on the biogenesis of ciliate
hydrogenosomes and little on the biogenesis of amoeboflagellate and fungal hydrogenosomes the review focusses on protein import to the hydrogenosome of T. vaginalis which has been studied to a greater extent. Where available, data on amoeboflagellate, ciliate or fungal hydrogenosomal import will be included. Elucidation of the mechanisms underlying organellar targeting in the early diverging trichomonads should help to broaden our understanding of the relationship between hydrogenosomes and mitochondria as well as to learn how protein import to organelles of endosymbiontic origin may have evolved.
Hydrogenosomal Proteins Given that trichomonad hydrogenosomes do not appear to contain DNA (Turner and Muller 1983), apparently due to a complete transfer of their genes into the nucleus of an ancestral host cell, the genes for all hydrogenosomal proteins are encoded in the nucleus. These proteins have been shown to be synthesized in the cytosol on free ribosomes which implies a posttranslational targeting route to their final hydrogenosomal destination (Lahti and Johnson 1991). A comparison of N-terminal sequence analyses of hydrogenosomal proteins and the genes which encode them has invariably shown that hydrogenosomal proteins are synthesized with an N-terminal extension which is not present in the mature protein isolated from the organelle (Bradley et al. 1997; Brondijk et al. 1996; Brul et al. 1994; van der Giezen et al. 1997a). These N-terminal extensions are 5-14 amino acids in length in T. vagina/is hydrogenosomal proteins, 8 amino acids long in the P. /anterna hydrogenosomal protein ferredoxin, and 27 amino acids long in the two Neocallimastix frontalis hydrogenosomal proteins analysed to date (Table 1). A comparison of the N-terminal extensions on all 15 hydrogenosomal proteins shows that they share several characteristics. They all have an Arg at position -2 or -3 relative to position + 1 of the mature protein and all but one have an Asp or Phe at the -1 position. In addition, the N-terminal extensions are generally rich in hydroxylated and hydrophobic amino acids, with a Leu following the start Met in all but two of the 15 hydrogenosomal extensions characterized. Closer examination of a number of these extensions reveals that they are capable of forming amphiphilic a-helices. The presence of these similar N-terminal extensions on newly synthesized hydrogenosomal proteins and their absence on the proteins isolated from the organelles paralleles the situation in mitochon-
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Table 1. N-terminal presequences of hydrogenosomal proteins.
Hydrogenosomal protein
Presequence
Start
Ref.
Trichomonas vaginalis: Adenylate kinase Ferredoxin Hsp60 Malic enzyme 1 Malic enzyme 2 Pyruvate:ferredoxin oxidoreductase 1 Pyruvate:ferredoxin oxidoreductase 2 u-Succinyl-CoA synthetase 1 u-Succinyl-CoA synthetase 2 u-Succinyl-CoA synthetase 3 P-Succinyl-CoA synthetase 1 ~-Succinyl-CoA synthetase 53
MLSGVSRN MLSQVCRF MSLlEAAKHFTRAF MLTSSVSVPVRN MLTSVSYPVRN MLRNF MLRSF MLAGDFSRN MLSSSFERN MLSSSFERN MLSASSNFARN MLSSSFARN
AART GTIT AKAR ICRA ICRS SKRV GKRI LKOP LHQP LHQP FNIL FNIL
1 1 1 1 1 1 1 1 1 1 1 1
Psalteriomonas lanterna: Ferredoxin
*MVSGVSRN
AART
2
Neocallimastix frontalis: Malic enzyme ~-Succinyl-CoA synthetase
MLAPIQTIARPVSSILPATGALAAKRT *MLANVTRSTSKAAPALASIAQTAQKRF
FFAP LSVH
3 4
Presequences are defined as sequences encoded by the gene, but absent from the N-terminus upon N-terminal sequencing of the protein isolated from the organelle. *Putative presequence determined by alignment of coding regions of the gene from several organisms. Start indicates the first four amino acids derived from N-terminal sequencing of the protein. References: 1) Bradley et al. 1997,2) Brul et al. 1994,3) van der Giezen et al. 1997a, 4) Brondijk et al. 1996.
dria and chloroplasts and therefore strongly suggests that these extensions may serve as presequences involved in targeting hydrogenosomal proteins to the organelle. To test this hypothesis, an in vitro protein import assay similar to ones used in mitochondrial import research was established with hydrogenosomes of T. vaginalis (Bradley et al. 1997). Using this assay, several parameters were investigated to determine the role of N-terminal extensions in hydrogenosomal protein targeting.
Hydrogenosomal Protein Import The hydrogenosomal in vitro import assay is described in Fig. 1 (for details refer to Bradley et al. 1997). The basic components of the assay are purified organelles, obtained by gradient centrifugation, and a radiolabelled precursor protein. Hydrogenosomal protein import experiments were predominantly carried out using the T. vaginalis hydrogenosomal protein ferredoxin as a precursor, which was simultaneously expressed and radiolabelled in E. coli. Ferredoxin is a 12 kDa hydrogenosomal matrix protein involved in electron transfer (Johnson et al.
1990). In vitro import assays using ferredoxin result in precursor binding to the surface of hydrogenosomes followed by import into the organelle upon which the precursor is cleaved to a size consistent with the mature protein thereby demonstrating the functionality of the assay (Bradley et al. 1997). Import assays carried-out with a protein lacking the 8 amino acid N-terminal extension of ferredoxin clearly show that this protein neither binds to the surface of hydrogenosomes nor is imported and cleaved (Bradley et al. 1997). These results confirm the hypothesis that the N-terminal extension serves as a presequence which is absolutely necessary for protein targeting to hydrogenosomes. As stated above, hydrogenosomal presequences share a number of common features. In particular the occurrence of a Leu directly following the start Met in all but two of the 15 hydrogenosomal presequences analysed so far indicates a potential targeting requirement for Leu at the +2 position. Indeed, a mutational analysis of the ferredoxin presequence in which the Leu was changed to Gly, another nonpolar amino acid, resulted in nearly complete abolition of binding and import of the altered ferredoxin precursor. These data demonstrate an intolerance for
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1. Protein import reaction
0+
----
+
purified 35S-labelled hydrogenosomes precursor
ATP, ATP-regenerating system and T. vaginalis-cytosol
2. Removal of unbound precursor by washing
rr- . . . . . . -
.--M
3. Protease protection assay
~
no protease
@ proteinase K
C
proteinase K + Triton X-100
4. Analysis by SDS-PAGE and f1uorography
Figure 1. Schematic of hydrogenosomal in vitro protein import and protease protection assay. The hydrogenosomal in vitro import assay consists of purified organelles and an 35S-radiolabelled precursor protein incubated in a cytosolic extract of T. vaginalis which has been supplemented with energy in the form of ATP and an ATP regenerating system. After 30 minutes of incubation at 37°C the organelles are washed free of unbound precursor and subdivided into three aliquots for a protease protection assay. One of these aliquots receives the protease proteinase K, the other proteinase K plus the detergent Triton X-100 while the third remains untreated as a control. After 5 minutes of incubation on ice protease digestion is stopped and the samples are subjected to 8DS-PAGE and fluorography to show size and localization of the precursor proteins. The untreated control is predicted to show the total amount of precursor associated with the organelle, inside and outside. On the other hand, proteinase K treatment of the sample results in the degradation of precursor bound to the outside of the organelle, while protein that has become localized inside the organelle should be protected from digestion. The latter is confirmed by destroying the integrity of the organelle with Triton X100 upon which all protein, whether inside or outside the organelle, should become accessible to proteinase K and degraded.
certain amino acids at this position (Bradley et al. 1997). Although Gly is not tolerated at the +2 position of the presequence, amino acids with bulky, hydrophobic side chains can functionally replace Leu in this position. Mutation of the +2 Leu to both lie and Val allows binding and import, albeit at reduced efficiency (Bradley and Johnson, unpublished).
Energy Requirements of Hydrogenosomal Import The translocation of a precursor protein from one side of a membrane to the other requires energy in the forms of temperature, ATP and, where it exists, a transmembrane electrochemical potential (Schatz and Dobberstein 1996). These requirements for translocation of proteins from the cytosol to the matrix of the hydrogenosomes have also been established (Bradley et al. 1997). In hydrogenosomal protein import assays performed at 0 °C, little precursor binding and no import are observed indicating that protein import is an active process. To test for the requirement of ATP, import reactions were depleted of ATP by incubation with the ADP-ATPase apyrase and omittance of the ATP regenerating system. Under these conditions precursor binding occurs but very little protein import can be detected consistent with a requirement of ATP-hydrolysis for protein translocation across membranes. The role ATP plays in translocation and whether ATP hydrolysis is necessary is not known. Based on similarities to other import systems, it is reasonable to predict that cytosolic ATP is required for chaperone-assisted maintenance of an import competent state of the precursor in the cytosol and its translocation through hydrogenosomal membranes. Hydrogenosomal ATP may also be required for refolding of the precursor in the hydrogenosomal matrix upon membrane passage, as demonstrated in detail for proteins that are imported into mitochondria (Horst et al. 1997; Neupert 1997; Pfanner et al. 1997). While the requirements for energy in the forms of temperature and ATP in hydrogenosomal protein binding and import could be directly demonstrated, evidence for the involvement of a hydrogenosomal membrane potential in protein import is more indirect. It was shown that hydrogenosomal protein import is sensitive to m-chlorophenyl-hydrazone (CCCP), a protonophore commonly used to investigate the effect of the absence of an electrochemical membrane potential on cellular processes. Thus a hydrogenosomal transmembrane electrochemical potential seems to be necessary for completion of protein import. It should be noted here that the existence of an electrochemical potential involved in protein translocation across membranes in other systems, especially across the mitochondrial inner membrane, the chloroplast thylakoid membrane and the bacterial cytoplasma membrane, is usually coupled to the existence of electron transport chains and FoF1 -ATPase activity (Bakker and Randall 1984; Neupert 1997; Robinson et al. 1993). Trichomonad hydrogenosomes, however, neither possess mem-
Protein Import and Origin of Hydrogenosomes
branous electron transporters nor an FoF1 -ATPase activity (Johnson et al. 1995; Muller 1993). Nevertheless, a weak electrochemical membrane potential across the hydrogenosomal membranes seems to be maintained (Yarlett et al. 1987), which could be due to ADP-ATP exchange as reported for mitochondrial rho- mutants of yeast (for discussion see Bradley et al. 1997).
Co-factor Requirements of Hydrogenosomal Import The hydrogenosomal in vitro import system contains a T. vaginalis cytosolic fraction. If this fraction is omitted, binding, but no import of precursors occurs, indicating the requirement for factors other than ATP in hydrogenosomal protein import. Nethylmaleimide (NEM) sensitivity of the cytosolic component needed for translocation suggests that at least one of these factors is a protein (Bradley et al. 1997). Transport of newly synthesized proteins to various subcellular locations has been shown to require proteinaceous cytosolic factors (Powers and Forbes 1994). These factors are best characterized in mitochondrial import studies where they have been shown to have two functions. Chaperones such as cytosolic Hsp70 support the loosely folded state of precursors which is necessary for import and to protect precursors from aggregation and degradation (Neupert 1997). Factors involved in targeting such as the mitochondrial import stimulating factor, MSF, exert a more specific function by binding to precursors and guiding them to the translocation machinery (Mihara and Omura 1996). Cytosolic chaperones are not only involved in mitochondrial targeting but also in targeting to many subcellular locations, including the endoplasmic reticulum, the chloroplast and the nucleus (Bukau et al. 1996). Thus, it seems probable that the NEM-sensitive component found in T. vagina/is cytosol acts as a chaperone to assist in specific targeting of proteins to the hydrogenosome. In this regard, it is interesting to note that antisera raised against mouse MSF, the NEM-sensitive cytosolic protein that stimulates the translocation of a subset of mitochondrial matrix proteins, strongly cross-reacts with a T. vagina/is cytosolic protein (our unpublished data).
Hydrogenosomal Protein Import Resembles Mitochondrial Import The data currently available show that protein import to hydrogenosomes occurs posttranslationally,
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is dependent on the presence of a cleavable N-terminal presequence, cytosolic protein(s) and energy in the forms of temperature, ATP and a transmembrane electrochemical potential. With these characteristics hydrogenosomal protein import most closely resembles protein import into chloroplasts and mitochondria, as opposed to protein translocation across other cellular membranes (Haucke and Schatz 1997; Schatz and Dobberstein 1996). A close examination of mitochondrial and chloroplast presequences reveals significant differences between these targeting signals (von Heijne et al. 1989). In contrast, mitochondrial and hydrogenosomal presequences closely resemble each other. Both types of presequences show a preference for a Leu directly following the start Met, while in chloroplast presequences an Ala is usually found at this position (Keegstra et al. 1989). An Arg is found in position -2 or -10 from the cleavage site in mitochondrial presequences and in position -2 or -3 from the cleavage site in hydrogenosomal presequences (von Heijne et al. 1989). In both types of presequences Arg, Leu and Ser are overrepresented and both have the ability to form amphiphilic a-helices in non-aqueous environments (Johnson et al. 1990; von Heijne 1986). Differences between chloroplast and mitochondrial presequences are indispensable to assure proper targeting to the respective organelle within a common cytosol. As hydrogenosomes and mitochondria are never found within the same cell it could be argued that presequences with the same characteristics evolved independently. However, the following should be considered: i) The unusually short presequences found on T. vaginalis, Parabasalia and P. /anterna, Percolozoa (Weekers et al. 1997) hydrogenosomal proteins closely resemble the short presequences (8-20 amino acids) found on mitochondrial proteins of kinetoplastids (Hausler et al. 1997). In this regard, it is noteworthy that kinetoplastids represent one of the earliest diverging eukaryotes with mitochondria. ii) Fungal hydrogenosomal presequences (27 amino acids) are considerably longer, as are fungal mitochondrial presequences (20-60 amino acids) (Neupert 1997). Thus presequence length seems to increase with increasing position in the eukaryotic tree. The similarity in length of presequences among mitochondria and hydrogenosome-bearing protists of similar systematic position, as well as the similarity in structure and function of presequences, strongly argue for the evolution of protein targeting sequences in a common progenitor organelle of the contemporary hydrogenosomes and mitochondria.
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Phylogenetic Studies Support a Common Origin for Hydrogenosomes and Mitochondria In addition to studies on hydrogenosomal protein import which support a common evolutionary origin for hydrogenosomes and mitochondria, further support for this hypothesis has been provided by molecular analyses of T. vaginalis hydrogenosomal heat shock proteins (Bui et al. 1996; Germot et al. 1996; Horner et al. 1996; Roger et al. 1996). Work from our laboratory has shown that T. vaginalis hydrogenosomes contain both Hsp70 and Hsp60, proteins that are otherwise found only within mitochondria or chloroplasts in eukaryotic cells (Bui et al. 1996). Moreover, we and others have sequenced and characterized T. vaginalis genes encoding Hsp70 (Bui et al. 1996; Germot et al. 1996), Hsp60 (Bui et al. 1996; Horner et al. 1996; Roger et al. 1996) and Hsp10 (Bui et al. 1996). These hydrogenosomal genes were chosen for phylogenetic analyses as they have been quite useful for determining bacterial phylogeny (eg. Gupta et al. 1994). The presence of Hsp70 and Hsp60 proteins in the hydrogenosome and the fact that both proteins are encoded by a single-copy gene indicates that the specific genes examined were originally derived from the organelle. The results of the phylogenetic analyses obtained using these gene sequences were striking. A variety of inference techniques consistently put hydrogenosomal Hsp70, Hsp60 and Hsp10 on the lineage that contains mitochondria (Bui et al. 1996; Germot et al. 1996; Horner et al. 1996; Roger et al. 1996). It should be noted that these analyses invariably included chloroplast Hsp sequences and in no instance did the T. vaginalis Hsps group with chloroplast homologues. These data provide strong evidence that hydrogenosomes and mitochondria have a common evolutionary origin. Interestingly, similar phylogenetic analyses on Hsp genes isolated from three protist lineages that lack both mitochondria and hydrogenosomes, entamoebids (Clark and Roger 1995), microsporidia (Germot et al. 1997) and diplomonads (Roger et al. 1998) also places these gene sequences in the clade composed of mitochondrial Hsps. This indicates that these organisms once contained an endosymbiont, most likely the one that contributed to the formation of mitochondria and hydrogenosomes, which was ultimately lost from the lineages that gave rise to Entamoeba, Nosema and Giardia. These recent studies on the origin of hydrogenosomes and the phylogeny of Hsps in Giardia and Entamoeba, as well as the observation that hydrogenosomes in free living ciliates are often found
in an intimate connection with endosymbiotic methanogens, recently culminated in a new hypothesis on eukaryote beginnings (Martin and Muller 1998). This hypothesis, coined the hydrogen hypothesis, takes into account the archaeal origin of the nuclear apparatus (Gupta and Golding 1996; Lake and Rivera 1994) versus the eubacterial origin of the core metabolism found in mitochondria, hydrogenosomes and the cytosol of eukaryotes lacking both hydrogenosomes and mitochondria. The hydrogen hypothesis states that the first eukaryote arose via a union between a methanogenic archaea which absolutely depended on molecular hydrogen for its metabolism and a eubacterium that evolved vast amounts of hydrogen under anaerobic conditions but was also able to respire. After the union was established and gene transfer from the eubacterium to the archaea had occurred, the primitive organelle of eubacterial origin was either lost as in the eukaryotes devoid of either mitochondria or hydrogenosomes or developed into hydrogenosomes under anaerobic conditions and into mitochondria under aerobic conditions. This hypothesis also takes into account structural and biochemical similarities between hydrogenosomes and mitochondria, such as the existence of two bounding membranes, the modus of division by binary fission (Benchimol et al. 1996) and the related enzymes involved in pyruvate degradation found in both organelles. By assuming that the eubacterium was able to live anaerobically as well as aerobically, it is proposed to have possessed both pyruvate: ferredoxin oxidoreductase and the pyruvate dehydrogenase complex. It is also proposed to have contained both hydrogenase and a respiratory chain, as would be predicted if a single endosymbiotic event gave rise to contemporary mitochondria and hydrogenosomes. The hydrogen hypothesis is elegantly simple and much of the evidence upon which it is based is solid. Clearly, compelling evidence exists that hydrogenosomes and mitochondria share a common ancestral history. However, strong evidence also exists indicating that the establishment of endosymbiotic relationships and horizontal gene transfer is a relatively frequent event (Hackstein et al. 1997). Thus, it is perhaps naive to envisage that all genes encoding proteins which are imported into either mitochondria or hydrogenosomes were derived from this single endosymbiotic event. As additional properties of the contemporary versions of these organelles are discovered, we may find that additional endosymbiotic and/or gene transfer events have contributed to the evolutionary history of these complex organelles.
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Future Prospects
anaerobic fungus Neocallimastix frontalis. FEMS Microbioi Lett 154: 277-282
To broaden our understanding of the relationship between hydrogenosomes and mitochondria it will be necessary to obtain additional information on the hydrogenosomal protein translocation machinery. In particular, comparison of hydrogenosomal import receptors and proteinaceous membrane pores to those found in mitochondria should be informative. In addition it will be interesting to learn more about other protein factors involved in hydrogenosomal protein targeting, such as cytosolic co-factors that stimulate translocation and the putative matrix peptidase. The evolutionary relationship between hydrogenosomes found in ciliates, fungi and flagellates presents a fundamental question for which there is little data. Characterization of hydrogenosomal protein import and phylogenetic analyses of proteins found in the organelles of these highly diverged species should shed light on this issue. Moreover, given the early divergence of trichomonads from the main trunk of eukaryotic evolution, information on T. vagina/is hydrogenosomal protein targeting may help elucidate how protein targeting itself evolved in the dawn of eukaryotic life.
Benchimol M, Johnson PJ, de Souza W (1996) Morphogenesis of the hydrogenosome: an ultrastructural study. Bioi Cell 87: 197-205
Acknowledgements The authors are grateful to Dr. Eckhard Meyer for helpful comments on the manuscript. This work was supported by National Institutes of Health grant AI27857 to PJJ, a postdoctoral fellowship (PI 218/11) from the Deutsche Forschungsgemeinschaft to EP and a predoctoral training grant (NIH A107323) to PJB. PJJ is a recipient of a Burroughs-WeUcome Scholar in Molecular Parasitology award.
Bradley PJ, Lahti CJ, Plumper E, Johnson PJ (1997) Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import. EMBO J 16: 3484-3493 Brondijk TH, Durand R, van der Giezen M, Gottschal JC, Prins RA, Fevre M (1996) scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis. Mol Gen Genet 253: 315-323 Brul S, Veltman RH, Lombardo MC, Vogels GO (1994) Molecular cloning of hydrogenosomal ferredoxin cDNA from the anaerobic amoeboflagellate Psalteriomonas lanterna. Biochim Biophys Acta 1183: 544-546 Bui ET, Bradley PJ, Johnson PJ (1996) A common evolutionary origin for mitochondria and hydrogenosomes. Proc Natl Acad Sci USA 93: 9651-9656 Bukau B, Hesterkamp T, Luirink J (1996) Growing up in a dangerous environment: a network of multiple targeting and folding pathways for nascent polypeptides in the cytosol. Trends Cell Bioi 6: 480-486 Cavalier-Smith T (1987) The simultaneous symbiotic origin of mitochondria, chloroplasts, and microbodies. Ann NY Acad Sci 503: 55-71 Clark CG, Roger AJ (1995) Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proc Natl Acad Sci USA 92: 6518-6521 Finlay BJ, Fenchel T (1989) Hydrogenosomes in some anaerobic protozoa resemble mitochondria. FEMS Microbiol Lett 65: 311-314
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Neupert W (1997) Protein import into mitochondria. Annu Rev Biochem 66: 863-917 Paul RG, Williams AG, Butler RD (1990) Hydrogenosomes in the rumen entodiniomorphid ciliate Po/yp/astron mu/tivesicu/atum. J Gen Microbiol136: 1981-1989 Pfanner N, Craig EA, Honlinger A (1997) Mitochondrial preprotein translocase. Annu Rev Cell Dev Bioi 13: 25-51 Powers MA, Forbes DJ (1994) Cytosolic factors in nuclear transport: what's importin? Cell 79: 931-934 Robinson C, Klosgen RB, Herrmann RG, Shackleton JB (1993) Protein translocation across the thylakoid membrane-a tale of two mechanisms. FEBS Lett 325: 67-69 Roger AJ, Clark CG, Doolittle WF (1996) A possible mitochondrial gene in the early-branching amitochondriate protist Trichomonas vaginalis. Proc Natl Acad Sci USA 93: 14618-14628 Roger AJ, Svard SG, Tovar J, Clark CG, Smith MW, Gillin FD, Sogin ML (1998) A mitochondrial-like chaperonin 60 gene in Giardia /amblia: evidence that diplomonads once harbored an endosymbiont related to the progenitor of mitochondria. Proc Natl Acad Sci USA 95: 229-234 Schatz G, Dobberstein B (1996) Common principles of protein translocation across membranes. Science 271 : 1519-1526 Sogin ML (1991) Early evolution and the origin of eukaryotes. Curr Opin Genet Dev 1: 457-463 Turner G, Muller M (1983) Failure to detect extrachromosomal DNA in Trichomonas vaginalis and Tritrichomonas foetus. J Parasitol69: 234-236 van der Giezen M, Rechinger KB, Svendsen I, Durand R, Hirt RP, Fevre M, Embley TM, Prins RA (1997a) A mitochondrial-like targeting signal on the hydrogenosomal malic enzyme from the anaerobic fungus Neocallimastix frontalis: support for the hypothesis that hydrogenosomes are modified mitochondria. Mol Microbiol 23: 11-21 van der Giezen M, Sjollema KA, Artz RRE, Alkema
W, Prins RA (1997b) Hydrogenosomes in the anaerobic fungus Neocallimastix frontalis have a double membrane but lack an associated organelle genome. FEBS Lett 408: 147-150 ' von Heijne G (1986) Mitochondrial targeting sequences may form amphiphilic helices. EMBO J 5: 1335-1342 von Heijne G, Steppuhn J, Herrmann RG (1989) Domain structure of mitochondrial and chloroplast targeting peptides. Eur J Biochem 180: 535-545 Weekers PH, Kleyn J, Vogels GD (1997) Phylogenetic position of Psalteriomonas /anterna deduced from the SSU rDNA sequence. J Euk Microbiol44: 467-470
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Whatley JM, John P, Whatley FR (1979) From extracellular to intracellular: the establishment of mitochondria and chloroplasts. Proc R Soc Lond B Bioi Sci B204: 165-187 Yarlett N, Rowlands CC, Evans JC, Yarlett NC, Lloyd D (1987) Nitroimidazole and oxygen derived radicals detected by electron spin resonance in hydrogenosomal and cytosolic fractions from Trichomonas vaginalis. Mol Biochem Parasitol24: 255-261
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Evelyn Plumper", Peter J. Bradleyb, and Patricia J. Johnson c ,1 alnstitut fOr Allgemeine Zoologie und Genetik, Schlossplatz 5, D - 48149 MOnster, Germany bDepartment of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA cDepartment of Microbiology and Immunology, University of California at Los Angeles, Los Angeles, CA 90095, USA 1Corresponding author; fax 1-310-206-5231 e-mail [email protected]
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Implications of Protein Import on the Origin of Hydrogenosomes Introduction Hydrogenosomes were first described 25 years ago in trichomonads, parasitic protists belonging to the phylum Parabasalia (Lindmark and Muller 1973). These double membrane-bounded redox organelles metabolize glycolytic pyruvate, generate ATP by substrate level phosphorylation and concomitantly evolve vast amounts of hydrogen due to the action of their marker enzyme hydrogenase (Muller 1993). Organelles with similar characteristics have since also been described in the amoeboflagellate Psalteriomonas lanterna, in rumen-dwelling and free living ciliates as well as in rumen-dwelling chytrid fungi (see Johnson et al. 1995; Muller 1993). These protists comprise a broad range of phylogenetically distant eukaryotes; they do, however, share two characteristics: they all live in anaerobic habitats and they all lack mitochondria. The origin of hydrogenosomes has been a matter of debate ever since their discovery. At a time when both morphological and biochemical data on fungal hydrogenosomes was very limited, Cavalier-Smith (1987) proposed that these organelles evolved from microbodies, such as peroxisomes. Later, data which was interpreted to support this hypothesis, was reported based on studies of the rumendwelling fungus Neocallimastix frontalis. These fungal hydrogenosomes were reported to most likely be bounded by only a single membrane, a microscopic property of peroxisomes that distinguishes these organelles from double-membrane bounded mitochondria. In addition, immunological crossreactivity of a fungal hydrogenosomal protein with antibodies against the three amino acid motif, Ser-Lys-Leu, that comprises a peroxisomal targeting signal (type 1) was reported (for details see Muller 1993). However, the presence of a hydrogenosomal protein with an epitope against such a simple sequence could occur by random chance and thus this observation alone cannot be interpreted to imply similar target-
ing mechanisms for fungal hydrogenosomes and peroxisomes. In fact, recent molecular analyses of two fungal hydrogenosomal proteins failed to reveal the existence of a C-terminal peroxisomal targeting sequence and re-examination of the Ultrastructure has clearly demonstrated the existence of two membranes surrounding fungal hydrogenosomes (Benchimol et al. 1997; Brondijk et al. 1996; van der Giezen et al. 1997a; van der Giezen et al. 1997b). Moreover, ciliate and trichomonad hydrogenosomes have also been shown to be surrounded by double membranes (Benchimol and De Souza 1983; Finlay and Fenchel 1989; Paul et al. 1990). Thus, at present, it appears that all examined hydrogenosomes are bounded by a double-membrane, contrary to that observed for microbody-derived organelles. The existence of two surrounding membranes as well as biochemical evidence indicating that trichomonad hydrogenosomes are anaerobic equivalents of mitochondria prompted two hypotheses to explain the origin of this organelle. Both hypotheses proposed an endosymbiotic origin. One took into account the biochemical differences between mitochondria and hydrogenosomes, whereas the other focussed on their similiarities. With regard to their differences, mitochondria lack hydrogenase, the marker enzmye of the hydrogenosome, and pyruvate:ferredoxin oxidoreductase, the hydrogenosomal counterpart to the mitochondrial pyruvate dehydrogenase complex. Furthermore, hydrogenosomes lack cytochromes, a respiratory chain, FoF1 -ATPase activity and DNA (see Johnson et al. 1995; Muller 1993) all of which are present in mitochondria. Based on these differences, one hypothesis proposed that hydrogenosomes arose via an endosymbiosis of an anaerobic bacterium with a eukaryotic host (Muller 1980; Whatley et al. 1979). In this scenario, hydrogenosomes and mitochondria would have evolved entirely independent of each other and
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their similarities would be accounted for by convergent evolution. The second hypothesis proposed that hydrogenosomes arose via the conversion of an established mitochondrion, based on the similiarities of these two organelles and their mutually exclusive appearance (Cavalier-Smith 1987). This hypothesis has since been supported by molecular analyses of a number of hydrogenosomal proteins which showed close homology to their mitochondrial counterparts, such as ferredoxin and succinyl-CoA synthetase (summarized in Johnson et al. 1995). According to this hypothesis, the biochemical differences between hydrogenosomes and mitochondria result from the anaerobic versus aerobic lifestyles of the respective hosts. This is not difficult to envisage as the majority of components that are found in mitochondria but that are absent in hydrogenosomes playa role in respiration, a property that is lacking in the anaerobic environment of the hydrogenosome. Interestingly, it is even possible to account for the lack of DNA in hydrogenosomes based on a lack of respiration. The proteins encoded in the mitochondrial DNA of most contemporary organisms are inner membrane proteins of the respiratory chain (Neupert 1997). As noted previously, since hydrogenosomes do not possess an active respiratory chain in their inner membranes, the maintenance of a mitochondrion-like genome in hydrogenosomes would be unnecessary (Johnson et al. 1993). In the late 80s and early 90s, phylogenetic analyses using rRNA sequences revealed that the lineage that gave rise to trichomonads diverged early from the main line of eukaryotic descent, prior to the advent of mitochondria-containing eukaryotes (Baroin et al. 1988; Sogin 1991). This phylogenetic position for trichomonads is in contrast to other hydrogenosome-containing organisms, such as ciliates and fungi, which evolved much later and belong to the crown group of eukaryotes. Based on the early divergence of the trichomonad lineage, the common origin hypothesis for mitochondria and hydrogenosomes was refined to state that hydrogenosomes and mitochondria evolved from a common progenitor organelle (Johnson et al. 1993) as opposed to the popular notion that mitochondria gave rise to hydrogenosomes. This refined hypothesis implies that the progenitor organelle was present in eukaryotes before the advent of true mitochondria or hydrogenosomes. This review is intended to summarize our current knowledge on the synthesis and targeting of hydrogenosomal proteins and to discuss what these data imply regarding the origin of hydrogenosomes. As almost nothing is known on the biogenesis of ciliate
hydrogenosomes and little on the biogenesis of amoeboflagellate and fungal hydrogenosomes the review focusses on protein import to the hydrogenosome of T. vaginalis which has been studied to a greater extent. Where available, data on amoeboflagellate, ciliate or fungal hydrogenosomal import will be included. Elucidation of the mechanisms underlying organellar targeting in the early diverging trichomonads should help to broaden our understanding of the relationship between hydrogenosomes and mitochondria as well as to learn how protein import to organelles of endosymbiontic origin may have evolved.
Hydrogenosomal Proteins Given that trichomonad hydrogenosomes do not appear to contain DNA (Turner and Muller 1983), apparently due to a complete transfer of their genes into the nucleus of an ancestral host cell, the genes for all hydrogenosomal proteins are encoded in the nucleus. These proteins have been shown to be synthesized in the cytosol on free ribosomes which implies a posttranslational targeting route to their final hydrogenosomal destination (Lahti and Johnson 1991). A comparison of N-terminal sequence analyses of hydrogenosomal proteins and the genes which encode them has invariably shown that hydrogenosomal proteins are synthesized with an N-terminal extension which is not present in the mature protein isolated from the organelle (Bradley et al. 1997; Brondijk et al. 1996; Brul et al. 1994; van der Giezen et al. 1997a). These N-terminal extensions are 5-14 amino acids in length in T. vagina/is hydrogenosomal proteins, 8 amino acids long in the P. /anterna hydrogenosomal protein ferredoxin, and 27 amino acids long in the two Neocallimastix frontalis hydrogenosomal proteins analysed to date (Table 1). A comparison of the N-terminal extensions on all 15 hydrogenosomal proteins shows that they share several characteristics. They all have an Arg at position -2 or -3 relative to position + 1 of the mature protein and all but one have an Asp or Phe at the -1 position. In addition, the N-terminal extensions are generally rich in hydroxylated and hydrophobic amino acids, with a Leu following the start Met in all but two of the 15 hydrogenosomal extensions characterized. Closer examination of a number of these extensions reveals that they are capable of forming amphiphilic a-helices. The presence of these similar N-terminal extensions on newly synthesized hydrogenosomal proteins and their absence on the proteins isolated from the organelles paralleles the situation in mitochon-
Protein Import and Origin of Hydrogenosomes
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Table 1. N-terminal presequences of hydrogenosomal proteins.
Hydrogenosomal protein
Presequence
Start
Ref.
Trichomonas vaginalis: Adenylate kinase Ferredoxin Hsp60 Malic enzyme 1 Malic enzyme 2 Pyruvate:ferredoxin oxidoreductase 1 Pyruvate:ferredoxin oxidoreductase 2 u-Succinyl-CoA synthetase 1 u-Succinyl-CoA synthetase 2 u-Succinyl-CoA synthetase 3 P-Succinyl-CoA synthetase 1 ~-Succinyl-CoA synthetase 53
MLSGVSRN MLSQVCRF MSLlEAAKHFTRAF MLTSSVSVPVRN MLTSVSYPVRN MLRNF MLRSF MLAGDFSRN MLSSSFERN MLSSSFERN MLSASSNFARN MLSSSFARN
AART GTIT AKAR ICRA ICRS SKRV GKRI LKOP LHQP LHQP FNIL FNIL
1 1 1 1 1 1 1 1 1 1 1 1
Psalteriomonas lanterna: Ferredoxin
*MVSGVSRN
AART
2
Neocallimastix frontalis: Malic enzyme ~-Succinyl-CoA synthetase
MLAPIQTIARPVSSILPATGALAAKRT *MLANVTRSTSKAAPALASIAQTAQKRF
FFAP LSVH
3 4
Presequences are defined as sequences encoded by the gene, but absent from the N-terminus upon N-terminal sequencing of the protein isolated from the organelle. *Putative presequence determined by alignment of coding regions of the gene from several organisms. Start indicates the first four amino acids derived from N-terminal sequencing of the protein. References: 1) Bradley et al. 1997,2) Brul et al. 1994,3) van der Giezen et al. 1997a, 4) Brondijk et al. 1996.
dria and chloroplasts and therefore strongly suggests that these extensions may serve as presequences involved in targeting hydrogenosomal proteins to the organelle. To test this hypothesis, an in vitro protein import assay similar to ones used in mitochondrial import research was established with hydrogenosomes of T. vaginalis (Bradley et al. 1997). Using this assay, several parameters were investigated to determine the role of N-terminal extensions in hydrogenosomal protein targeting.
Hydrogenosomal Protein Import The hydrogenosomal in vitro import assay is described in Fig. 1 (for details refer to Bradley et al. 1997). The basic components of the assay are purified organelles, obtained by gradient centrifugation, and a radiolabelled precursor protein. Hydrogenosomal protein import experiments were predominantly carried out using the T. vaginalis hydrogenosomal protein ferredoxin as a precursor, which was simultaneously expressed and radiolabelled in E. coli. Ferredoxin is a 12 kDa hydrogenosomal matrix protein involved in electron transfer (Johnson et al.
1990). In vitro import assays using ferredoxin result in precursor binding to the surface of hydrogenosomes followed by import into the organelle upon which the precursor is cleaved to a size consistent with the mature protein thereby demonstrating the functionality of the assay (Bradley et al. 1997). Import assays carried-out with a protein lacking the 8 amino acid N-terminal extension of ferredoxin clearly show that this protein neither binds to the surface of hydrogenosomes nor is imported and cleaved (Bradley et al. 1997). These results confirm the hypothesis that the N-terminal extension serves as a presequence which is absolutely necessary for protein targeting to hydrogenosomes. As stated above, hydrogenosomal presequences share a number of common features. In particular the occurrence of a Leu directly following the start Met in all but two of the 15 hydrogenosomal presequences analysed so far indicates a potential targeting requirement for Leu at the +2 position. Indeed, a mutational analysis of the ferredoxin presequence in which the Leu was changed to Gly, another nonpolar amino acid, resulted in nearly complete abolition of binding and import of the altered ferredoxin precursor. These data demonstrate an intolerance for
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Plumper, P. J. Bradley, and P. J. Johnson
1. Protein import reaction
0+
----
+
purified 35S-labelled hydrogenosomes precursor
ATP, ATP-regenerating system and T. vaginalis-cytosol
2. Removal of unbound precursor by washing
rr- . . . . . . -
.--M
3. Protease protection assay
~
no protease
@ proteinase K
C
proteinase K + Triton X-100
4. Analysis by SDS-PAGE and f1uorography
Figure 1. Schematic of hydrogenosomal in vitro protein import and protease protection assay. The hydrogenosomal in vitro import assay consists of purified organelles and an 35S-radiolabelled precursor protein incubated in a cytosolic extract of T. vaginalis which has been supplemented with energy in the form of ATP and an ATP regenerating system. After 30 minutes of incubation at 37°C the organelles are washed free of unbound precursor and subdivided into three aliquots for a protease protection assay. One of these aliquots receives the protease proteinase K, the other proteinase K plus the detergent Triton X-100 while the third remains untreated as a control. After 5 minutes of incubation on ice protease digestion is stopped and the samples are subjected to 8DS-PAGE and fluorography to show size and localization of the precursor proteins. The untreated control is predicted to show the total amount of precursor associated with the organelle, inside and outside. On the other hand, proteinase K treatment of the sample results in the degradation of precursor bound to the outside of the organelle, while protein that has become localized inside the organelle should be protected from digestion. The latter is confirmed by destroying the integrity of the organelle with Triton X100 upon which all protein, whether inside or outside the organelle, should become accessible to proteinase K and degraded.
certain amino acids at this position (Bradley et al. 1997). Although Gly is not tolerated at the +2 position of the presequence, amino acids with bulky, hydrophobic side chains can functionally replace Leu in this position. Mutation of the +2 Leu to both lie and Val allows binding and import, albeit at reduced efficiency (Bradley and Johnson, unpublished).
Energy Requirements of Hydrogenosomal Import The translocation of a precursor protein from one side of a membrane to the other requires energy in the forms of temperature, ATP and, where it exists, a transmembrane electrochemical potential (Schatz and Dobberstein 1996). These requirements for translocation of proteins from the cytosol to the matrix of the hydrogenosomes have also been established (Bradley et al. 1997). In hydrogenosomal protein import assays performed at 0 °C, little precursor binding and no import are observed indicating that protein import is an active process. To test for the requirement of ATP, import reactions were depleted of ATP by incubation with the ADP-ATPase apyrase and omittance of the ATP regenerating system. Under these conditions precursor binding occurs but very little protein import can be detected consistent with a requirement of ATP-hydrolysis for protein translocation across membranes. The role ATP plays in translocation and whether ATP hydrolysis is necessary is not known. Based on similarities to other import systems, it is reasonable to predict that cytosolic ATP is required for chaperone-assisted maintenance of an import competent state of the precursor in the cytosol and its translocation through hydrogenosomal membranes. Hydrogenosomal ATP may also be required for refolding of the precursor in the hydrogenosomal matrix upon membrane passage, as demonstrated in detail for proteins that are imported into mitochondria (Horst et al. 1997; Neupert 1997; Pfanner et al. 1997). While the requirements for energy in the forms of temperature and ATP in hydrogenosomal protein binding and import could be directly demonstrated, evidence for the involvement of a hydrogenosomal membrane potential in protein import is more indirect. It was shown that hydrogenosomal protein import is sensitive to m-chlorophenyl-hydrazone (CCCP), a protonophore commonly used to investigate the effect of the absence of an electrochemical membrane potential on cellular processes. Thus a hydrogenosomal transmembrane electrochemical potential seems to be necessary for completion of protein import. It should be noted here that the existence of an electrochemical potential involved in protein translocation across membranes in other systems, especially across the mitochondrial inner membrane, the chloroplast thylakoid membrane and the bacterial cytoplasma membrane, is usually coupled to the existence of electron transport chains and FoF1 -ATPase activity (Bakker and Randall 1984; Neupert 1997; Robinson et al. 1993). Trichomonad hydrogenosomes, however, neither possess mem-
Protein Import and Origin of Hydrogenosomes
branous electron transporters nor an FoF1 -ATPase activity (Johnson et al. 1995; Muller 1993). Nevertheless, a weak electrochemical membrane potential across the hydrogenosomal membranes seems to be maintained (Yarlett et al. 1987), which could be due to ADP-ATP exchange as reported for mitochondrial rho- mutants of yeast (for discussion see Bradley et al. 1997).
Co-factor Requirements of Hydrogenosomal Import The hydrogenosomal in vitro import system contains a T. vaginalis cytosolic fraction. If this fraction is omitted, binding, but no import of precursors occurs, indicating the requirement for factors other than ATP in hydrogenosomal protein import. Nethylmaleimide (NEM) sensitivity of the cytosolic component needed for translocation suggests that at least one of these factors is a protein (Bradley et al. 1997). Transport of newly synthesized proteins to various subcellular locations has been shown to require proteinaceous cytosolic factors (Powers and Forbes 1994). These factors are best characterized in mitochondrial import studies where they have been shown to have two functions. Chaperones such as cytosolic Hsp70 support the loosely folded state of precursors which is necessary for import and to protect precursors from aggregation and degradation (Neupert 1997). Factors involved in targeting such as the mitochondrial import stimulating factor, MSF, exert a more specific function by binding to precursors and guiding them to the translocation machinery (Mihara and Omura 1996). Cytosolic chaperones are not only involved in mitochondrial targeting but also in targeting to many subcellular locations, including the endoplasmic reticulum, the chloroplast and the nucleus (Bukau et al. 1996). Thus, it seems probable that the NEM-sensitive component found in T. vagina/is cytosol acts as a chaperone to assist in specific targeting of proteins to the hydrogenosome. In this regard, it is interesting to note that antisera raised against mouse MSF, the NEM-sensitive cytosolic protein that stimulates the translocation of a subset of mitochondrial matrix proteins, strongly cross-reacts with a T. vagina/is cytosolic protein (our unpublished data).
Hydrogenosomal Protein Import Resembles Mitochondrial Import The data currently available show that protein import to hydrogenosomes occurs posttranslationally,
307
is dependent on the presence of a cleavable N-terminal presequence, cytosolic protein(s) and energy in the forms of temperature, ATP and a transmembrane electrochemical potential. With these characteristics hydrogenosomal protein import most closely resembles protein import into chloroplasts and mitochondria, as opposed to protein translocation across other cellular membranes (Haucke and Schatz 1997; Schatz and Dobberstein 1996). A close examination of mitochondrial and chloroplast presequences reveals significant differences between these targeting signals (von Heijne et al. 1989). In contrast, mitochondrial and hydrogenosomal presequences closely resemble each other. Both types of presequences show a preference for a Leu directly following the start Met, while in chloroplast presequences an Ala is usually found at this position (Keegstra et al. 1989). An Arg is found in position -2 or -10 from the cleavage site in mitochondrial presequences and in position -2 or -3 from the cleavage site in hydrogenosomal presequences (von Heijne et al. 1989). In both types of presequences Arg, Leu and Ser are overrepresented and both have the ability to form amphiphilic a-helices in non-aqueous environments (Johnson et al. 1990; von Heijne 1986). Differences between chloroplast and mitochondrial presequences are indispensable to assure proper targeting to the respective organelle within a common cytosol. As hydrogenosomes and mitochondria are never found within the same cell it could be argued that presequences with the same characteristics evolved independently. However, the following should be considered: i) The unusually short presequences found on T. vaginalis, Parabasalia and P. /anterna, Percolozoa (Weekers et al. 1997) hydrogenosomal proteins closely resemble the short presequences (8-20 amino acids) found on mitochondrial proteins of kinetoplastids (Hausler et al. 1997). In this regard, it is noteworthy that kinetoplastids represent one of the earliest diverging eukaryotes with mitochondria. ii) Fungal hydrogenosomal presequences (27 amino acids) are considerably longer, as are fungal mitochondrial presequences (20-60 amino acids) (Neupert 1997). Thus presequence length seems to increase with increasing position in the eukaryotic tree. The similarity in length of presequences among mitochondria and hydrogenosome-bearing protists of similar systematic position, as well as the similarity in structure and function of presequences, strongly argue for the evolution of protein targeting sequences in a common progenitor organelle of the contemporary hydrogenosomes and mitochondria.
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Phylogenetic Studies Support a Common Origin for Hydrogenosomes and Mitochondria In addition to studies on hydrogenosomal protein import which support a common evolutionary origin for hydrogenosomes and mitochondria, further support for this hypothesis has been provided by molecular analyses of T. vaginalis hydrogenosomal heat shock proteins (Bui et al. 1996; Germot et al. 1996; Horner et al. 1996; Roger et al. 1996). Work from our laboratory has shown that T. vaginalis hydrogenosomes contain both Hsp70 and Hsp60, proteins that are otherwise found only within mitochondria or chloroplasts in eukaryotic cells (Bui et al. 1996). Moreover, we and others have sequenced and characterized T. vaginalis genes encoding Hsp70 (Bui et al. 1996; Germot et al. 1996), Hsp60 (Bui et al. 1996; Horner et al. 1996; Roger et al. 1996) and Hsp10 (Bui et al. 1996). These hydrogenosomal genes were chosen for phylogenetic analyses as they have been quite useful for determining bacterial phylogeny (eg. Gupta et al. 1994). The presence of Hsp70 and Hsp60 proteins in the hydrogenosome and the fact that both proteins are encoded by a single-copy gene indicates that the specific genes examined were originally derived from the organelle. The results of the phylogenetic analyses obtained using these gene sequences were striking. A variety of inference techniques consistently put hydrogenosomal Hsp70, Hsp60 and Hsp10 on the lineage that contains mitochondria (Bui et al. 1996; Germot et al. 1996; Horner et al. 1996; Roger et al. 1996). It should be noted that these analyses invariably included chloroplast Hsp sequences and in no instance did the T. vaginalis Hsps group with chloroplast homologues. These data provide strong evidence that hydrogenosomes and mitochondria have a common evolutionary origin. Interestingly, similar phylogenetic analyses on Hsp genes isolated from three protist lineages that lack both mitochondria and hydrogenosomes, entamoebids (Clark and Roger 1995), microsporidia (Germot et al. 1997) and diplomonads (Roger et al. 1998) also places these gene sequences in the clade composed of mitochondrial Hsps. This indicates that these organisms once contained an endosymbiont, most likely the one that contributed to the formation of mitochondria and hydrogenosomes, which was ultimately lost from the lineages that gave rise to Entamoeba, Nosema and Giardia. These recent studies on the origin of hydrogenosomes and the phylogeny of Hsps in Giardia and Entamoeba, as well as the observation that hydrogenosomes in free living ciliates are often found
in an intimate connection with endosymbiotic methanogens, recently culminated in a new hypothesis on eukaryote beginnings (Martin and Muller 1998). This hypothesis, coined the hydrogen hypothesis, takes into account the archaeal origin of the nuclear apparatus (Gupta and Golding 1996; Lake and Rivera 1994) versus the eubacterial origin of the core metabolism found in mitochondria, hydrogenosomes and the cytosol of eukaryotes lacking both hydrogenosomes and mitochondria. The hydrogen hypothesis states that the first eukaryote arose via a union between a methanogenic archaea which absolutely depended on molecular hydrogen for its metabolism and a eubacterium that evolved vast amounts of hydrogen under anaerobic conditions but was also able to respire. After the union was established and gene transfer from the eubacterium to the archaea had occurred, the primitive organelle of eubacterial origin was either lost as in the eukaryotes devoid of either mitochondria or hydrogenosomes or developed into hydrogenosomes under anaerobic conditions and into mitochondria under aerobic conditions. This hypothesis also takes into account structural and biochemical similarities between hydrogenosomes and mitochondria, such as the existence of two bounding membranes, the modus of division by binary fission (Benchimol et al. 1996) and the related enzymes involved in pyruvate degradation found in both organelles. By assuming that the eubacterium was able to live anaerobically as well as aerobically, it is proposed to have possessed both pyruvate: ferredoxin oxidoreductase and the pyruvate dehydrogenase complex. It is also proposed to have contained both hydrogenase and a respiratory chain, as would be predicted if a single endosymbiotic event gave rise to contemporary mitochondria and hydrogenosomes. The hydrogen hypothesis is elegantly simple and much of the evidence upon which it is based is solid. Clearly, compelling evidence exists that hydrogenosomes and mitochondria share a common ancestral history. However, strong evidence also exists indicating that the establishment of endosymbiotic relationships and horizontal gene transfer is a relatively frequent event (Hackstein et al. 1997). Thus, it is perhaps naive to envisage that all genes encoding proteins which are imported into either mitochondria or hydrogenosomes were derived from this single endosymbiotic event. As additional properties of the contemporary versions of these organelles are discovered, we may find that additional endosymbiotic and/or gene transfer events have contributed to the evolutionary history of these complex organelles.
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Future Prospects
anaerobic fungus Neocallimastix frontalis. FEMS Microbioi Lett 154: 277-282
To broaden our understanding of the relationship between hydrogenosomes and mitochondria it will be necessary to obtain additional information on the hydrogenosomal protein translocation machinery. In particular, comparison of hydrogenosomal import receptors and proteinaceous membrane pores to those found in mitochondria should be informative. In addition it will be interesting to learn more about other protein factors involved in hydrogenosomal protein targeting, such as cytosolic co-factors that stimulate translocation and the putative matrix peptidase. The evolutionary relationship between hydrogenosomes found in ciliates, fungi and flagellates presents a fundamental question for which there is little data. Characterization of hydrogenosomal protein import and phylogenetic analyses of proteins found in the organelles of these highly diverged species should shed light on this issue. Moreover, given the early divergence of trichomonads from the main trunk of eukaryotic evolution, information on T. vagina/is hydrogenosomal protein targeting may help elucidate how protein targeting itself evolved in the dawn of eukaryotic life.
Benchimol M, Johnson PJ, de Souza W (1996) Morphogenesis of the hydrogenosome: an ultrastructural study. Bioi Cell 87: 197-205
Acknowledgements The authors are grateful to Dr. Eckhard Meyer for helpful comments on the manuscript. This work was supported by National Institutes of Health grant AI27857 to PJJ, a postdoctoral fellowship (PI 218/11) from the Deutsche Forschungsgemeinschaft to EP and a predoctoral training grant (NIH A107323) to PJB. PJJ is a recipient of a Burroughs-WeUcome Scholar in Molecular Parasitology award.
Bradley PJ, Lahti CJ, Plumper E, Johnson PJ (1997) Targeting and translocation of proteins into the hydrogenosome of the protist Trichomonas: similarities with mitochondrial protein import. EMBO J 16: 3484-3493 Brondijk TH, Durand R, van der Giezen M, Gottschal JC, Prins RA, Fevre M (1996) scsB, a cDNA encoding the hydrogenosomal beta subunit of succinyl-CoA synthetase from the anaerobic fungus Neocallimastix frontalis. Mol Gen Genet 253: 315-323 Brul S, Veltman RH, Lombardo MC, Vogels GO (1994) Molecular cloning of hydrogenosomal ferredoxin cDNA from the anaerobic amoeboflagellate Psalteriomonas lanterna. Biochim Biophys Acta 1183: 544-546 Bui ET, Bradley PJ, Johnson PJ (1996) A common evolutionary origin for mitochondria and hydrogenosomes. Proc Natl Acad Sci USA 93: 9651-9656 Bukau B, Hesterkamp T, Luirink J (1996) Growing up in a dangerous environment: a network of multiple targeting and folding pathways for nascent polypeptides in the cytosol. Trends Cell Bioi 6: 480-486 Cavalier-Smith T (1987) The simultaneous symbiotic origin of mitochondria, chloroplasts, and microbodies. Ann NY Acad Sci 503: 55-71 Clark CG, Roger AJ (1995) Direct evidence for secondary loss of mitochondria in Entamoeba histolytica. Proc Natl Acad Sci USA 92: 6518-6521 Finlay BJ, Fenchel T (1989) Hydrogenosomes in some anaerobic protozoa resemble mitochondria. FEMS Microbiol Lett 65: 311-314
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Evelyn Plumper", Peter J. Bradleyb, and Patricia J. Johnson c ,1 alnstitut fOr Allgemeine Zoologie und Genetik, Schlossplatz 5, D - 48149 MOnster, Germany bDepartment of Microbiology and Immunology, Stanford University, Stanford, CA 94305, USA cDepartment of Microbiology and Immunology, University of California at Los Angeles, Los Angeles, CA 90095, USA 1Corresponding author; fax 1-310-206-5231 e-mail [email protected]