Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 1365-2958Blackwell Publishing Ltd, 2003512307317Original ArticleP. Hamel et al.Isoforms of the mitochondrial phosphate carrier
Molecular Microbiology (2004) 51(2), 307–317
doi:10.1046/j.1365-2958.2003.03810.x
Redundancy in the function of mitochondrial
phosphate transport in Saccharomyces cerevisiae
and Arabidopsis thaliana
Patrice Hamel,1† Yann Saint-Georges,1
Brigida de Pinto,2 Nicole Lachacinski,1
Nicola Altamura2 and Geneviève Dujardin1*
1
Centre de Génétique Moléculaire, Avenue de la Terrasse,
91198- Gif sur Yvette, France.
2
Consiglio Nazionale delle Ricerche, Istituto di
Biomembrane e Bioenergetica, Via Amendola 165/A,
70126- Bari, Italy.
Summary
Most cellular ATP is produced within the mitochondria
from ADP and Pi which are delivered across the innermembrane by specific nuclearly encoded polytopic
carriers. In Saccharomyces cerevisiae, some of these
carriers and in particular the ADP/ATP carrier, are
represented by several related isoforms that are distinct in their pattern of expression. Until now, only one
mitochondrial Pi carrier (mPic) form, encoded by the
MIR1 gene in S. cerevisiae, has been described. Here
we show that the gene product encoded by the
YER053C ORF also participates in the delivery of
phosphate to the mitochondria. We have called this
gene PIC2 for Pi carrier isoform 2. Overexpression of
PIC2 compensates for the mitochondrial defect of the
double mutant Dmir1 Dpic2 and restores phosphate
transport activity in mitochondria swelling experiments. The existence of two isoforms of mPic does
not seem to be restricted to S. cerevisiae as two Arabidopsis thaliana cDNAs encoding two different mPiclike proteins are also able to complement the double
mutant Dmir1 Dpic2. Finally, we demonstrate that
Pic2p is a mitochondrial protein and that its steady
state level increases at high temperature. We propose
that Pic2p is a minor form of mPic which plays a role
under specific stress conditions.
Introduction
One of the main functions of mitochondria is the producAccepted 1 September, 2003. *For correspondence. E-mail
[email protected]; Tel. (+33) 1 69 82 31 69; Fax (+33)
1 69 82 31 50. †Present address: UCLA, Department of Chemistry
and Biochemistry, 607 Charles E Young Drive, 90095–1569, Los
Angeles, USA.
© 2003 Blackwell Publishing Ltd
tion of most cellular energy via oxidative phosphorylation.
This process requires the four respiratory complexes that
transfer electrons from NADH or succinate to molecular
oxygen and the ATP synthase that utilizes the proton
gradient generated by the electron flow to produce ATP.
The synthesis of ATP is controlled by the supply of cytosolic inorganic phosphate (Pi) and ADP to the mitochondrial matrix. Specific nuclearly encoded carriers deliver Pi
and ADP to the mitochondria. These carriers are polytopic
proteins imported from the cytosol and inserted within the
mitochondrial inner membrane. They belong to a large
eukaryote-specific family of related mitochondrial carriers
(MCF) that catalyse the shuttling of various metabolites
across the mitochondrial inner-membrane. Mitochondrial
carrier members are not very well conserved at the level
of sequence identity (15–20%) but share a putative common topology with the presence of six transmembrane
segments.
According to in silico analysis, there are about 35 ORFs
whose gene products are predicted to belong to the MCF
in Saccharomyces cerevisiae (El Moualij et al., 1997;
Paulsen et al., 1998; Belenkiy et al., 2000; Palmieri et al.,
2000), the function of most of these ORFs is still largely
unknown. Remarkably, some of these carriers are represented by several related isoforms which are highly identical at the level of amino acid sequence but distinct in
their pattern of expression. For instance, the three isoforms of the S. cerevisiae ADP/ATP translocator (AAC1,
2 and 3) are 80% identical and two of them were shown
to be differentially regulated (Gawaz et al., 1990; Kolarov
et al., 1990; Fiore et al., 1998 for review). Likewise, three
isoforms of the ADP/ATP carrier also exist in human and
are distinguished by their tissues specificities (Lunardi
et al., 1992; Stepien et al., 1992; Giraud et al., 1998).
The mitochondrial phosphate carrier (mPic) catalyses
the proton co-transport of phosphate into the mitochondrial matrix. The S. cerevisiae mPic was purified from
mitochondria (Guérin et al., 1990) and its gene, called
MIR1 (originally for mitochondrial import receptor), identified (Murakami et al., 1990; Phelps et al., 1991). In S.
cerevisiae, a defect in phosphate transport impairs growth
on non-fermentable substrates and is associated with a
reduction in the membrane potential and consequently
with an inhibition of mitochondrial protein import (Zara
et al., 1996). Site-directed mutagenesis experiments iden-
308 P. Hamel et al.
tified residues of Mir1p that are important for the structure
and phosphate/proton transport activity (Briggs et al.,
1999; Phelps et al., 2001; Wohlrab et al., 2002 and see
Supplementary material Fig. S1). BLAST analysis reveals
that another S. cerevisiae ORF, YER053C, encodes a
protein sharing the characteristic features of MCF proteins
and which is 40% identical to Mir1p and to mammalian
mPics. This sequence similarity with other mPic family
members suggests that the protein encoded by YER053C
could be an isoform of Mir1p. Indeed, MIR1 and YER053C
have a similar expression pattern in diauxic shift experiments and are both glucose-repressed, a common trait of
numerous genes involved in mitochondria biogenesis
(DeRisi et al., 1997; Belenkiy et al., 2000; Takabatake
et al., 2001). However, conflicting results came from Takabatake et al. (2001) who have shown that the yer053cnull mutant has no phenotype at 28∞C and that a tagged
form of Yer053p is found in vacuoles, presumably because
the presence of the epitope has targeted the protein to
the vacuolar compartment for degradation. Wohlrab et al.
(2002) have also ruled out a role of Yer053p in phosphate
transport based on the observation that some functionally
significant residues present in Mir1p are missing in
Yer053p. Finally, the purified protein fails to mediate phosphate uptake activity in a liposome reconstitution experiment (Takabatake et al., 2001; Wohlrab et al., 2002). In
mammals, there is only one mitochondrial transporter
encoding gene but two isoforms (A and B) are generated
by alternative splicing from the same transcript. The A
form is highly expressed and distributed in heart and
muscle whereas the B form is present in low amount in
all the tissues examined (Dolce et al., 1994; Fiermonte
et al., 1998; Huizing et al., 1998). In the Arabidopsis
thaliana genome, three genes encoding different mPiclike proteins are found but their function in phosphate
uptake has not been investigated.
In order to determine whether isoforms of the mPic
coexist in S. cerevisiae and plants and establish their
respective contributions in the delivery of phosphate to
mitochondria, we have taken advantage of yeast genetics
and reinvestigated thoroughly the phenotypes of mir1-null
and yer053c-null and double mutants. In this paper, we
show a synergistic effect in the double mutant suggesting
that Yer053p is involved in the same mitochondrial function
than Mir1p, and have named the gene PIC2 for Pi carrier
isoform 2, accordingly. That Pic2p is a mitochondrial phosphate carrier was established by showing that overexpression of the PIC2 gene compensates for the mitochondrial
defect of the double mutant and restores phosphate transport activity in mitochondria swelling experiments. Finally,
we assigned unambiguously a mitochondrial localization
for Pic2p by combining biochemical fractionation and fluorescence microscopy techniques. The existence of two
isoforms of mPic does not seem to be restricted to S.
cerevisiae as two A. thaliana cDNAs encoding two different
mPic-like proteins are also able to complement the double
mutant Dmir1 Dpic2 and restore phosphate transport function in S. cerevisiae. The study of the relative accumulation
of the two proteins suggests that Pic2p is the minor isoform
at 28∞C but could be recruited when yeast is challenged
by high temperature conditions.
Results
Synergistic effect of the double mutant Dmir1 Dpic2
In order to determine the role of the PIC2 gene, we have
constructed a strain carrying a complete deletion of the
wild-type allele (Dpic2). In agreement with the results of
Takabatake et al. (2001), we found that the Dpic2 strain is
viable. However, its doubling time in non-fermentable
medium at 28∞C is 7 h compared to 3 h for wild-type strain
and its growth on non-fermentable substrate is more
severely impaired at 36∞C, giving rise to only papillae
growth (Fig. 1). This finding suggests that a mitochondrial
function is compromised in the absence of Pic2p, particularly at high temperature.
In order to analyse the functional relationship existing
between Pic2p and Mir1p, we have constructed a Dmir1
mutant and a Dmir1 Dpic2 double mutant. As shown in
Fig. 1, the growth on a non-fermentable substrate of the
Dmir1 mutant is severely impaired at 28∞C and completely
blocked at 36∞C. The duplication time of the Dmir1 mutant
in ethanol medium at 28∞C is about 15 h versus 3 h for
the wild-type control. Moreover, as previously reported by
Zara et al. (1996), the deletion of the MIR1 gene leads to
a high production of mitochondrial rho- mutants that have
Fig. 1. Synergistic effect of the Dmir1 and Dpic2 mutations.
The Dmir1 and Dpic2 single mutants, the Dmir1 Dpic2 double mutant
and isogenic wild-type strains (+) were grown one day on glucose
containing medium (Glu), replica-plated on a medium containing glycerol (Gly) as a non-fermentable substrate and incubated for 3 days
at 28∞ or 36∞C. The percentage of rho- mutants accumulating in
glucose medium at 28∞C after 15 generations was estimated by
plating the cells on a medium containing 0.1% glucose, 2% glycerol
and is indicated in the right part of the figure.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
Isoforms of the mitochondrial phosphate carrier 309
lost mitochondrial DNA and thus caused an inactivation
of the respiratory chain. Interestingly enough, the double
mutant Dmir1 Dpic2 did not grow at all on non-fermentable
medium at 28∞ or 36∞C and led to a higher production of
rho- mutants than the single mutant Dmir1 in a fermentable medium. These results evidenced a synergistic effect
in the double mutant, suggesting that Pic2p is involved in
the same mitochondrial function than Mir1p.
Overexpression of PIC2 compensates for the
mitochondrial defect of the double mutant Dmir1 Dpic2
In order to establish if Pic2p overlaps in function with
Mir1p, we have asked whether the overexpression of the
PIC2 gene could compensate for the lack of growth on
non-fermentable substrate observed in the double mutant
Dmir1 Dpic2. For this purpose we have taken advantage
of low and high copy expression vectors (centromeric
versus 2m) carrying the strong constitutive promoter of the
PGK gene encoding phosphoglycerate kinase. According
to Nacken et al. (1996), the PGK promoter allows a 40fold increase in the expression of a reporter gene as
compared to the CYC1 promoter, controlling the transcription of mitochondrial cytochrome c. In the same study, the
authors also report that the expression level of the
reporter gene from a multicopy plasmid is about 20-fold
higher than from a centromere based plasmid.
We have cloned the PCR amplified PIC2 gene in the
high or low copy expression vector (pFL61 or pFL61cen),
in the sense and antisense orientation relative to the PGK
promoter as we had previously noticed the existence of a
weak promoter located in the PGK terminator (Bonnefoy
et al., 1996 and unpublished). The double mutant Dmir1
Dpic2 was transformed with these various constructions
and the ability of the transformants to grow on a nonfermentable substrate was tested. The expression of the
wild-type PIC2 gene cloned on a high copy vector is able
to restore growth on glycerol at 28∞C and 36∞C, regardless
of the orientation of the insert (Fig. 2A and data not
shown). The doubling time in non-fermentable medium of
the transformants expressing PIC2 is similar to those
expressing the MIR1 gene and they produce a low percentage of rho- mutants (<1%). The low copy plasmid
carrying the PIC2 ORF was also able to restore growth
on glycerol of the double mutant Dmir1 Dpic2 but only
when PIC2 was cloned in the sense orientation (Fig. 2A).
Altogether, these results show that PIC2 is able to compensate for the absence of Mir1p when it is overexpressed, suggesting that Pic2p is also probably active in
mitochondrial phosphate transport.
Phosphate transport function of Pic2p
The mitochondrial import of phosphate can be measured
by following the swelling of mitochondria when these are
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
Fig. 2. Overexpression of the S. cerevisiae PIC2 or the A. thaliana
AT5 and AT3 compensates for the absence of Mir1p and Pic2p. The
various transformants were grown two days on minimal glucose
medium (Glu), replica-plated on glycerol medium (Gly) and incubated
three days (A) or a week (B) at 28∞C.
A. The double mutant Dmir1 Dpic2 was transformed by the plasmid
carrying MIR1 (YEpMK6), the 2m control vector (pFL61), or various
plasmids carrying PIC2 cloned in the high (2m, YEp) or low (cen, YCp)
copy vectors and in the sense (YEpY53 and YCp56) and antisense
orientations (YEpY55 and YCp57) with respect to the PGK promoter
(see Experimental procedures).
B. The double mutant Dmir1 Dpic2 was transformed with the control
vector (pFL61) and the plasmids carrying MIR1 (YEpMK6), AT5
(YepAT5) or AT3 (YEpAT3) (see Experimental procedures and
Results for plasmid selection).
suspended in phosphate salt solution. Swelling is the
osmotic result of the increase of the mitochondrial matrix
solute concentration which causes water uptake by mitochondria. This can be monitored by recording the
decrease of the mitochondrial suspension absorbance. By
using this technique, Zara et al. (1996) have shown that
mitochondria isolated from the single mutant Dmir1 are
unable to swell in presence of phosphate. As expected,
the phosphate uptake is also blocked in the double mutant
310 P. Hamel et al.
Dmir1Dpic2 (Fig. 3A) but the introduction of the YEpY53
plasmid overexpressing the PIC2 gene restores phosphate-induced swelling essentially to the same rate as
that observed in the transformants overexpressing the
MIR1 gene. In both cases, the uptake of phosphate is
sensitive to mersalyl, a sulfhydryl reagent inhibitor of mPic
(Guérin et al., 1990; Zara et al., 1996). In the Pic2p
sequence, there is a cysteine at position 44 that is absent
in Mir1p and present at position 42 in mammalian phosphate carriers (see Supplementary material, Fig. S1 and
data not shown). This cysteine 42 was shown to react with
N-ethylmaleimide (NEM) and the phosphate transport
activity in mammalian mitochondria is thus sensitive to
NEM (Kolbe and Wohlrab, 1985). Therefore, we have
checked whether the phosphate transport catalysed by
Pic2p is also sensitive to NEM. Figure 3A shows that the
swelling of mitochondria from cells expressing only Mir1p
is insensitive to NEM as expected from the work of Guérin
et al. (1990) whereas the swelling of mitochondria from
cells expressing only Pic2p appears sensitive to NEM.
Thus, as in the case of the mammalian mitochondrial
phosphate carrier, the Pic2p-dependent phosphate transport activity appears sensitive to NEM and this sensitivity
correlates with the presence of the cysteine 44.
In conclusion, Pic2p is a second mitochondrial phosphate carrier that can substitute for Mir1p in the delivery
of phosphate to the mitochondria.
Pic2p is a mitochondrial protein
Fig. 3. Phosphate-induced swelling in mitochondria from Dmir1 Dpic2
transformants overexpressing the S. cerevisiae genes MIR1 and PIC2
or the A. thaliana cDNAs AT5 and AT3. Mitochondria were purified
from Dmir1 Dpic2 transformants carrying the plasmids YEpMK6
(MIR1), YEpY53 (PIC2) (A) or YEpAT3 (AT3), YepAT5 (AT5) (B).
Phosphate transport was monitored on fresh mitochondria by following mitochondria swelling essentially as described by Manon and
Guérin (1988). Mitochondria were suspended at a concentration of
0.1 mg ml-1 mitochondrial proteins in 1 ml buffer containing 0.2 M
potassium phosphate (pH 7.4), 38 mM oligomycin, 0.2 mM antimycin,
0.036 mg valinomycin. 0.5 mM mersalyl or 2 mM NEM was added
when indicated. The absorbance decrease was monitored at 546 nm
as a function of time.
The subcellular localization of Pic2p in S. cerevisiae cells
was examined by biochemical fractionation and fluorescence microscopy approaches.
First, mitochondria were purified from a strain expressing a functional c-myc-tagged form of the protein (Fig. 4A).
A c-myc immunoreactive protein of about 50 kDa, the
predicted size for the Pic2p-cmyc, is highly enriched in the
mitochondrial fraction compared to the post-mitochondrial
supernatant. As expected, two bona fide mitochondria
resident proteins, Qcr9p, a subunit of the respiratory complex III, also tagged with a c-myc epitope and Atp2p, a
subunit of the ATP synthase were mainly detected in the
mitochondrial fraction. We have also shown that the mitochondrial fraction is only slightly contaminated with vacuoles, using an antibody specific for the vacuolar
carboxypeptidase Y.
Second, cells expressing a Pic2p-gfp fusion protein
were examined by fluorescence microscopy. As shown in
Fig. 4B, the fusion protein is located in fluorescent speckles also stained with a specific mitochondrial dye, the
Mitotracker probe. The overlay of GFP fluorescence and
MitoTracker labelling (Merge) indicates that the tagged
protein mostly co-localized with the mitochondria. However, in a few percent of cells, large vacuoles are present
and exhibit GFP fluorescence (data not shown), suggesting that some degradation of the fusion protein occurs.
This could explain the vacuolar localization of Pic2p previously reported using electron microscopy techniques
(Takabatake et al., 2001). Altogether, these results establish that Pic2p is located within the mitochondria.
Cloning of the Arabidopsis thaliana orthologues of the
yeast MIR1/PIC2 genes
Inspection of the bovine and human sequence data© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
Isoforms of the mitochondrial phosphate carrier 311
Fig. 4. Mitochondrial localization of Pic2p and comparative accumulation of Mir1p and Pic2p at 28∞C and 36∞C. Pic2p was tagged with a c-myc
epitope (strain YST9) or with the GFP protein (strain YST20) as described in Experimental procedures.
A. Mitochondria were purified from wild-type, PIC2-C-MYC and QCR9-C-MYC cells grown in complete galactose medium at 28∞C. Forty
micrograms of mitochondrial protein pellet and 80 micrograms of postmitochondrial supernatant were separated by SDS-PAGE and immunoblotted
with antibodies directed against c-myc (upper panel), the mitochondrial ATPase subunit Atp2p and the vacuolar protein Cpyp. WT: CW30; PIC2C-MYC: YST9; QCR9-C-MYC: YST6 strain carrying the tagged Qcr9p-c-myc (Saint-Georges et al., 2001).
B. Cells expressing the Pic2p-gfp fusion protein (YST20) were grown in complete medium at 28∞C, washed once and incubated with 0.05 mM
Mitotracker red CMX ROS (MT, Molecular Probes) for 10 min. After two additional washes cells were observed in fluorescence microscopy. The
merge at the right of the figures represents the superposition of both GFP fluorescence and red fluorescence of MitoTracker.
C. Mitochondria were extracted from PIC2-C-MYC (YST9) and Dmir1 strains grown in complete galactose medium at 28∞C or 36∞C, separated
by SDS-PAGE and immunoblotted with antibodies directed against c-myc that reveals the tagged form Pic2p-c-myc, Atp2p as control and mPic
(Mir1p). The construction of the Dmir1 strain is described in Experimental procedures.
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
312 P. Hamel et al.
bases revealed the presence of only one gene encoding
for the mitochondrial phosphate transport (Dolce et al.,
1994 and data not shown). In order to determine
whether other organisms besides S. cerevisiae may also
possess more than one phosphate carrier gene, we
have performed in silico analysis and found that there
are two to four genes encoding mitochondrial phosphate
carrier-like proteins in Drosophila melanogaster, A.
thaliana and Caenorhabditis elegans (see Fig. 5). These
phosphate carrier-like proteins share about 35% identity
with Pic2p and Mir1p.
To identify functional mitochondrial phosphate carriers
in A. thaliana, we have taken advantage of the heterologous complementation approach. For this aim, we have
transformed the Dmir1 Dpic2 strain with the A. thaliana
cDNA library constructed in the high copy expression vector pFL61 (Minet et al., 1992) and selected five transformants able to grow on a non-fermentable substrate at
28∞C. All the plasmids isolated from these transformants
carry the same cDNA derived from the gene located on
chromosome V (accession number ath:At5g14040) and
we have called it AT5. As shown in Fig. 2B, the AT5
transformants are growing slower on glycerol medium
(doubling time ~ 10 h) than the transformants expressing
Mir1p (doubling time ~ 3 h), and the production of rhomutants was estimated to be about 30%.
In order to test the function of the second A. thaliana
protein exhibiting 66%, 38% and 33% identity with AT5,
Pic2p and Mir1p, respectively, we have amplified and
cloned the corresponding cDNA into the yeast expression
vector pFL61. The gene is located on chromosome III
(accession number ath:At3g48850) and we have called it
AT3. The double mutant Dmir1 Dpic2 was transformed with
the plasmid expressing the AT3 cDNA. As shown in
Fig. 2B, the transformants were able to grow on a nonfermentable substrate, demonstrating that AT3 is also able
to partially compensate for the absence of both yeast
mitochondrial phosphate carriers. However, as with the
AT5 cDNA, the heterologous complementation is only partial. Indeed, we found that the doubling time and the
production of rho- mutants are similar in the transformants
expressing AT3 or AT5 (data not shown).
To assess whether Dmir1 Dpic2 transformants expressing either the AT5 or AT3 proteins have recovered phosphate uptake into mitochondria, we have performed
phosphate-induced swelling experiments in mitochondria
purified from both transformants. As shown in Fig. 3B, the
introduction of plasmids overexpressing AT5 or AT3 in the
double mutant Dmir1 Dpic2 restore some phosphateinduced swelling that is inhibited by mersalyl. A third
putative ORF (AT2, accession number ath:At2g17270),
identified through the in silico analysis, encodes a protein
sharing 43% identity to AT5 and AT3 proteins. Despite
many attempts, we were unable to amplify the corresponding cDNA from different cDNA libraries (data not
shown).
Our results suggest that the existence of two isoforms
of mPic is not a specificity of S. cerevisiae as we have
shown that at least two functional mitochondrial phosphate carriers do also exist in A. thaliana.
Comparison of the accumulation of Pic2p and Mir1p
Fig. 5. Phylogenic tree of mPics.
The tree was constructed using a multiple sequence alignment with
hierarchical clustering as described by Corpet (1988) taking the
Aac1p ADP/ATP carrier as an out-group sequence (accession number: NP_013772). The Mir1p and Pic2p were aligned with the mPic
from A. thaliana (AT2, AT3 and AT5, this work), H. sapiens (Q00325,
Dolce et al., 1994), and mPic-like proteins from S. pombe
(NP_596208), D. melanogaster (1: NP_611468 and 2: NP_524069)
and C. elegans (1: NP_502087; 2: NP_494870; 3: NM_060160; 4:
NP_506148). Remarkably, mPic is encoded by only one gene in S.
pombe.
We have shown that Mir1p and Pic2p are able to transport
phosphate to mitochondria in S. cerevisiae. Previous studies reported that the two S. cerevisiae genes are coordinately regulated in aerobiosis/anaerobiosis conditions
(Takabatake et al., 2001) and display a similar expression
pattern in diauxic shift experiments (DeRisi et al., 1997;
Belenkiy et al., 2000). In order to understand the relative
role of each carrier in the distribution of phosphate to the
mitochondria, we have monitored the expression of PIC2
and MIR1 genes at the mRNA and protein levels in various conditions.
Polyadenylated mRNAs from the wild-type strain grown
in glucose or galactose medium were hybridized to PIC2
and MIR1 specific probes. The expression of the two
genes is clearly glucose-repressed as already described
in previous studies and PIC2 appears to be three- to
fourfold less expressed than MIR1 (data not shown). Thus,
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
Isoforms of the mitochondrial phosphate carrier 313
the low phosphate transport activity of Pic2p could be
related to its low abundance and might account for the
leaky growth on non-fermentable substrate of the mir1null strain. Since, we have shown that the growth on nonfermentable substrate of the Dpic2 mutant was affected at
36∞C, we have used the c-myc tagged protein to monitor
the accumulation of Pic2p as a function of temperature in
protein extracts from cells grown in galactose at 28∞C and
36∞C. Figure 4C shows that there is a threefold increase
of Pic2p abundance in mitochondria from cells grown at
36∞C compared to mitochondria from cells grown at 28∞C.
This prompted us to check whether the accumulation of
Mir1p level was also dependent on temperature. To monitor the level of Mir1p, we have used an antibody raised
against S. cerevisiae mPic (Zara et al., 1996). We have
shown that this antibody is highly specific for Mir1p and
does not cross-react with Pic2p as no immunoreactive
species is detected in the Dmir1 mitochondria (Fig. 4C,
lane 3). The steady state level of Mir1p appears identical
in mitochondria purified from cells grown at 28∞C or 36∞C.
These results indicate that the amount of Pic2p relative to
Mir1p varies upon temperature growth conditions. Interestingly enough, genomic expression programs (Gasch
et al., 2000; Causton et al., 2001) have shown that the
PIC2 mRNA (and not the MIR1 mRNA) is also increased
in some environmental or stress conditions, in particular
after heat-shock. However, this threefold increase of Pic2p
level is probably not sufficient for the Pi delivery at 36∞C
because in the Dmir1 strain does not grow at all on a nonfermentable substrate at 36∞C.
In conclusion, Mirp is the major mPic at 28∞C in S.
cerevisiae but the steady state level of Pic2p is increased
at high temperature.
Discussion
In the present study, we provided evidence for a novel
mitochondrial phosphate carrier in S. cerevisiae, encoded
by the PIC2 gene, in addition to the well known mPic,
Mir1p. The analysis of mRNAs suggests that PIC2 is less
expressed than MIR1, a finding consistent with the codon
usage: the Codon Adaptation Index (CAI) is 0.362 for
MIR1 and 0.158 for PIC2. These observations could
account for the fact that we did not retrieve PIC2 in our
complementation experiments with yeast cDNA libraries
(see Experimental procedures). However, that overexpression of Pic2p can clearly compensate for the absence
of Mir1p establishes that Pic2p is a mitochondrial phosphate carrier. Interestingly enough, the transport of Pi due
to Pic2p is sensitive to the specific inhibitor NEM whereas
that due to Mir1p is not. Mir1p and Pic2p share about 40%
identity and out of the 22 residues important for the carrier
activity, 18 are identical or conserved (see Supplementary
material, Fig. S1). The five absolute consensus residues
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
of the mitochondrial carriers with established transport
function are conserved in Pic2p, in particular the lysine at
position 43 that appears critical for the phosphate transport (Briggs et al., 1999 and Supplementary material,
Fig. S1). However, when expressed as bacterial inclusion
bodies and reconstituted in vitro into liposomes, Pic2p
shows no phosphate transport activity (Takabatake et al.,
2001; Wohlrab et al., 2002). We have no expertise in this
domain but these negative results could be due to various
reasons linked to in vitro techniques, e.g. to the fact that
the protein has been made in E. coli and not in yeast.
However, an interesting possibility would be that Pic2p
needs to interact with another unidentified yeast protein,
present in our in vivo complementation assays, to become
fully active.
Sequence comparisons with mPics of other organisms
reveal that Pic2p is more related to heterologous mPics
than to S. cerevisiae Mir1p (Fig. 5 and data not shown).
There is clear molecular evidence for ancient duplications
of numerous S. cerevisiae genes (Wolfe and Shields,
1997) and sequence comparisons between the duplicated
proteins generally reveal that they are more related to
each other than to their heterologous counterparts from
other species. It would be interesting to study in detail the
relationship between the conservation of the various
domains of the S. cerevisiae mPic and their role in the
phosphate transport activity in order to understand this
rapid divergence of Mir1p.
Of the two functional isoforms we have identified in A.
thaliana, AT5 is probably the major one as about 50 ESTs
of AT5 are found in the databases and none for AT3.
Interestingly enough, AT3 is more related to Pic2p than to
Mir1p and would also correspond to a minor form. The
AT5 isoform presents about 79% and 75% sequence
identity with the Glycine max and Lotus japonicus mPics
which both exhibit high phosphate transport activity when
reconstituted into liposomes (Takabatake et al., 1999;
Nakamori et al., 2002). Several genes coding mPic-like
proteins are also present in D. melanogaster and C. elegans genomes (see Fig. 5) and in mammals, there are
two isoforms A and B resulting from alternative splicing
(Dolce et al., 1994; Fiermonte et al., 1998). Thus, several
isoforms of mPic appear in numerous organisms indicating that the occurrence of several isoforms of mPic is a
common feature of eukaryotic organisms. Another example of functional isoforms in both S. cerevisiae and human
is exemplified by the ADP/ATP carrier (Fiore et al., 1998
for review). In A. thaliana, four genes encoding putative
mitochondrial ADP/ATP carriers are also found in the
genome, three of which encode functional carriers with
established translocation activity in a bacterial heterologous system (Haferkamp et al., 2002). Altogether, these
observations suggest that eukaryotic cells would often
retain in their proteome several isoforms of the two carri-
314 P. Hamel et al.
ers directly involved in ATP generation by oxidative
phosphorylation.
We have shown that the two A. thaliana cDNAs complement the phosphate transport deficiency of the yeast
mutant. Four human carriers, the hMRS3/4 carrier and
the three human mitochondrial ADP/ATP carriers, are all
able to complement the deficiency as a result of the lack
of the endogenous carriers in yeast (Li et al., 2001; De
Marcos Lousa et al., 2002). Thus the function of mitochondrial carriers seems conserved through evolution.
Sequence alignments of Mir1p and Pic2p with their A.
thaliana, human and bovine homologues show that the
A. thaliana and the mammalian proteins display a Nterminal extension that is absent in the S. cerevisiae
proteins (see Fig. S1 Supplementary material). It was
shown previously that the presequence of the human
mPic was not essential for the import within mammalian
mitochondria but interferes with import into yeast mitochondria (Pratt et al., 1991; Zara et al., 1992). Thus, we
have constructed an at5 mutant expressing a protein
without the N-terminal extension and tested this construction in our complementation tests. Both forms of the
protein, with and without presequence, complement
equally well the yeast double mutant Dmir1 Dpic2 (data
not shown). We concluded that the presequence of AT5
does not seem to interfere with the yeast import process
in our experimental conditions. The precise role of these
presequences in the biogenesis of mitochondrial carriers
remains to be elucidated
Analysis of the steady state level of c-myc-tagged Pic2p
and Mir1p in mitochondria of cells grown at 28∞C or 36∞C
shows that the abundance of Pic2p is enhanced at 36∞C,
while the abundance of Mir1p remains unchanged. A twofold increase in the accumulation of PIC2 mRNA was also
observed after heat shock (Gasch et al., 2000; Causton
et al., 2001). The growth on glycerol medium of the Dpic2
mutant is severely impaired at 36∞C but the overexpression of Mir1p restores a normal growth at high temperature. Altogether these results suggest that in wild-type
cells grown at 36∞C, Mir1p would not suffice to deliver Pi
and that Pic2p would become more important. Because
the abundance of Mir1p does not vary at 28∞C and 36∞C,
it would be interesting in the future to determine if Mir1p
becomes less active at 36∞C. The study of the yeast
genome expression in response to environmental
changes suggests that in pairs of homologous genes, a
member of the pair would be expressed in normal conditions whereas the expression of the other member could
play a particular important role in special environmental
conditions (Gasch et al., 2000; Causton et al., 2001). Similarly, whereas the 50 ESTs of AT5 were found in plants
grown in various conditions, the three ESTs of the A.
thaliana mPic-like protein AT2 were all found in mRNAs
extracted from seeds (White et al., 2000) or from plants
grown in dehydration conditions suggesting that this protein could be more expressed and play a specific role in
dryness conditions. Thus, these minor forms of mPic in S.
cerevisiae and in A. thaliana would become important in
specific stress conditions.
Experimental procedures
Strains, media, genetic methods and plasmids
BMA64 is a diploid homozygous for ade2–1, ura3–1, his3–
11,-15, Dtrp1, leu 2–3,-112. CW30 is mat alpha, ade2–1,
ura3–1, his3–11,-15, trp1–1, leu 2–3,-112. Escherichia coli
techniques have been described in Sambrook et al. (1989),
yeast media and genetic methods in Dujardin et al. (1980).
Glucose and galactose were used as fermentable substrates,
glycerol and ethanol as non-fermentable substrates. Yeast
cells were transformed by the lithium acetate procedure of
Schiestl and Gietz (1989) for library screening and by the
one-step technique (Chen et al., 1992) for purified plasmids.
The yeast high copy expression vector pFL61 contains the
replication origin of the yeast 2m plasmid, the URA3 gene and
the promoter and terminator of the phosphoglycerate kinase
gene (PGK; Minet et al., 1992). The low copy expression
vector pFL61cen was constructed by replacing the 520 bp
ClaI fragment carrying the 2m replication origin by a 810-bp
ClaI fragment carrying CENIV and an Ars replication origin.
Construction of Dmir1, Dpic2 and Dmir1Dpic2 mutants
For MIR1, a 933-bp HincII fragment was replaced by a
1162 bp PvuII fragment carrying the TRP1 gene. For PIC2,
a 913 bp BglII-PstI fragment was replaced by a 2751 bp
NsiI-BamHI fragment carrying the LEU2 gene. The two constructions were used to transform the diploid strain BMA64
to tryptophan or leucine prototrophy. The Trp + or Leu + diploids were sporulated and microdissected to obtain the haploid strains Dpic2 or Dmir1 respectively. The double mutant
Dmir1 Dpic2 was constructed by crossing the two single
mutants.
Cloning of the wild-type MIR1 and PIC2 genes
The MIR1 cDNA was cloned by transforming the Dmir1 Dpic2
strain with a yeast cDNA library constructed in pFL61 (F.
Lacroute, unpubl.). Out of 5 ¥ 104 uracil prototrophic transformants, eight transformants able to grow on non-fermentable
medium were selected at 28∞C. All the plasmids (e.g.
YEpMK6) extracted from these transformants carry the MIR1
ORF. YEpMK6 carries a 1209 bp cDNA with the complete
MIR1 ORF as well as 25 bp upstream the AUG initiator codon
and 248 bp downstream the stop codon.
The PIC2 ORF was PCR-amplified from yeast genomic
DNA using Advantage 2 polymerase (Clontech) with specific
oligonucleotides (5¢-GGCAGTCAATGACACATTGTTAACTG;
3¢-CCTCTAGCACAGAGTGTTTACG). The 1057 bp PCR
product was first cloned in the pGEM-T Easy vector
(Promega) and then at the NotI sites of pFL61 in the sense
orientation with respect to the PGK promoter. The resulting
plasmid YEpY53 carries a wild-type PIC2 ORF which was
© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
Isoforms of the mitochondrial phosphate carrier 315
confirmed by sequencing. The NotI insert was then cloned in
the antisense orientation of pFL61 (YEpY55) and in the
pFL61cen vector in the sense (YCpY56) and antisense orientation (YCpY57) with respect to the PGK promoter.
bodies raised against Atp2p (Dr J. Velours, IBGC, Bordeaux,
France), Cpyp (Dr H. Riezman, Basel, Switzerland), Mir1p
(Dr N. Pfanner, Freiburg, Germany), c-myc (Dr JM. Galan,
Institut J. Monod, Paris, France).
Cloning of the A. thaliana orthologues
Acknowledgements
The A. thaliana cDNA AT5 was cloned by transforming the
Dmir1 Dpic2 strain with an A. thaliana cDNA library constructed in pFL61 (Minet et al., 1992). Out of 3 ¥ 104 Ura +
transformants, five were growing on glycerol medium after
10 days of incubation at 28∞C. All the plasmids extracted from
these transformants (e.g. YepAT5) were sequenced and
shown to correspond to AT5 cDNAs. YepAT5 is 1538 bp long
and carries the complete AT5 ORF as well as 73 bp upstream
the AUG initiator codon and 337 bp downstream the stop
codon.
The A. thaliana AT3 ORF was PCR-amplified from DNA
extracted from the A. thaliana cDNA library using a highfidelity
polymerase
(DyNAzyme
EXT
polymerase,
Finnzymes) and specific oligonucleotides(5¢-ATGTCTGACT
CAAGCAGATCGCTGATCC; 3¢-GCGCGGCCGGTAATTTT
ATGCACTTACAGATGG). The 1108 bp PCR product was first
cloned in the pGEM-T Easy vector (Promega) and then at
the NotI sites of pFL61 to give YEpAT3. The NotI insert of
YEpAT3 exhibits a wild-type AT3 sequence.
We thank M. Kermorgant, C. Germain and C. Tendeng for
their contribution to this work, Drs J. Velours, H. Riezman, N.
Pfanner and J.-M. Galan for the gift of antisera, Rosa
Castaldo for the excellent technical assistance, C.J. Herbert
and A.-M. Becam for help in the GFP experiment, Dr G.
Balavoine for help in the analysis of phylogenic trees and D.
Bernard and Prof C. Clarke for critical reading of the manuscript. This work is part of a common research project CNRS/
CNR and was supported by grants from the Association
Française contre les Myopathies and by a grant from the
MIUR-Cluster C03-Progetto 2, L.488/92. P.H. was supported
by a MENRT and Ligue Contre le Cancer de l’Essonne fellowship and in part by an American Heart Association postdoctoral fellowhip (0120100Y). Y. S.-G. was supported by a
MENRT fellowship.
C-myc and GFP tagging of Pic2p
Pic2p was tagged at its C-terminus with 13 c-myc epitopes,
using HIS3 as a marker gene, according to Longtine et al.
(1998). A Pic2p-gfp fusion protein was constructed using a
green fluorescent protein that is optimized for expression in
fungi and KanR as a marker gene, as described by Knop
et al. (1999). The PIC2-C-MYC and PIC2-GFP constructions were both integrated at the PIC2 locus and are therefore expressed under the control of the PIC2 regulatory
sequences. Amplified DNAs were used to transform the
strain CW30 to histidine prototrophy or kanamycin resistance. The His + strain YST9, expressing Pic2p-c-myc and
the KanR strain YST20, expressing Pic2p-gfp, are both able
to grow on fermentable medium at 36∞C showing that the
tagged proteins are fully functional. As a control, we have
also tagged Mir1p with an HA-epitope and confirmed a
mitochondrial localization (data not shown). Fluorescence
observations were performed with a Zeiss HBO
microscope.
Mitochondrial protein purification and Western blots
Cells were grown on complete or minimal galactosecontaining medium. Galactose medium (2% galactose, 0.1%
glucose) was used because it is a fermentable substrate that
does not repress mitochondrial functions and enable the
growth of our control strain Dmir1 Dpic2. Mitochondria were
purified following classic differential-centrifugation procedures after digestion of the cells par Zymolyase-100T (Seikagaku Kogyo) as described in Kermorgant et al. (1997). The
post-mitochondrial supernatant was concentrated by TCA
precipitation. Western blots were probed with specific anti© 2003 Blackwell Publishing Ltd, Molecular Microbiology, 51, 307–317
Supplementary material
The following material is available from
http://www.blackwellpublishing.com/products/journals/
suppmat/mmi/mmi3810/mmi3810sm.htm
Fig. S1. Alignment of functional and putative mitochondrial
phosphate carriers.
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Mir1p_S.cerevisiae
Pic2p_S.cerevisiae
mPicA_H.sapiens
mPicA_B.taurus
mPicAt5_A.thaliana
mPicAt3_A.thaliana
mPicAt2_A.thaliana
Mir1p_S.cerevisiae
Pic2p_S.cerevisiae
mPicA_H.sapiens
mPicA_B.taurus
mPicAt5_A.thaliana
mPicAt3_A.thaliana
mPicAt2_A.thaliana
Mir1p_S.cerevisiae
Pic2p_S.cerevisiae
mPicA_H.sapiens
mPicA_B.taurus
mPicAt5_A.thaliana
mPicAt3_A.thaliana
mPicAt2_A.thaliana
Mir1p_S.cerevisiae
Pic2p_S.cerevisiae
mPicA_H.sapiens
mPicA_B.taurus
mPicAt5_A.thaliana
mPicAt3_A.thaliana
mPicAt2_A.thaliana
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LEATRIRLVS.QPQFANGLVGGFSRILK.EEGIGSFYSGFTPILFKQIPYNIAKFLVFERASEFYYG..FAGPKEKLSSTSTTLLNLLSGLTAGLAAA
FEAIKVKQQTTMPPFCNNVVDGWKKMYAESGGMKAFYKGIVPLWCRQIPYTMCKFTSFEKIVQKIYS.VLPKKKEEMNALQQISVSFVGGYLAGILCA
MEAAKVRIQT.QPGYANTLRDAAPKMYK.EEGLKAFYKGVAPLWMRQIPYTMMKFACFERTVEALYKFVVPKPRSECSKPEQLVVTFVAGYIAGVFCA
MEAAKVRIQT.QPGYANTLRDAAPKMYK.EEGLKAFYKGVAPLWMRQIPYTMMKFACFERTVEALYKFVVPKPRSECSKPEQLVVTFVAGYIAGVFCA
FEAVKVRVQT.QPGFARGMSDGFPKFIK.SEGYGGLYKGLAPLWGRQIPYTMMKFASFETIVEMIYKYAIPNPKSECSKGLQLGVSFAGGYVAGVFCA
MEAVKVRVQT.QPGFARGLSDGLPKIIK.SEGFRGLHKGLVPLWGRQIPYTMMKFATFENTVELIYKKVMPTPKEECSKPVQLGVSFAGGYIAGIFCA
FEAIKVRVQT.QPMFAKGLLDGFPRVYR.SEGLAGFHRGLFPLWCRNLPFSMVMFSTFEQSVEFIYQKIIQKRKQDCSKAQQLGVTCLAGYTAGAVGT
300
*
320
*
340
*
360
*
380
IVSQPADTLLSKVNKTKKAPGQSTVGLLAQLAKQLGFFGSFA.GLPTRLVMVGTLTSLQFGIYGSLKSTLGCPPTIEIGGGGH.........
AVSHPADVMVSKINSERKANESMSVASKRIYQ.KIGFTGLWN.GLMVRIVMIGTLTSFQWLIYDSFKAYVGLPTTG................
IVSHPADSVVSVLNKEKGSSASLVLK.......RLGFKGVWK.GLFARIIMIGTLTALQWFIYDSVKVYFRLPRPPPPEMPESLKKKLGLTQ
IVSHPADSVVSVLNKEKGSSASEVLK.......RLGFRGVWK.GLFARIIMIGTLTALQWFIYDSVKVYFRLPRPPPPEMPESLKKKLGYTQ
IVSHPADNLVSFLNNAKGATVGDAVK.......KIGMVGLFTRGLPLRIVMIGTLTGAQWGLYDAFKVFVGLPTTGGVAPAPAIAATEAKA.
IISHPADNLVSFLNNSKGATVADAVK.......RLGLWGMLTRGLPLRIFMIGTLTGAQWVIYDAVKVLAGLPTTGGASPATALAPSVSA..
IISNPADVVLSSLYNNKAKNVLQAVR.......NIGFVGLFTRSLPVRITIVGPVITLQWFFYDAIKVLSGFPTSGGVKKPVDAAKLSV...
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100
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IDVVKTRIQLEPTVYNKGMVGSFKQIIAGEGAGALLTGFGPTLLGYSIQGAFKFGGYEVFKKFFIDNLGYDTASRYKNSVYMGSAAMAEFLADIALCP
LDLVKCRLQVNPKLYTS.NLQGFRKIIANEGWKKVYTGFGATFVGYSLQGAGKYGGYEYFKHLYSSWLSP......GVTVYLMASATAEFLADIMLCP
LDLVKCRMQVDPQKYKG.IFNGFSVTLKEDGVRGLAKGWAPTFLGYSMQGLCKFGFYEVFKVLYSNMLGEENTYLWRTSLYLAASASAEFFADIALAP
LDLVKCRMQVDPQKYKS.IFNGFSVTLKEDGFRGLAKGWAPTFIGYSLQGLCKFGFYEVFKVLYSNMLGEENAYLWRTSLYLAASASAEFFADIALAP
LDLVKCNMQIDPAKYKS.ISSGFGILLKEQGVKGFFRGWVPTLLGYSAQGACKFGFYEYFKKTYSDLAGPEYTAKYKTLIYLAGSASAEIIADIALCP
LDVIKCNMQIDPLKYKN.ITSAFKTTIKEQGLKGFTRGWSPTLLGYSAQGAFKYGLYEYAKKYYSDIVGPEYAAKYKTLIYLAGSASAEIVADVALCP
LDVLKVNMQVNPVKYNS.IPSGFSTLLREHGHSYLWRGWSGKLLGYGVQGGCRFGLYEYFKTLYSDVLP....NHNRTSIYFLSSASAQIFADMALCP
*
20
*
40
*
60
*
80
*
............................MSVSAAP.................................AIPQYSVSDYMKFALAGAIGCGSTHSSMVP
............................MESNKQP...........RK.....................IQLYTKEFYATCTLGGIIACGPTHSSITP
MFSSVAHLARANPFNTPHLQ..LVHDGLGDLRSSSP...GPTGQPRRP........RNLAAAAVEEQYSCDYGSGRFFILCGLGGIISCGTTHTALVP
MYSSVVHLARANPFNAPHLQ..LVHDGLAGPRSDPA...GPPGPPRRS........RNLAAAAVEEQYSCDYGSGRFFILCGLGGIISCGTTHTALVP
MESPKNSLIPSFLYSSSSSPRSFLLDQVLNSNSNAAFEKSPSPAPRSSPTSMISRKNFLIASPTEPGKGIEMYSPAFYAACTFGGILSCGLTHMTVTP
MSDSSRSLIPSFLYSSDHR...LFQATTMSTHLKSQPLISPTNSSVSS....N.GTSFAIATPNEK...VEMYSPAYFAACTVAGMLSCGITHTAITP
.............................MTRVKS.......KLD..E......................ELSSPWFYTVCTMGGMLSAGTTHLAITP
lys43 cys44
229
226
278
278
291
280
227
135
129
182
182
195
184
131
37
38
85
85
98
87
38
Functional mPics from Saccharomyces cerevisiae: Mir1p: (S12318), Pic2p (S50556), Homo sapiens: isoform A, (A53737) Bos Taurus: isoform A
(C53737), Arabidopsis thaliana At5 (BAB08283) At3 (CAB87913) and putative mPic from Arabidopsis thaliana: At2 (B84550) were aligned using
CLUSTALW algorithm (Blosum62 scoring matrix) in Bioedit software. The alignment was edited in the GeneDoc multiple alignment editor software.
Amino- acids which are identical or conserved in all sequences are shaded black and those conserved in 5 out of 7 sequences are shaded grey. The 22
residues higlighted in color correspond to amino-acids which were found critical or important for the phosphate transport activity of Mir1p on the basis of
mutagenesis studies (Phelps et al., 1991; Briggs et al., 1999; Phelps et al., 2001; Wohlrab et al., 2002 and references therein). Residues are highlighted in
yellow when the amino-acid in Mir1p is conserved in all carriers or only in the functional carriers. Residues are higlighted in blue when the amino-acid in
Mir1p is not conserved in all the functional carriers. The positions of the lysine 43 and the cysteine 44 in Pic2p are indicated by downward arrows. Position
1 corresponds to the first amino-acid of the Pic2p ORF. Mammalian PiCs display a N-terminal presequence which is cleaved off upon import into the
mitochondria.
Figure Supplemental data: Alignment of functional and putative mitochondrial phosphate carriers.