Download Protein Import, Replication, and Inheritance of a Vestigial

THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 280, No. 34, Issue of August 26, pp. 30557–30563, 2005
Printed in U.S.A.
Protein Import, Replication, and Inheritance of a
S
Vestigial Mitochondrion*□
Received for publication, January 21, 2005, and in revised form, April 18, 2005
Published, JBC Papers in Press, June 28, 2005, DOI 10.1074/jbc.M500787200
Attila Regoes‡§, Danai Zourmpanou§¶, Gloria León-Avila¶储, Mark van der Giezen¶**,
Jorge Tovar ¶‡‡, and Adrian B. Hehl‡§§
From the ‡Institute of Parasitology, University of Zürich, Winterthurerstrasse 266a, CH-8057 Zürich, Switzerland and the
¶School of Biological Sciences, Royal Holloway University of London, Egham Hill, Egham TW20 0EX, United Kingdom
Mitochondrial remnant organelles (mitosomes) that
exist in a range of “amitochondrial” eukaryotic organisms represent ideal models for the study of mitochondrial evolution and for the establishment of the minimal
set of proteins required for the biogenesis of an endosymbiosis-derived organelle. Giardia intestinalis, often
described as the earliest branching eukaryote, contains
double membrane-bounded structures involved in ironsulfur cluster biosynthesis, an essential function of mitochondria. Here we present evidence that Giardia mitosomes also harbor Cpn60, mtHsp70, and ferredoxin
and that despite their advanced state of reductive evolution they have retained vestiges of presequence-dependent and -independent protein import pathways
akin to those that operate in mammalian mitochondria.
Although import of IscU and ferredoxin is still reliant on
their amino-terminal presequences, targeting of Giardia Cpn60, IscS, or mtHsp70 into mitosomes no longer
requires cleavable presequences, a derived feature from
their mitochondrial homologues. In addition, we found
that division and segregation of a single centrally positioned mitosome tightly associated with the microtubular cytoskeleton is coordinated with the cell cycle,
whereas peripherally located mitosomes are inherited
into daughter cells stochastically.
Highly derived but functional mitochondrial remnant organelles (mitosomes) are found in “amitochondrial” eukaryotes
as diverse as Entamoeba histolytica, Trachipleistophora hominis, Cryptosporidium parvum, Blastocystis hominis, and Giardia intestinalis (1–5). Although able to metabolize small
amounts of oxygen, these organisms live mostly in oxygendeprived environments and have lost their capacity for ATP
biosynthesis linked to oxidative phosphorylation, one of the
main functions of aerobic mitochondria (6). Investigating the
biology of these vestigial organelles is thus central to our un* This work was supported in part by Swiss National Science Foundation Grant 3100A0-100270 (to A. B. H.) and by Wellcome Trust Grant
059845 (to J. T.). The costs of publication of this article were defrayed in
part by the payment of page charges. This article must therefore be
hereby marked “advertisement” in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
□
S The on-line version of this article (available at http://www.jbc.org)
contains four supplemental tables.
§ Both authors contributed equally to this work.
储 Present address: Departamento de Genética y Biologı́a Molecular,
CINVESTAV IPN, 07360 México DF, México.
** Present address: School of Biological Sciences, Queen Mary University of London, Mile End Rd., London E1 4NS, United Kingdom.
‡‡ To whom correspondence may be addressed. Tel.: 44-1784-414159;
Fax: 44-1784-434326; E-mail: [email protected].
§§ To whom correspondence may be addressed. Tel.: 41-44-635-8526/
30; Fax: 41-44-635-8907; E-mail: [email protected].
This paper is available on line at http://www.jbc.org
derstanding of mitochondrial evolution and of the minimal set
of proteins required for the biogenesis and inheritance of an
endosymbiosis-derived organelle (7–9).
Giardia lamblia is a unicellular protozoan parasite of the
small intestine in vertebrates and a leading cause of diarrheal
disease worldwide (10). It may also represent the most basal
branch of eukaryotic evolution, although its phylogenetic placing remains controversial (11–13). The presence of a poorly
developed endomembrane system and its unusual genomic organization make Giardia an ideal model for the study of early
genome organization and cell biology (14, 15). Indirect evidence
that Giardia once harbored a mitochondrion (16) appeared to
be confirmed by the discovery of small (⬃100 nm) double membrane-bounded organelles distributed throughout the cytoplasm that harbor enzymes that participate in iron-sulfur cluster assembly, an essential function of mitochondria (3, 17).
However, attempts to localize the mitochondrial marker Cpn60
using heterologous anti-Cpn60 antibodies proved controversial,
as the observed label did not seem to be associated with biological membranes in electron microscopy micrographs and did
not co-localize with Isc proteins in confocal microscopy images
(3, 18). These observations were inconsistent with the postulated mitochondrial ancestry of mitosomes.
Here, we have used specific homologous antibodies and
epitope-tagged mitochondrial proteins conditionally expressed
in transgenic parasites to demonstrate unequivocally that the
key mitochondrial marker proteins Cpn60, mtHsp70, ferredoxin (Fd),1 IscS, and IscU reside in Giardia mitosomes. In vivo
organelle targeting experiments showed that only IscU and Fd
require the presence of amino-terminal targeting presequences, which are cleavable and functionally conserved with
those of mammalian mitochondria. Finally, we found that a
single central mitosome actively divides and segregates during
mitosis, whereas peripheral organelles are not coordinately
replicated and segregate into daughter cells stochastically.
EXPERIMENTAL PROCEDURES
Parasite Cultivation and Transfection—Trophozoites of the G. lamblia strain WB were grown in TYI-S-33 medium supplemented with
10% adult bovine serum and bovine bile as described previously (3, 59).
Incubations with nocodazole or cytochalasin D (10 ␮g/ml) added as
100⫻ stock solutions in Me2SO to normal growth medium were done for
up to 2 h at 37 °C. Parasites were harvested by chilling culture tubes on
ice for 30 min to detach adhering cells and centrifugation at 800 ⫻ g.
Transgenic Giardia lines were generated by electroporation in the
presence of indicated plasmid DNA and appropriate drug selection (60).
Construction of Expression Vectors—Standard recombinant DNA
techniques were used for nucleic acid preparation and analysis. For full
details on the construction of conditional and constitutive recombinant
1
The abbreviations used are: Fd, ferrodoxin; PBS, phosphate-buffered saline; HA, hemagglutinin; GFP, green fluorescent protein; PM,
peripheral mitosome; CM, central mitosome.
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Characterization of Giardia Mitosomes
expression vectors used in this study see supplemental data.
Cultivation and Transfection of Mammalian Cells—Human embryonic kidney 293 cells were maintained in Dulbecco’s modified Eagle’s
medium supplemented with 10% fetal calf serum at 37 °C in a humidified 5% CO2 atmosphere. Cells of 20 –30% confluence in 6-well plates
were transfected with plasmid DNA using FuGENE 6 transfection
reagent (Roche Applied Science) according to the manufacturer’s protocol. Transfected cells were incubated for 18 h and subsequently
treated with 0.25% trypsin in TDE buffer (136 mM NaCl, 50 mM KCl, 50
mM EDTA, 25 mM Tris, pH 7.5) for 2 min, detached by resuspension,
centrifuged at 500 ⫻ g for 10 min, and resuspended in 1.2 ml of fresh
Dulbecco’s modified Eagle’s complete medium. Cells (8.0 ⫻ 104) were
applied on each glass slide and attached to the surface for 5 h at 37 °C
in a 5% CO2 incubator. Following removal of medium and addition of
500 ␮l of fresh medium containing 125 nM MitoTracker Red (Molecular
Probes), slides were incubated at 37 °C for 15 min, washed twice in
PBS, fixed in 4% paraformaldehyde for 30 min at 37 °C, and processed
for microscopy as described below.
Laser Scanning Confocal Immunofluorescence Microscopy—After
treatments, human embryonic kidney 293 cell-containing slides were
washed five times in PBS and mounted with VectaShield mounting
medium. Slides were observed under a scanning confocal microscope
(Radiance 2100; Bio-Rad). Images were collected using LaserSharp
software and further processed with Adobe Photoshop 7.0. In the case of
parasites, chilled cells were harvested as described above, washed with
ice-cold PBS, and fixed with 3% formaldehyde for 40 min at room
temperature, followed by a 5-min incubation with 0.1 M glycine in PBS.
Fixed cells were permeabilized with 0.2% Triton X-100 in PBS for 20
min and blocked ⬎2 h in 2% bovine serum albumin in PBS. Incubations
with titrated primary or secondary antibodies were performed in 2%
bovine serum albumin/0.2% Triton X-100 in PBS. Cells were washed
with PBS between incubations and embedded with Vectashield containing DAPI (Vector Labs). Image stacks were collected on a Leica SP2
AOBS confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany) using the appropriate settings. Color merging was done
using the Leica software or the Metaview software package (Visitron
Systems, Puchheim Germany). In some cases images were further
processed using the Imaris software (Bitplane) in conjunction with the
Huygens package for deconvolution.
Production of Polyclonal Antisera in Mice—Polyclonal antisera
against GiCpn60 were produced as described previously (20). For details see supplemental data.
RESULTS
Localization of Cpn60 and mtHsp70 Corroborates the Endosymbiotic Origin of the Relict Organelles of Giardia—The cellular localization of mitochondrial marker proteins such as
Cpn60 and mtHsp70 has not been demonstrated unequivocally
in Giardia. The apparent extra-mitosomal distribution of
Cpn60, as determined by confocal microscopy with heterologous anti-Cpn60 antibodies, is highly controversial (3, 18, 19).
We have used specific homologous antibodies raised against a
bacterially expressed GiCpn60 fusion protein and ectopic expression of an epitope-tagged variant of Cpn60 (Cpn60-HA) to
establish the cellular distribution of this protein in parasite
trophozoites by confocal immunofluorescence microscopy (IFA).
Mouse sera that reacted specifically with a single band of the
predicted size in Western blots of crude Giardia extracts (Fig.
1A) showed typical staining for Giardia mitosomes in confocal
micrographs, i.e. 40 –50 small spherical signals distributed
throughout the cytoplasm and a distinct rod-like signature
signal associated with the basal body complex in all cells (Fig.
1B). Co-localization of Cpn60 with the mitosomal marker protein GiIscS using homologous antibodies demonstrated that
this mitochondrial marker protein is indeed targeted into Giardia mitosomes (Fig. 1C). Moreover, conditional ectopic expression of hemagglutinin (HA)-tagged Cpn60 (Cpn60-HA)
(Fig. 1D) and of mtHsp70-HA (Fig. 1E) mimicked the distribution of native Cpn60 and IscS, providing further confirmation
that all these mitochondrial marker proteins are targeted
into mitosomes.
To ascertain that mitosomal marker proteins were not present in other membrane compartments, we performed co-local-
FIG. 1. Giardia Cpn60 is a mitosomal protein. A, Western blot of
Giardia Triton X-100 soluble proteins separated by SDS-PAGE and
probed with a polyclonal antibody against recombinant GiCPN60 revealed a single band of ⬃60 kDa. Relative masses of marker proteins
are indicated in kilodaltons. B–G, subcellular localization of endogenous and recombinant mitosome matrix proteins by immunofluorescence. Nuclear DNA is stained with DAPI and appears blue. All micrographs were produced with a confocal laser scanning microscope. Scale
bars are 2 ␮m. B, giardial Cpn60 (red) detected by the anti-Cpn60
antibody shows the typical distribution of mitosome proteins, including
labeling of the elongated organelle associated with the basal body
complex (arrowhead). C, co-localization of GiCpn60 (green) and the
mitosome marker IscS (red). D and E, ectopically expressed Cpn60-HA
or GimtHSP70 co-localize with IscS in mitosomes. F and G, mitosomes
do not overlap with the two other major membrane compartments, the
peripheral vesicles and the endoplasmic reticulum. Peripheral vesicles
are defined by peripherally associated clathrin; the endoplasmic reticulum is labeled with antibodies against a giardial protein disulfide
isomerase 2.
ization studies using antibodies against established markers
for peripheral vesicles (clathrin, GiCLH) (Fig. 1F) and endoplasmic reticulum (protein disulfide isomerase 2, GiPDI2) (Fig.
1G). Analysis by confocal microscopy confirmed that mitosomes
are independent organelles not related to the endoplasmic reticulum or the endosomal-lysosomal membrane system (20).
These data, together with the phylogeny of the giardial Cpn60,
mtHsp70, and IscS (16, 21–24), strongly support the mitochondrial ancestry of Giardia mitosomes.
Conserved and Diverged Features of Protein Import Pathways in Giardia Mitosomes—In contrast to their homologues
from other eukaryotes, giardial Cpn60, mtHsp70, and IscS lack
Characterization of Giardia Mitosomes
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FIG. 2. Analysis of protein import into mitosomes by confocal
microscopy. A, subcellular localization of recombinant IscU-HA. B,
the identical protein lacking a pre-peptide (⌬p-IscU-HA) was completely
excluded from mitosomes and remained in the cytoplasm. Excess cytoplasmic protein associates also with cytoskeletal elements in an unspecific manner. C, conversely, deletion of 5 N-terminal amino acids in
GiCpn60 (⌬N-Cpn60-HA) does not abolish import. Single confocal sections and three-dimensional reconstructed images from entire stacks
are shown. Scale bars are 2 ␮m.
putative amino-terminal targeting presequences (16, 21–23).
Their mitosomal localization, however, suggests that these proteins are imported into the organelle via non-cleavable targeting signals that may be equivalent to those from a number of
luminal mitochondrial proteins such as oxoacyl-CoA thiolase
and chaperonin 10 (25–27). In contrast, putative organelle targeting presequences have been identified in two Giardia proteins, IscU and Fd, but their functionality has not been demonstrated (3, 28). We tested the requirement for these putative
targeting presequences using tagged variants of IscU and Fd
fused either to HA or to green fluorescent protein (GFP). Removal of amino acids 2–27 (Thr2-Glu27) from GiIscU-HA led to
the accumulation of truncated protein in the cytoplasm and
was completely excluded from mitosomes (Fig. 2, A and B).
Similarly, deletion of amino acids 2–26 (Thr2-Asp26) from
GiIscU䡠GFP and of amino acids 2–18 (Ser2-Gln18) from
GiFd䡠GFP led to accumulation of protein in the cytosolic fraction (Fig. 3A). Deletion of amino acids 2–5 (Leu2-Thr6) from the
amino terminus of Cpn60-HA, which contains no predicted
targeting presequence and is only 2 amino acids longer than
that of its bacterial homologues (16), had no effect on its mitosomal localization (Fig. 2C). Together, these data demonstrate
that presequence-dependent and -independent protein import
pathways still operate in Giardia mitosomes, despite their
advanced state of reductive evolution.
Processing of IscU and Fd Amino-terminal Presequences—A
functional feature of mitochondrial and hydrogenosomal protein import is the proteolytic removal of targeting peptides
upon organelle import (29, 30). To investigate whether the
amino-terminal peptides of Giardia IscU and Fd are cleaved
upon import into mitosomes, the initial 26 and 18 amino acids
of IscU (Met1-Asp26) and of Fd (Met1-Gln18), respectively, were
fused to the passenger protein GFP. Transfection of these constructs into Giardia trophozoites resulted in very high levels of
GFP expression, which appeared evenly distributed through-
FIG. 3. Processing of the IscU and Fd amino-terminal presequences and their requirement for organelle import. The subcellular distribution of IscU and of indicated GFP fusion proteins in
Giardia trophozoites was monitored by confocal microscopy and Western blotting using specific antibodies against GFP and GiIscU. A, removal of the IscU organelle targeting signal redirects the IscU䡠GFP
fusion protein from mitosomes (upper panel) to the cytosol (middle
panel); similarly, signal-less Fd accumulates in the cytosol. B, aminoterminal presequences from IscU and Fd both target GFP into the
organellar fraction. The great majority of precursor proteins remain in
the cytosol, whereas mature protein (27 kDa) is found exclusively in the
organellar fraction. C, confocal image showing that IscU is targeted into
mitosomes in Giardia trophozoites (central panel). IscU partitions
mostly to the mixed membrane fraction where the 17-kDa precursor
(arrow) and the ⬃14.4-kDa mature (asterisk) proteins are observed.
Blots in panels A and B were probed with an anti-GFP monoclonal
antibody; blot in panel C was probed with a specific anti-GiIscU polyclonal antibody. The size of molecular markers in kilodaltons is shown
on the right. MMF, mixed membrane fraction sedimentable at highspeed; C, cytosolic fraction. Green box, coding region of the GFP gene;
gray box, sequence encoding the amino-terminal peptide of IscU; orange
box, sequence encoding the amino-terminal peptide of Fd.
out the cell in confocal images (not shown). However, cell fractionation experiments clearly demonstrated the accumulation
of the full-length 5⬘-IscU䡠GFP and 5⬘-Fd䡠GFP fusion proteins in
the cytosolic fraction, whereas mature GFP, which co-migrated
with GFP from control parasite lines, was found exclusively in
the organellar fraction (Fig. 3B). Such distribution of precursor
and processed proteins strongly suggests that the amino-terminal targeting signals of Giardia IscU and Fd are removed
upon organelle import and that in vivo protein import into
mitosomes is an active saturable process. Western blot analysis
of parasite subcellular fractions using specific anti-IscU antibodies confirmed that the native Giardia protein is indeed
processed upon mitosome import (Fig. 3C) as indicated by the
presence of a major band of ⬃14.5 kDa (mature protein) and a
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Characterization of Giardia Mitosomes
FIG. 4. Conserved function of mitosomal and mitochondrial amino-terminal targeting peptides. Human embryonic kidney 293 cells transiently
transfected with 5⬘-Fd䡠GFP fusion (upper
panels) and GFP control (lower panels)
plasmid constructs were incubated in the
presence of MitoTracker Red, fixed with
paraformaldehyde, and viewed under a
confocal microscope. GFP is targeted into
mitochondria exclusively when fused to
the mitosomal targeting signal of Giardia
ferredoxin; control cells accumulate GFP
in the cytosol. DIC, differential interference contrast.
minor band of ⬃17 kDa (precursor protein) consistent with the
removal of the targeting peptide from the precursor protein.
Together, these data demonstrate that mitosome protein import, like protein import into mitochondria and hydrogenosomes, is a saturable process that requires, at least in part, the
presence of cleavable amino-terminal presequences.
Functional Conservation of Targeting Signals in Giardia
Mitosomes and Mammalian Mitochondria—The requirement
of cleavable targeting presequences for mitosomal protein import suggests that the mechanisms of giardial mitosome protein import could be functionally conserved with those of mitochondria. To test this hypothesis we cloned the 5⬘-Fd䡠GFP
fusion construct into a mammalian expression vector and transiently transfected human embryonic kidney cells. The passenger GFP protein was efficiently targeted into mammalian mitochondria by the Giardia Fd presequence, as demonstrated by
the co-localization of recombinant GFP with the mitochondrial
marker dye MitoTracker Red (Fig. 4, upper panels). In the
absence of the mitosomal targeting presequence, GFP accumulated in the cytosol (lower panels). These data demonstrate the
functional conservation of targeting presequences between
Giardia mitosomes and mammalian mitochondria and suggest the existence of common protein import pathways in these
organelles.
Organelle Replication and Inheritance—In confocal microscopy images Giardia mitosomes appear as ⬃100-nm spherical
compartments randomly distributed throughout the cytoplasm
with concomitant staining of a distinct rod-like structure invariably localized in the medial axis between the nuclei. Based
on their cellular distribution we have dubbed these structures
peripheral mitosomes (PMs) and central mitosomes (CMs), respectively. To investigate how PMs and CMs replicate and
segregate, fluorescence microscopy was used to analyze their
distribution at different stages of the cell cycle. The single CM
localized exclusively to the basal body region between the nuclei during interphase (Fig. 5A). In mitotic cells, however, doubling and segregation of CMs was consistently observed as an
early event prior to the onset of karyokinesis (Fig. 5, B and D),
suggesting that replication and inheritance of CMs is coordinated in a cell cycle-dependent manner. To assess the behavior
of PMs during the cell cycle, photomicrographs of interphase
and of mitotic cells were used to quantify the number of PMs
present in each cell. Statistical analysis showed that the mean
number of PMs was slightly higher in mitotic cells than in
interphase trophozoites (mean values 57.6 and 50.9/cell, respectively, n ⫽ 40) (Fig. 5C). Although significant (p ⬍0.05), the
difference is far too small to account for the number of organelles that would be expected in mitotic cells if replication of
PMs were coordinated with the cell cycle. Rather, our data
suggest that PMs are not coordinately replicated and that they
segregate during cell division stochastically.
The close association of CMs with the basal body region
suggested the involvement of the cytoskeleton in organelle
positioning, division, and inheritance. To investigate the contribution of the cytoskeleton to these processes, trophozoites
were treated for 2 h with cytochalasin D or with nocodazole,
specific inhibitors of actin filaments and microtubule assembly,
respectively. Cytochalasin D did not change the appearance
and localization of CMs, although PMs appeared to be more
abundant in these cells (Fig. 5B). Because actin filaments have
never been detected in Giardia, the reason for this effect remains unexplained. Conversely, addition of nocodazole to the
culture medium resulted in de-localization of the CMs, whereas
the distribution of PMs appeared unaffected (Fig. 5B). These
data demonstrate a direct physical connection between CMs
and the microtubular cytoskeleton.
We established a sequence of events for CM duplication and
segregation during mitosis using Cpn60 as a marker of mitosomes (Fig. 5D). In agreement with a recent investigation (31),
we found that cell division follows a ventral-dorsal pattern with
preservation of orientation and handedness in the daughter
cells. The cell in the left panel (Fig. 5D) represents the earliest
detectable stage of mitosis (stage I). Typical features were the
beginning assembly of a daughter ventral disk and elongated
nuclei with the DNA appearing condensed into 8 –10 lumps.
The CM was duplicated very early, and the two organelles were
separated but still in close proximity. During stage II sequential karyokinesis of both nuclei was observed (32), and both the
basal bodies of the axonemes and CMs became clearly segregated into daughter cells. In stage III cytokinesis was completed and daughter cells received one CM and a set of PMs
each. Such close association between CMs and the cytoskeleton
during the cell cycle suggests similarities with mitochondrial
replication and inheritance in Trypanosoma brucei, as discussed below.
DISCUSSION
Until the recent discovery of mitosomes, Giardia had been
believed to be devoid of organelles of endosymbiotic origin,
either through secondary loss or because branching of the
diplomonad ancestor had occurred before the establishment of
mitochondria in the eukaryotic lineage. Contrary to the proposed mitochondrial ancestry of mitosomes, however, electron
and confocal microscopy images obtained using mammalianand Entamoeba-specific anti-Cpn60 antibodies suggested that
this key marker of mitochondria might not co-localize with Isc
proteins in Giardia mitosomes or even be associated with biological membranes (3, 18). Data presented in this report settle
the controversy (19) by demonstrating unequivocally that all
these mitochondrial markers do indeed reside in mitosomes.
Our findings substantiate the hypothesis that these organelles
represent vestigial mitochondria and demonstrate the impor-
Characterization of Giardia Mitosomes
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FIG. 5. Subcellular localization and inheritance of the central mitosome during the cell cycle. A, close-up differential interference
contrast (DIC) and fluorescence images of a CM stained with anti-GiCpn60 showing association with the basal body complex of Giardia. CF, caudal
flagella; VF, ventral flagella; BB, basal bodies of the six posterior flagella; N, nuclei; CM, central mitosome; PM, peripheral mitosomes. B,
distribution and subcellular localization of all PMs and the CMs in a projection image of optical sections. Left panel, interphase cell. Second panel,
postmitotic cell with divided CM. Third panel, treatment with the actin-depolymerizing agent cytochalasin D does not affect the subcellular
localization of the CM. Right panel, the CM become delocalized in cells treated with the microtubule-depolymerizing drug nocodazole, but PMs
appear unchanged. The outlines of the nuclei are shown in red. Blue arrowheads, CM localization. C, statistical analysis of PM numbers in dividing
cells. Gray bars, mean number of PMs in dividing and interphase cells (n ⫽ 40); line bars, standard deviation. D, representative images showing
CM localization (yellow arrowheads) during three stages of mitosis and cell division (I-III). Micrographs show three-dimensional reconstructed
confocal images (25 optical sections/stack). Nuclei are stained with DAPI and are depicted in green for enhanced contrast. DIC images, layer 12/25.
Scale bars are 2 ␮m.
tance of using homologous antibodies when working with
highly derived organisms such as Giardia.
The biosynthesis and assembly of iron-sulfur clusters is carried out in the lumen of mitochondria, hydrogenosomes, and
Giardia mitosomes. Most of the proteins that reside and function in the lumen of mitochondria and hydrogenosomes are
imported into their respective organelles via positively charged
and cleavable amino-terminal targeting presequences (29, 30).
Targeting of GiIscU and GiFd into Giardia mitosomes was also
found to be dependent on their amino-terminal presequences,
which are cleaved off from precursor proteins upon import into
mitosomes. Furthermore, mitosomal targeting presequences
are readily recognized by the protein import machinery of
mitochondria, thus demonstrating a conservation of function
between protein import pathways in both organelles. Despite
their highly degenerate nature and despite the difficulty in
identifying homologues of the protein translocases of mitochon-
dria in Giardia mitosomes (see below), these organelles have
clearly retained the molecular components required to translocate proteins from the cytosol in a presequence-dependent
manner. Evidence for the functional conservation of mitosomal
and mitochondrial targeting signals has also been obtained in
Entamoeba and Cryptosporidium (2, 33–38). Such apparent
widespread conservation of function in mitosomal and mitochondrial protein targeting, together with the mutually exclusive distribution of mitosomes and mitochondria in eukaryotic
lineages, strongly supports the mitochondrial ancestry of
mitosomes.
Mitochondrial and hydrogenosomal IscS, mtHsp70, and
Cpn60 all require cleavable amino-terminal targeting presequences for import into their respective organelles (39 – 43). In
Giardia, however, we found that this feature is no longer conserved as all three proteins are efficiently targeted into mitosomes, alone or as fusion proteins, in the absence of amino-
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Characterization of Giardia Mitosomes
FIG. 6. Graphical representation of the in silico effort to uncover components of the mitosome protein import apparatus in
the Giardia Genome Database. Searched subunits of conserved complexes of the translocase of the outer (TOM) or inner (TIM) membrane and
other proteins associated with the import machinery are depicted with
their full or numerical designation (e.g. 40 for TOM40). OM, outer membrane; IM, inner membrane. Subunits depicted in shaded light gray have
not been found in the Giardia Genome Database; subunits in solid black
are confirmed as orthologues and as imported mitosome matrix proteins
in the case of GiHsp70 and GiCpn60; subunits in solid dark gray have
produced significant hits in the Giardia Genome Database and are subject
to confirmation. For details see supplemental information.
terminal targeting presequences (16, 21–23). Further, removal
of the 5 initial amino acids of GiCpn60 did not preclude its
import into mitosomes, demonstrating that Giardia Cpn60 is
not constrained by the amino-terminal peptide requirement
displayed by its mitochondrial and hydrogenosomal homologues. Together with the identification of only two proteins
with cleavable amino-terminal targeting presequences in Giardia (IscU and Fd), these data illustrate the extent of reductive
evolution already imposed on the presequence-dependent protein import pathway of Giardia mitosomes. Import of a
mtHsp70 protein without an obvious organelle targeting presequence has also been observed in T. hominis, another mitosome-containing organism (1). Such a derived feature resembles the presequence-independent import mechanism observed
in a number of luminal mitochondrial proteins such as rhodanese, 3-oxoacyl-CoA thiolase, and chaperonin 10 (27). This
might represent an ancestral feature of organelle protein import as some bacterial proteins appear to have a natural predisposition for organelle targeting (44).
The protein import machinery of mitochondria is known to
contain dozens of proteins, including cytosolic chaperones,
membrane-bound protein translocases, and accessory luminal
mitochondrial proteins such as molecular chaperonins and
processing peptidases (29). The number of cellular proteins
required for hydrogenosomal protein import is as yet unknown,
but this process shares many of the physiological requirements
of mitochondrial protein import (30, 45, 46). It is now evident
that Giardia must also possess a considerable number of proteins that participate in mitosome protein import and organelle
division, yet the number of putative mitosomal proteins
glanced from the fully sequenced Giardia genome remains
pitifully low (47). Fig. 6 is a working model and provides a
graphical representation of a data base search for components
of the protein translocation machinery (for experimental details see supplemental data). Each of the subunits detailed in
Fig. 6 was searched in the Giardia Genome Database using
query sequences from a wide variety of organisms, ranging
from protozoa to man, and including plant sequences (supplemental information, Tables S2-S4). Although two candidate
components were tentatively identified, i.e. the GiTOM70 subunit (GenBankTM EAA41006) and a mitochondrial protein peptidase homologue (GenBankTM EAA39560), the genomic data
alone were insufficient to reconstruct protein import machin-
ery derived from a mitochondrial endosymbiont. The use of
more refined bioinformatics tools and of alternative biochemical and biophysical protein identification methods, including
organelle proteomics and mass spectrometry, will be required
for the identification of the molecular components that participate in mitosome protein import and organelle replication.
A unique feature of Giardia mitosomes is their distinct morphological manifestations within a cell, i.e. a unique centrally
located rod-shaped structure and the more abundant spherical
organelles distributed throughout the cytoplasm. Interestingly,
only the CM is actively inherited in a cell cycle-dependent
manner, while the peripheral organelles appear to be maintained at relatively constant numbers and segregate into
daughter cells stochastically. Exactly how PMs of Giardia are
replicated and maintained in constant numbers in the cell is at
present unclear. One possibility is that PMs might arise from
the ends of CMs in a process analogous to the fission events
observed in yeast and mammalian mitochondria where,
through the participation of dynamin-related mechanoenzymes, complex mitochondrial networks can give rise to individual organelles (48 –50). A second possibility is that individual PMs may divide uncoordinatedly by binary fission or
septation, as observed in trichomonad hydrogenosomes (51).
Further research is required to unequivocally differentiate between these possibilities.
CMs are tightly associated with the basal bodies of flagellar
axonemes, which are known to replicate early during mitosis
(52), and depend on the integrity of the microtubular cytoskeleton for positioning and replication as demonstrated by their
dramatic de-localization in nocodazole-treated trophozoites.
These features of mitosome replication and inheritance are
shared with those of T. brucei mitochondria, where a single
organelle partitions by association with the basal bodies of the
two daughter flagella (53). Such similarities in mitosomal and
mitochondrial biogenesis further suggest the evolutionary relatedness between these organelles.
The absence of a remnant organellar genome in mitosomes
has thus far prevented a direct genetic demonstration of their
mitochondrial ancestry, but the reported presence of DNA in
the mitosomes of Blastocystis is a promising development (4, 5,
54, 55). The genetic demonstration that Nyctotherus hydrogenosomes are modified mitochondria suggests that reductive
evolution of the original mitochondrial endosymbiont may have
given rise to aerobic and anaerobic mitochondria, hydrogenosomes, and, in all likelihood, to mitosomes as predicted by the
hydrogen hypothesis for the first eukaryote (8, 56 –58). The
confirmed cellular localization of mitochondrial marker proteins in Giardia mitosomes, their functionally conserved protein import mechanisms, as well as their conserved features of
organelle replication and inheritance are all evidence that Giardia mitosomes represent vestigial anaerobic mitochondria.
Acknowledgments—We thank the Giardia Genome Project for making sequence data available before publication. We thank
Drs. A. Schneider and M. Marti for critical reading of the manuscript,
Drs. A. Harvey and M. Crompton for help with mammalian cell
transfections, and T. Michel and N. Sommerville for excellent technical assistance.
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