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Eukaryotic Cell, April 2006, p. 784-787, Vol. 5, No. 4
1535-9778/06/$08.00+0     doi:10.1128/EC.5.4.784-787.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Cardiolipin in Hydrogenosomes: Evidence of Symbiotic Origin

Ivone de Andrade Rosa,¹ Marcelo Einicker-Lamas,² Róbson Roney Bernardo,³ Lucia Mendonça Previatto,² Ronaldo Mohana-Borges,² José Andrés Morgado-Díaz,⁴ and Marlene Benchimol^1*

Laboratório de Ultraestrutura Celular, Universidade Santa Úrsula, Rio de Janeiro, Brazil,¹ Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil,² Faculdade de Farmácia, Universidade Estácio de Sá, Rio de Janeiro, Brazil,³ INCA-Grupo de Biologia Estrutural, Divisão de Biologia Celular, Instituto Nacional de Cncer, Rio de Janeiro, Brazil⁴

Received 18 November 2005/ Accepted 18 January 2006

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Hydrogenosomes are found in organisms that lack typical mitochondria. Cardiolipin is a phospholipid located exclusively in bacterial membranes and the inner membrane of mitochondria. Here we show, by cell fractionation, thin-layer chromatography, high-pressure liquid chromatography, and matrix-assisted laser desorption ionization-time of flight mass spectrometry that hydrogenosomes of Tritrichomonas foetus, a cattle vaginal parasite, contain cardiolipin, which is strong evidence for its endosymbiotic origin.

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The hydrogenosome is an unusual organelle found in several trichomonad species and other protists living in low-oxygen or even anoxic environments. The hydrogenosome-containing microorganisms do not present typical mitochondria (11, 18, 20). Interestingly, this organelle shares some biochemical similarities with mitochondria; for example, hydrogenosomes contain enzymes that play roles in pyruvate metabolism and ATP synthesis.

The origin of the hydrogenosome has been the subject of intense discussion, since it also shares some structural and morphological features with mitochondria; for example, it is enveloped by two membranes (2), divides autonomously by fission (3), imports proteins posttranslationally (16), and produces ATP (18). In addition, targeting and translocation of proteins into the hydrogenosomes present similarities to mitochondrial protein import (4), and a member of the mitochondrial carrier family was found in hydrogenosomes (9). However, there is also evidence that hydrogenosomes and mitochondria are not so close in terms of their origin. Some of these differences are the lack of a genome, with the possible exception of hydrogenosomes from Nyctotherus ovalis (1), and the lack of a respiratory chain, cytochromes, the F_0-F₁ ATPase, the tricarboxylic acid cycle, and oxidative phosphorylation (6, 20).

At least two different hypotheses have been proposed for the origin of the trichomonad hydrogenosome: independent endosymbiosis of an ancestral anaerobic eubacterium with a eukaryotic host (19) and conversion of an established mitochondrion which was adapted to an anaerobic lifestyle (12). The common hypothesis for mitochondrial and hydrogenosome origin was refined to state that both organelles evolved from a common progenitor structure present in eukaryotes before the advent of true mitochondria or hydrogenosomes (10).

Cardiolipin is a typical component of the bacterial cytoplasmic membrane and the inner mitochondrial membrane. Cardiolipin is unique among phospholipids because it consists of two molecules of phosphatidic acid linked by a glycerol. It has long been associated with proteins that conduct oxidative phosphorylation. In eukaryotes it is the only lipid that is synthesized in the mitochondrion. In addition, it is almost exclusively present in membranes designed to generate an electrochemical potential gradient for ATP synthesis, including the mitochondrial inner membrane and bacterial plasma membrane (17). Cardiolipin is also called diphosphatidylglycerol and plays several other functions in mitochondria, such as a structural and functional role in many multimeric complexes associated with mitochondrial membranes. Cardiolipin also participates in protein-protein contacts, resulting in the formation of highly ordered membrane structure. It also has a specific role in the import of proteins into mitochondria, and it binds in a highly specific way to the DNA in chromatin. Thus, cardiolipin appears to have a functional role in the regulation of gene expression.

Hydrogenosomes do not present oxidative phosphorylation, membrane respiratory complexes, or DNA. However, cardiolipin could participate in trichomonads in other processes, such as membrane structure, import of proteins, or apoptosis, as previously described for mitochondria (23).

The presence of cardiolipin in hydrogenosomes would be an additional argument for the hypothesis of greater similarity to mitochondria and the symbiotic origin of this organelle. The endosymbiotic event in an -proteobacterium that gave rise to mitochondria and related organelles is of great interest because this event might represent the moment of origin of the eukaryotic cell itself (8, 11, 12). Cerkasovová et al. (5) published that the hydrogenosomes of Tritrichomonas foetus contained cardiolipin, detected by thin-layer chromatography (TLC) and quantitative phosphorus measurement. However, this finding was subsequently refuted by Paltauf and Meingassner (21). These authors reported that cardiolipin was absent from both T. foetus and Trichomonas vaginalis, which led them to state that there were no evolutionary connections between hydrogenosomes and mitochondria, thus somehow frustrating the endosymbiotic hypothesis for the origin of this organelle.

Since contradictory evidence for the presence of this lipid in hydrogenosomes has been raised, we decided to reexamine the presence of cardiolipin in isolated hydrogenosomes using a more sensitive methodology.

A highly purified hydrogenosomal fraction was obtained from T. foetus following the protocol of Díaz and De Souza (7) and incubated in 10-N-nonyl-acridine orange, a fluorescent cardiolipin marker (13). It was analyzed by fluorescence microscopy, and positive staining was found in structures resembling hydrogenosomes by their localization in the cell, size, and shape (not shown). Thin-layer chromatography (TLC) was performed using lipid extracts obtained from whole-cell homogenates of T. foetus as well as mitochondrial and hydrogenosomal fractions according to the method described by Horwitz and Perlman (15). It was observed that a representative TLC plate after exposure to iodine vapors (Fig. 1) showed all the major phospholipids (phosphatidylserine, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine) were present in all the samples tested.

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FIG. 1. TLC for cardiolipin detection. Thin-layer chromatograph of lipid extracts. Lanes: H, hydrogenosomal fraction of T. foetus; Tf, cell homogenate of T. foetus; Std, commercial cardiolipin standard; M, mitochondrial fraction obtained from Crithidia deanei. CLP, cardiolipin; O, origin.

The cardiolipin spot was easily identified in the lipid extract derived from mitochondria, although it was very weak in T. foetus after iodine vapor exposure. We needed to confirm the identity of the lipid obtained from T. foetus and the hydrogenosome fraction as cardiolipin. For this reason, the spot identified as cardiolipin in all samples was analyzed by high-pressure liquid chromatography (HPLC). Figure 2A shows that the cardiolipin spots identified in the whole-cell and hydrogenosome fractions were really cardiolipin, as judged from their having the same retention times after HPLC analyses.

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FIG. 2. A. HPLC for cardiolipin detection. Lipid samples eluted from TLC plates were analyzed by HPLC. A commercial cardiolipin standard (Avanti Polar Lipids) was used as the reference. a. HPLC profile of the cardiolipin derived from whole-cell homogenates of T. foetus (see text). The retention time was the same for both the sample lipid and the commercial standard. b. HPLC profile of the cardiolipin derived from the purified hydrogenosomal fraction. The spot corresponding to cardiolipin (CLP), identified by its relative mobility in the solvent system used, was scraped from the TLC plate and cardiolipin was extracted from the silica after three washes with chloroform. The cardiolipin-containing sample was dried under N2 and used for HPLC and mass spectrometry analysis. B. Negative-mode MALDI-TOF mass spectrometry spectrum of purified cardiolipin from T. foetus. The mass spectra were acquired in a Voyager DE-PRO MALDI-TOF spectrometer (Applied Biosystems). The experiments were carried out in negative reflected mode at 20,000 V to ionize the samples and using 250 scans for each spectrum. Cardiolipin (1,472 Da) from Avanti Polar Lipids was used as the standard. Both the sample and the standard were dissolved in chloroform and mixed with 5% norhormane (dissolved in 50% acetonitrile and methanol) in a 1:1 ratio before application to the plate. The spectra were processed using the package program Data Explorer version 4.4 (Applied Biosystems). The m/z of 1,469.9464 corresponds to 3 glycerol, 2 phosphate, and 4 linoleic acid groups plus 1 sodium ion. The m/z of 1,448.4124 corresponds to the same compound without the sodium ion.

When we coinjected the lipid obtained from our samples with the commercial cardiolipin standard, we detected a single peak after HPLC analysis (data not shown). In agreement with the chromatographic analysis, the negative-ion matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrum of the purified cardiolipin contained two major peaks, one [M+Na⁺-2H]^– at m/z 1,469.9464 and the other [M-H]^– at m/z 1,448.4124 (Fig. 2B). These pseudomolecular ions are consistent with diphosphatidylglycerol (cardiolipin) composed of 3 glycerol, 2 phosphate, and 4 linoleic acid groups.

As the T. vaginalis genome project is almost complete and available, we decided to search for biosynthetic enzymes such as cardiolipin synthase in the trichomonad genome. Similar sequences were found in the trichomonad genome with a significant degree of homology to those of Candida albicans, Saccharomyces cerevisiae, and Cryptococcus neoformans.

Together, these results provide evidence that the hydrogenosomes of T. foetus contain cardiolipin, a typical mitochondrial and bacterial membrane lipid. The presence of cardiolipin argues for an endosymbiotic origin of hydrogenosomes. The localization of the heat shock proteins Hsp60 and Hsp70 in Trichomonas hydrogenosomes (14) provided the first strong data that the organelle itself shared a common ancestry with mitochondria. In addition, Trichomonas hydrogenosomes, like mitochondria, divide by segmentation and partition (3). The discovery of homologues of the mitochondrial complex in Trichomonas spp. added an important piece to the hydrogenosome evolution hypothesis (11).

Recently, Dolezal et al. (8) identified proteins related to the translocase in Giardia and Trichomonas in the mitochondrial inner membrane. In addition, Sutak et al. (22) found a mitochondrial-type assembly of FeS centers in the hydrogenosomes of T. vaginalis. These findings led these authors to the hypothesis that mitosomes, hydrogenosomes, and mitochondria may represent different forms of the same fundamental organelle.

Much progress has been made in the past few years in reconstructing the history of mitochondria and hydrogenosomes. It is now apparent that these organelles shared a common ancestor at some point. Some groups have suggested that mitochondria and hydrogenosomes are two forms of the same fundamental organelle (12).

Here, with the use of more sensitive approaches and analytical techniques, we clearly demonstrate that cardiolipin is present in hydrogenosomes from T. foetus, which sheds new light on the discussion of the endosymbiotic origin of hydrogenosomes.

 
This work was supported by CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), PRONEX (Programa de Núcleo de Excelência), FAPERJ (Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro), and AUSU (Associação Universitária Santa Úrsula).

 
* Corresponding author. Mailing address: Rua Jornalista Orlando Dantas, 59, CEP 222-31-010, Botafogo, Rio de Janeiro, Brazil. Phone and fax: 55-21-2553-1615. E-mail: marleneben{at}uol.com.br.

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Eukaryotic Cell, April 2006, p. 784-787, Vol. 5, No. 4
1535-9778/06/$08.00+0     doi:10.1128/EC.5.4.784-787.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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• Schlame, M. (2008). Thematic Review Series: Glycerolipids. Cardiolipin synthesis for the assembly of bacterial and mitochondrial membranes. J. Lipid Res. 49: 1607-1620 [Abstract] [Full Text]  

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Reprints and Permissions Copyright Information Books from ASM Press MicrobeWorld Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by de Andrade Rosa, I. Articles by Benchimol, M. Search for Related Content PubMed PubMed Citation Articles by de Andrade Rosa, I. Articles by Benchimol, M.