From the Department of Biochemistry and Molecular Biology, University of Texas Medical School, Houston, Texas 77225
Received for publication, April 25, 2001, and in revised form, May 2, 2001
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ABSTRACT |
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Reduction of mitochondrial cardiolipin (CL)
levels has been postulated to compromise directly the function of
several essential enzymes and processes of the mitochondria. There
is limited genetic evidence for the critical roles with which CL and
its precursor phosphatidylglycerol (PG) have been associated. A null
allele of the PGS1 gene from Saccharomyces
cerevisiae, which encodes the enzyme responsible for the
synthesis of the CL precursor PG phosphate, was created in a yeast
strain in which PGS1 expression is exogenously regulated by
doxycycline. The addition of increasing concentrations of doxycycline
to the growth medium causes a proportional decrease to undetectable
levels of PGS1 transcript, PG phosphate synthase activity,
and PG plus CL. The doubling time of this strain with increasing
doxycycline increases to senescence in non-fermentable carbon sources
or at high temperatures, conditions that do not support growth of the
pgs1 In eukaryotic cells, cardiolipin
(CL)1 and its precursor
phosphatidylglycerol (PG) are found almost exclusively in the inner mitochondrial membrane, where CL is the major anionic phospholipid (1).
Due to their intracellular distribution, these anionic phospholipids
have been postulated to be an essential component in many mitochondrial
processes such as electron transport, ion permeability, membrane
integrity, protein import, and solute transport (2, 3).
Mitochondria house the primary oxygen utilization centers of cells and
are therefore the major source of oxidative damage to tissue. Reduction
of CL levels is correlated with a compromise in terminal oxidation and
an increase in oxidative damage to tissue through the accumulation of
highly reactive intermediates generated by the electron transport chain
(ETC) (4, 5). Oxidative damage to CL appears to be a major factor in
aging (6, 7). Because of the high level of unsaturation of its fatty
acyl groups, CL is the primary and specific target of lipid
peroxidation during ischemia and reperfusion (8, 9). Alterations in
mitochondrial functions reliant on CL also are associated with
Alzheimer's and several other neurodegenerative diseases (7, 10).
One of the most important mitochondrial processes with which anionic
phospholipids are thought to participate is protein import (11). This
function is inhibited by adriamycin, an antibiotic that specifically
binds anionic phospholipids (12). The 25 amino acid presequence of
cytochrome c oxidase (Cox) subunit IV (Cox4p), widely
utilized as a model peptide for mitochondrial targeting sequences, is
both stabilized and properly oriented by CL (13). Another unique
property of CL is its ability to adopt non-bilayer structures in the
presence of divalent cations such as calcium (14, 15) which is a known
regulator of mitochondrial function. These structures are believed
necessary for protein import (16, 17). Anionic phospholipids have also
been shown to provide the membrane organization sites in
Escherichia coli for complexes composed of both integral and
peripheral membrane proteins that are responsible for initiation of DNA
replication (18) and protein translocation across membranes (19).
A widely studied phospholipid/protein interaction is that of CL with
Cox (20). Many studies have demonstrated the necessity of CL for the
in vitro function of this enzyme complex (21). Adriamycin
inhibits its function (22), and decreased CL levels are associated with
a decline in the function of this enzyme in aged cells (23). In yeast,
CL synthase activity is proportional to Cox activity at different
stages of the cell cycle (24), and Cox activity has been shown to be
diminished in mutants (crd1 Only a few reports appear to demonstrate a requirement for CL in
vivo at the molecular level for any of the above processes. The
most direct evidence for the essential role of PG and CL in mammalian
cells comes from Chinese hamster ovary cells with a mutation (26) in
the gene encoding the PG phosphate synthase (PGPS). The enzyme
catalyzes the committed step of CL biosynthesis, and the strain,
therefore, has reduced levels of both PG and CL (27, 28). These mutants
have defects in electron transport and ATP production, show reduced
oxygen utilization, possess gross alterations in mitochondrial
morphology, are temperature-sensitive at 37 °C, and eventually lose
viability even when grown with glucose. A yeast model system based on a
null mutant of CL synthase has also been studied (25). However, the
phenotypic changes brought about by this mutation are not dramatic
(29-31) suggesting the major roles of CL are substituted by an
increase in PG. More severe mitochondrial dysfunction occurs when PG
levels are also reduced by growth of this mutant on glucose rather than
a non-fermentable carbon source. However, any conclusions drawn from
these experiments are compromised by the extreme effects changes in
carbon source have on growth characteristics of cells in general and
mitochondrial development and function in particular.
In the present study, all mitochondrial PG and CL were depleted by
disruption of PGS1, which encodes PGPS (32). Null mutants in
PGS1 are temperature-sensitive, cannot grow on
non-fermentable carbon sources, and are petite lethal (32-34). A
regulatable PGS1 gene was introduced on a plasmid into the
pgs1 Strains, Plasmids, and Growth Conditions--
The wild type
yeast strain utilized in this study is YPH499 (ade2-101,
his3
Because fluorescence microscopy is difficult in an ade2
strain due to the build up of a red pigment, ADE2 revertants
were created in both the wild type parental and pgs1
The doxycycline-regulated plasmid, pDO292 (CEN4,
ARS1, URA3, CMVp/TetR DNA Binding
Domain/VP16 Activation Domain,
TetO/CYC1p/PGS1/CYC1t), was
constructed as follows. Cloning of PGS1 was accomplished by PCR amplification from genomic DNA isolated (38) from strain DL1
(39) with primers
5'-ataggatccATGACGACTCGTTTGCTCCAACTCACTCGTCCTC-3' and
5'-atagcggccgcCTAAAGTTTTTTACCCAAAATGGAGGTAGC-3'.
(Capital letters refer to regions of PGS1 homology
with the start and stop codons underlined, respectively, and lowercase
letters refer to restriction sites and filler DNA.) In order to insert
the gene 3' of the tetracycline-regulated promoter, the 1.59-kilobase
pair amplicon was cut with BamHI and NotI and
ligated into plasmid pCM189 (40) cut with the same restriction enzymes.
Because of problems associated with the stability of PGS1
plasmids in E. coli, ligations were transformed into and
plasmids stored in yeast. Yeast spheroplast transformations of
pgs1 Quantitative Reverse Transcriptase (RT) PCR--
Yeast strains
were grown in YEP medium with 2% sucrose to the mid-exponential phase
of growth. Total RNA was isolated using Trizol (Life Technologies,
Inc.) with 0.5-mm zirconia/silica beads and a Mini BeadBeater (BioSpec
Products). RNA was quantified by spectroscopy. Primers (see Table I for
a list used) were selected to amplify transcribed regions from 500 to
800 base pairs in length by Oligo Primer Analysis Software. RT-PCR was
accomplished using the Access System (Promega). Primers used for
COX1 and 21 S RNA were designed to detect only properly
spliced messages. Quantification was accomplished by varying either the
number of cycles or RNA concentrations. Care was taken to ensure that
the amplification had not plateaued and that the reactions produced
bands of similar intensities upon replication (no variability in
starting exponential PCR amplification). Each preparation of RNA was
standardized using primers for either COX4, COB,
or ACT1. Standard reactions used 25 cycles, 50 pmol of each
primer, and 100 ng/µl RNA in 50 µl. Amplicons were separated by
agarose electrophoresis (Seakem, FMC), visualized with Sybr Green
(Molecular Probes), and quantified using a Fluor-S MultiImager
(Bio-Rad).
PGPS Enzymatic Assays and Phospholipid Quantification--
Yeast
strains were grown to the mid-exponential phase of growth in YEP medium
with 2% sucrose. Crude mitochondrial preparations were isolated (42)
using a Mini BeadBeater (BioSpec Products) and differential
centrifugation. PGPS assays were performed essentially as described
previously (32) by conversion of [14C]glycerol
3-phosphate (PerkinElmer Life Sciences) to [14C]PG
phosphate dependent on CDP-diacylglycerol (Avanti, dioleoyl). The
resulting mixture of [14C]PG phosphate and
[14C]PG was isolated and quantified in a liquid
scintillation counter (Amersham Pharmacia Biotech). One unit is
equivalent to the formation of 1 nmol of PG phosphate plus PG per min
per mg of protein.
Phospholipid quantification was performed essentially as described
previously (32). Cells were labeled for 12 h with 50 µCi of
[32P]Pi (Amersham Pharmacia Biotech) in
synthetic defined media (36) with 2% sucrose to the mid-exponential
phase of growth. Cells were homogenized with a solution of 10:5:4
CH3OH, CHCl3, 0.1 N HCl in a Mini
BeadBeater (BioSpec Products). Lipids were isolated by acid organic
extraction (half volumes each of CHCl3 and 0.1 N HCl, 0.5 M NaCl), dried, and resuspended in
an appropriate volume of CHCl3. Phospholipids were
separated by thin layer chromatography using silica gel plates
(Whatman) (43), and individual species were identified by
co-migration of standards (Sigma). Phospholipid quantification was
accomplished using both one- and two-dimensional thin layer
chromatography. The spots were quantified by phosphorimaging using
either an Instant Imager (Packard Instrument Co.) or a Molecular Imager
FX (Bio-Rad) and normalized to the total amount of organic soluble
32P.
Fluorescent Dye Staining of Mitochondria--
The wild type and
psg1-regulatable strains were grown with no or 10 µg/ml
doxycycline in YEP medium with 2% sucrose to the mid-exponential phase
of growth. Cells were stained for 30 min with 50 nM
10-N-nonyl acridine orange (NAO) (44) or 20 µM
2-(4-dimethylaminostyryl)-1-methylpyridinium iodide (DASPMI) (45) and
visualized with a 100× objective using a FITC filter. 50 µM carbonyl cyanide
p-trifluoromethoxyphenylhydrazone (FCCP) was added to
DASPMI-stained cells grown without doxycycline to demonstrate complete
dissipation of mitochondrial membrane potential.
Mitochondrial Isolation--
The isolation of mitochondria was
accomplished essentially by the method of Glick and Pon (46). Cells
were grown for 3 days in increasing volumes of YEP media with 2%
sucrose to the mid-exponential phase of growth. Cells were harvested
and spheroplasts prepared with zymolyase (ICN). Spheroplasted cells
were lysed in hypotonic media by Dounce homogenization or with an
Omnimixer (Omni). A crude mitochondrial preparation (pellet) was
separated from the endoplasmic reticulum fraction (supernatant) by
differential centrifugation. Highly purified mitochondria were obtained
by sedimentation through a sucrose step gradient (47). A 70% sucrose
cushion was employed when isolating mitochondria from the
psg1-regulatable strain grown under repressing conditions.
The mitochondrial associated microsomal membranes (MAM) fraction was
isolated from the upper quarter of the gradient (48). Proteins from
each fraction were concentrated using a TL100 ultracentrifuge
(Beckman Instruments).
Proteolipid isolation of ATPase subunits was accomplished as
described (49) by organic extraction of sonicated highly purified mitochondria. ATP6p, ATP8p, and ATP9p were separated by
SDS-polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining.
In Vitro Transcription/Translation and Mitochondrial Protein
Import Assays--
COX4 and COX6 were cloned
into plasmid pCITE (CAP-independent translation enhancer, Novagen)
by amplification of genomic DNA from strain DL1 using primer
pairs 5'-ataccATGgTTTCACTACGTCAATCTATAA-3' plus
5'-atagcatgcAGAAGGTAAAAAGTAAAAGAGAAAC-3' and
5'-ataccATGgTATCAAGGGCCATATTCAGAA-3' plus
5'-atagcatgcTTGTGTGGTAGCTTTTTCCTTATTA-3', respectively. The amplicons
and parent vector were cut with NcoI and SphI and
ligated producing plasmids pDO290 (COX4) and pDO291
(COX6) in which the genes were expressed from the plasmid T7
promoter. Constructs were verified by sequencing. In vitro
transcription/translation was accomplished using the STP3 kit (Novagen)
with 500 ng of plasmid DNA in each 100-µl transcription/translation
reaction. Ribosomes were removed by centrifugation, and the
biotinylated protein products were denatured with 5 M urea.
The proteins were then prepared for import assays by 10-fold dilution.
Mitochondrial protein import assays were accomplished as described
(50). Highly purified mitochondria were thawed on ice and potentiated
with ATP. The assays were performed with 50 µg of mitochondrial
protein and 20 µl of the diluted biotinylation reaction. The
reactions were performed for the times indicated and stopped with
valinomycin (Sigma). Mitochondria were treated with proteinase K,
extensively washed, resuspended in protein loading reagent (Bio-Rad),
and subjected to SDS-PAGE in 5-20% gradient Ready gels (Bio-Rad).
Protein products were detected by Western blotting using
streptavidin-linked horseradish peroxidase and chemiluminescence
(Novagen) and quantified using either autoradiography and densitometry
or a Molecular Imager FX (Bio-Rad).
Western Blotting Analysis--
Protein samples were subjected to
SDS-PAGE in 5-20% gradient Ready gels and electrophoretically
transferred to nitrocellulose sheets. Sheets were probed with the
antibodies listed in Table II and detected using horseradish peroxidase
secondary antibody and chemiluminescence (Amersham Pharmacia Biotech)
as described above.
Pulse-Chase Protein Labeling--
Yeast strains were grown in
sulfate-deficient growth medium (0.3% yeast extract, 1 g/liter
NH4Cl, 1 g/liter KH2PO4, 600 mg/liter MgCl2, 500 mg/liter NaCl, 400 mg/liter
CaCl2, 5 mg/liter FeCl2) with 2% sucrose to
the mid-exponential phase of growth. Proteins were labeled with 50 µCi of [35S]methionine and [35S]cysteine
(Amersham Pharmacia Biotech) in spheroplasts for 10 min as described
(51). The labeling was chased with cold amino acids, and the reactions
were stopped at the times indicated. Protein was precipitated with
trichloroacetic acid and neutralized with NH4OH prior to
immunoprecipitation. Immunoprecipitations were optimized and performed
as described (52) using Pansorbin heat-killed Staphylococcus
aureus (Calbiochem), separated by SDS-PAGE, and the gels dried.
Detection and quantification were accomplished using a Molecular Imager
FX (Bio-Rad).
Construction of a Yeast System for the Exogenous Regulation of
Mitochondrial Anionic Phospholipids--
A complete PGS1
null mutant was created in a multiply auxotrophic laboratory strain.
This was accomplished by substituting the PGS1 open reading
frame with the TRP1 selectable marker by homologous
recombination. Only a very short stretch of the promoter and terminator
of the gene were excised, and the entire open reading frame was
deleted. Genomic PCR and Southern analysis confirmed the deletion (data
not shown).
All of the phenotypes observed with the previously described
psg1
A low copy "tet-off" plasmid system, pDO292, was employed to afford
exogenous regulation of the PGS1 gene. (A high copy version of the plasmid was not fully repressible.) All phenotypes were observed
to return to wild type when strains containing this plasmid were grown
without the tetracycline derivative doxycycline. The following
experiments were performed in the two the psg1
In order to test the effect of doxycycline on the regulation of the
PGS1 plasmid in the pgs1
In order to test the effect of doxycycline on PGPS activity in the
psg1-regulatable strain, enzyme assays were employed with crude mitochondrial fractions from the strain grown in different concentrations of doxycycline. There is a well regulated decrease to
undetectable levels of PGPS specific activity as the concentration of
doxycycline is increased (Fig. 2B). The specific activity of PGPS in the parental strain is ~1.3 nmol/min/mg. Therefore, the enzyme activity of this strain grown under fully derepressing conditions (without doxycycline) is ~33% above wild type levels.
In order to test the effect of doxycycline on the relative levels of PG
and CL in the psg1-regulatable strain, the strain was grown
in different concentrations of doxycycline in the presence of
[32P]Pi. Lipids were extracted and separated
by thin layer chromatography. Phospholipids were detected by
autoradiography and quantified with a PhosphorImager. All of the
phospholipids except PG and CL remained unchanged with different
concentrations of doxycycline (Fig. 3).
There is a well regulated decrease to undetectable levels of both PG
and CL as the concentration of doxycycline is increased. The relative
percentage of PG in wild type cells is ~0.5% and for CL it is 1.5%,
showing no discernible increase for these phospholipids in the
fully derepressed strain.
Another method of CL detection is fluorescent staining with the
CL-specific dye NAO (44). Fig. 4 shows
mitochondrial staining of the psg1-regulatable cells with
and without doxycycline. Without doxycycline, the mitochondrial network
is clearly stained. When the strain is grown with doxycycline, no
staining of CL is detected even when the image is overexposed.
psg1
In order to determine the effect of doxycycline on overall
mitochondrial membrane potential, the fluorescent dye DASPMI, a vital
stain sensitive to mitochondrial membrane potential (45), was used to
stain cells in a growing culture. The mitochondrial potential, which is
observed for the psg1-regulatable cells in the absence of
doxycycline, is indistinguishable from that of the wild type parental
cells (Fig. 6). The mitochondrial network is clearly stained in these cells. However, no energized mitochondria are detectable when the cells are grown with doxycycline even when the
image is overexposed. This lack of staining is indistinguishable from
that of the strain grown without doxycycline when the mitochondrial membrane potential is collapsed with the uncoupler FCCP.
Analysis of Protein Levels in Anionic Phospholipid-depleted
Mitochondria--
In order to test protein import into mitochondria
lacking the major anionic phospholipids, highly purified mitochondria
are required. Specifically, the amount of contaminating microsomal fraction must be minimized. Crude mitochondrial preparations were purified by sedimentation through a sucrose gradient that separates purified mitochondria from MAM (47). Immunoblotting was utilized to
determine the purity of the respective fractions. The amount of signal
from antibodies to porin (the outer mitochondrial membrane voltage
dependent anion channel) increased on a per mg of protein basis as the
mitochondrial fraction was purified (Fig.
7A). However, a significant
amount of this protein was observed in the MAM fraction. In order to
determine the amount of microsomal protein contaminating the purified
mitochondrial fraction, antibody to
dolichyl-phosphate-
Because of the possibility that the mitochondrial isolation protocol
was significantly decreasing the mitochondrial yield, immunoblotting
was performed to determine the amount of mitochondrial inner membrane
protein associated with the MAM fraction. Insignificant amounts of
Cox2p can be seen in this fraction (Fig. 7C, lane 2). However, significant amounts of outer membrane proteins are again observed (Fig. 7D, lane 2). As discussed below, Cox2p is
only detected in cell fractions from cells containing PG and CL (Fig. 7C, lanes 1 and 2 versus lanes
3 and 4), whereas porin is present independent of
anionic phospholipid content (Fig. 7D).
As a first step in determining if the loss of anionic phospholipids had
any effect on protein import, purified mitochondria were used to study
the relative amounts of various mitochondrial proteins. Proteins
(integral membrane, peripheral, and soluble) from all four compartments
(outer membrane, intermembrane space, inner membrane, and matrix), both
nuclear and mitochondria encoded, were quantified by immunoblot
analysis. The majority of proteins tested in doxycycline-grown
cells was not altered in amount from cells grown without doxycycline
(see Table II for summary of proteins tested); the results with porin
(Fig. 8F) are representative
of this group of proteins. The only proteins found to be in lower abundance, indeed practically absent, in the doxycycline-grown psg1-regulatable strain were cytochrome b and the
largest four subunits of Cox (Fig. 8, A-E). Cox subunits 5, 6, and 7 were present at reduced levels from wild type but clearly well
above the levels of the larger subunits of Cox. Densitometry analyses
of immunoblots with different preparations of isolated mitochondria
were used to obtain relative measures of these proteins in cells grown
with and without doxycycline. Cox1p is reduced 6-fold in
doxycycline-grown psg1-regulatable cells, whereas Cox3p is
reduced 15-fold. Cox2p, Cox4p, and Cobp are all reduced more than
25-fold. These results were also confirmed by comparison of protein
amounts in the psg1
In order to determine if the amount of these ETC proteins is in
proportion to the amount of anionic phospholipid present in mitochondria, immunoblots were performed on mitochondria isolated from
the psg1-regulatable strain grown with different doxycycline concentrations. When the strain is grown under derepressing conditions, the amount of Cox4p is indistinguishable from that found in the PGS1 parental strain (Fig. 8G). However, the
amount of Cox4p diminishes with increasing repression of
PGS1 expression until no protein is detectable at
doxycycline concentrations consistent with complete repression of the
PGS1 gene expression. A proportional decrease is also
observed for the mitochondrial encoded protein Cox2p (Fig. 8H).
A common mechanism for regulation of protein levels in cells occurs at
the level of transcription. In order to determine if lack of these
proteins in the doxycycline-grown psg1-regulatable strain is
due to reduced transcription of their respective genes, quantitative
RT-PCR was employed to determine the amount of message expressed from
these genes (see Table I for primers
used). In no case were message levels affected by doxycycline
concentration except the PGS1 message that is clearly
observed to decrease with increasing concentrations of doxycycline
(Fig. 9, A and B).
No change is observed for either nuclear encoded COX4 (Fig.
9A) or mitochondrial encoded COB (Fig.
9B) message. RT-PCR experiments with COX2,
ATP6, and 21 S rRNA show that mitochondrial gene
transcription is unaffected and that proper splicing occurs (Fig.
9C).
Of the five proteins found absent in the doxycycline-grown
psg1-regulatable strain, four are encoded by mitochondrial
DNA. In order to determine if the other four mitochondrial encoded proteins (55) are down-regulated, it was first necessary to determine
their presence in the pgs1 Mechanism of Mitochondrial ETC Protein Down-regulation--
In
order to test directly protein import into anionic
phospholipid-depleted mitochondria, in vitro mitochondrial
protein import assays were performed with highly purified mitochondria
isolated from both the psg1
In order to determine whether the proteins found to be missing in
pgs1
Mitochondrial encoded proteins are individually regulated at the level
of translation by specific nuclear encoded mitochondrial translation
activation factors (NEMTAFs) (58). In order to determine if the lack of
translation observed was caused by repression of these factors,
quantitative RT-PCR was performed with primers specific for messages of
these factors. In every case, message levels were found somewhat lower
in the psg1 The construction of a "biological reagent" to probe the role
of anionic phospholipids in mitochondrial function has allowed us to
determine the molecular basis for the dependence of pgs1 Not surprising cells repressed for expression of PGS1 lack
components of the ETC such as mitochondrial encoded Cobp, Cox1p, Cox2p,
and Cox3p. Prior to identifying the PEL1 (renamed
PGS1) gene product as PGPS, pel1 mutants were
shown to be respiratory-deficient and lacking the spectra associated
with Cox and cytochrome b (34, 59). The surprising finding
is that the molecular basis for lack of these proteins is failure to
translate their respective messages that are present in normal amounts.
In addition, nuclear encoded Cox4p is missing for the same reason.
However, other nuclear encoded proteins found in all sub-compartments
of the mitochondria as well as the remaining mitochondrial encoded
proteins are present in normal amounts.
The lack of the mitochondrial encoded components of the ETC cannot be
attributed to the lack of Cox4p. In cox4 Some mitochondrial encoded proteins are inserted into the membrane
co-translationally and utilize nuclear encoded proteins that must
themselves be imported into the mitochondria (62). Cox4p may also be
co-translationally imported (63). Therefore, lack of the machinery for
co-translational insertion of proteins or a dependence on anionic
phospholipids to couple translation with insertion might result in the
lack of their detection at both steady state and in pulse-chase
experiments. Since Cox4p is still posttranslationally imported in
vitro into PG/CL-depleted mitochondria and no precursor is
detected in vivo, the anionic phospholipid-dependent step is at the level of translation.
Because general protein insertion of most nuclear and mitochondrial
encoded proteins seems unaffected without PG and CL, perhaps the lack of translation possibly coupled with insertion is specific for mitochondrial encoded proteins of the ETC and for Cox4p. However, lack
of the Oxa1p, which is required for membrane insertion of Cox2p and
Cox3p, results in accumulation of these proteins in the matrix and not
in lack of translation (64, 65). The bacterial Sec-dependent membrane translocation system shows a strong
dependence on anionic phospholipids for function (19). Although there
are no homologues of the Sec system in the mitochondria, mitochondria may possess similar uncharacterized systems that are dependent on
anionic phospholipids for organization of functional membrane associated complexes.
Although lack of DASPMI staining indicates a low membrane potential in
the absence of anionic phospholipid, there must be sufficient membrane
potential to support the minimal requirements of protein import (50)
that were inhibited in vitro by addition of an ionophore.
Our results indicate there is little or no dependence of protein import
on anionic phospholipids. A previous study showed there was a
detectable reduction in protein import when a crd1 Since lack of anionic phospholipids results in failure to translate
components of the ETC, our biological reagent cannot be used to test
whether components of the ETC require these lipids for function (2, 3).
However, in vitro reconstitution experiments show a
necessity for CL for ATP synthase activity (67) and particularly the
ADP/ATP translocase (68, 69). Activation of mitochondria from
psg1 In summary, we have constructed a biological reagent that has proven
useful in defining the molecular basis for the dysfunction of
mitochondria lacking PG and CL. This reagent should be useful in
determining the apparent requirement of these lipids in the translation
of a subset of proteins integral to ETC function. Such studies may lead
to uncovering additional factors involved in the complex process of
translocation of proteins across membranes and their insertion into the
membrane bilayer.
strain. Doxycycline addition also causes
mitochondrial abnormalities as observed by fluorescence microscopy.
Products of four mitochondrial encoded genes (COX1, COX2, COX3, and COB) and one
nuclear encoded gene (COX4) associated with the
mitochondrial inner membrane are not present when PGS1 expression is fully repressed. No translation of these proteins can be
detected in cells lacking the PGS1 gene product, although transcription and splicing appear unaffected. Protein import of other
nuclear encoded proteins remains unaffected. The remaining proteins
encoded by mitochondrial DNA are expressed and translated normally.
Thus, the molecular basis for the lack of mitochondrial function in
pgs1
cells is the failure to translate gene products essential to the electron transport chain.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) lacking CL synthase
(25).
strain in order to study the effects of attenuating
anionic phospholipids without the need to alter growth conditions or
compare different strains. The resulting strain was used to determine
the molecular basis for dysfunction of mitochondria lacking these phospholipids.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
200, leu2
1,
lys2-801, trp1
63,
ura3-52, MATa) (35). Disruption of
PGS1 was accomplished by homologous recombination with a
polymerase chain reaction (PCR) product of TRP1 carried on
plasmid pRS304 (35) by amplification with primers
5'-GCAGCCCACACAAGAAAGTAGATATAATGTAGGACACCCAGCTTGTCCATAATTGCTAATAGCATACTCAGGATAggtattttctccttacgcatctgtg-3' and
5'-GCTCTTCATCTCTTGTAATAATGAGAGCATTCGATTCAAATCTAATGAATACGCCCTTCTCGTATAGTTTGAAGagagtgcaccaaacgacattactat-3'. (Capital letters refer to regions of PGS1 homology
(promoter for the first primer and terminator for the second), and
lowercase letters refer to regions of TRP1 homology.) The
resulting PCR product contained the TRP1 gene flanked by DNA
homologous to regions 5' and 3' to PGS1. PGS1 null strains
were selected on tryptophan dropout minimal media glucose plates (36);
pgs1
strains were identified by PCR analysis of isolated
genomic DNA as described below. Many of the experiments were duplicated
in strain YCD4 (his3-11, 15, leu2-3, 112, pgs1
::HIS3, ura3-251, 328, 372, MATa), another pgs1
strain, (32) together with its PGS1 wild type parent, DL1,
completely unrelated to YPH499, as described under "Results."
Because of problems associated with the recovery of pgs1
cells from stationary phase and the high mortality resulting from
freezing, only mid-log phase cells were used for frozen stocks, and
fresh culture plates were prepared every 2-3 weeks.
strains. The gene was amplified from plasmid pRS402 (35) with
primers 5'-TTTTCTTAAAAGAATCAAAGACAGATAAAA-3' and
5'-CACACCGCATAGATCTTATGTATGAAATTC-3', and this product was used
to transform the strains to white colonies on adenine dropout media.
The ADE2 derivatives of YPH499 and its pgs1
derivative were named SDO224 (referred to as the "wild type"
strain) and SDO225 (referred to as the "psg1
" strain), respectively. These were the strains used in this study.
0 derivatives of the wild type strain were created with
ethidium bromide as described previously (37) as a control of the
inability to create
0 derivatives of the
psg1
strain.
strains were accomplished as described (41).
Transformants were selected on yeast nitrogen base uracil dropout
medium containing ethanol and glycerol (36). The construct was verified
by sequencing PCR products obtained by amplification of DNA isolated
from the strain. The psg1
strain carrying this plasmid is
referred to throughout the text as the "psg1-regulatable" strain and was maintained at 10 µg/ml doxycycline (fully repressed) on YEP (1% yeast extract, 2%
peptone) medium with 2% sucrose. Derepression was accomplished by
growth for at least 24 h in lower doxycycline concentrations.
Strain YCD4 (also a psg1
strain) was also transformed
with the plasmid and tested in several experiments as described under
"Results."
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
strain, YCD4 (32), an incomplete disruption of the
open reading frame, were verified in the new genetic background; the psg1
cells have no detectable PG or CL (see Fig. 3). This
was true whether grown in glucose or sucrose. The strain is
temperature-sensitive at 37 °C and grows with the same doubling time
as its wild type parent on YEP media containing glucose (Fig.
1A). The strain can grow on
defined (minimal) media only if supplemented appropriately. The
psg1
strain grows slowly on sucrose (Fig. 1B)
and not at all on galactose, maltose, melibiose, ethanol, or glycerol.
The strain enters stationary phase at a lower cell density than its wild type parent and has difficulty recovering from late stationary phase. The strain is also petite lethal, i.e. it cannot be
induced to lose its mtDNA as observed previously (53). Although growth of the psg1
strain on sucrose was slower than on glucose,
the former carbon source was selected for subsequent experiments to avoid the complications of catabolite repression of mitochondrial biogenesis and function induced by glucose (54).
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Fig. 1.
Glucose and sucrose growth curves.
SDO224 (wild type, WT) and SDO225 (pgs1 ) were
grown for eight generations in rich media cultures using carbon sources
of either 2% glucose (A) or sucrose (B). Late
logarithmic cultures were used to inoculate media of the same
composition at an A540 of 0.02 (A) or 0.005 (B). Optical density readings were
taken at the indicated times. The experiment was performed in duplicate
at least three times, and the points shown have less than 5% standard
deviation. The results for SDO225 (pgs1
) are
indistinguishable from those obtained with strain YCD4
(pgs1
) and represent averages of data obtained with both
strains. Doubling times were calculated by finding the least squares
best line fit to the points in the exponential phase of growth. The
doubling time in glucose of SDO224 pgs1
(wild type) is
1.52 ± 0.11 h, and SDO225 (pgs1
0 is 1.51 ± 0.11 h; in sucrose, SDO224 (wild type) is 2.45 ± 0.08 h, and SDO225 (pgs1
) is 3.10 ± 0.14 h.
strains SDO225 and YCD4 (32).
strain, quantitative
RT-PCR was employed with RNA isolated from the strain grown in
different concentrations of doxycycline. There is a well regulated
decrease to undetectable levels of PGS1 mRNA as the
concentration of doxycycline is increased (Fig.
2A, see also Fig. 9,
A and B). The amount of PGS1 message
without doxycycline is ~30% higher than the amount in the parental
strain.
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Fig. 2.
Effect of doxycycline on PGS1
transcript level and PGPS enzymatic activity. A,
SDO225/pDO292 (pgs1-regulatable strain) was grown for eight
generations in rich media with 2% sucrose with the indicated
concentrations of doxycycline to the mid-exponential phase of growth.
Total RNA was isolated and subjected to quantitative RT-PCR with
PGS1-specific primers (see Table I and Fig. 9, A
and B). The results are expressed as percentage of fully
derepressed levels. The experiments were performed in duplicate on at
least two occasions with two distinct RNA preparations. The results are
indistinguishable from those obtained with strain YCD4/pDO292
(pgs1-regulatable strain) and represent averages and
standard deviations of data obtained with both strains. B,
SDO225/pDO292 (pgs1-regulatable strain) was grown for eight
generations in rich media with 2% sucrose with the indicated
concentrations of doxycycline to the mid-exponential phase of growth.
Crude mitochondrial preparations were isolated and PGPS assays
performed. The assays were performed in duplicate on two occasions with
at least three distinct membrane preparations. The results are
indistinguishable from those obtained with strain YCD4/pDO292
(pgs1-regulatable strain) and represent averages and
standard deviations of data obtained with both strains.
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Fig. 3.
Phospholipid analysis. SDO225/pDO292
(pgs1-regulatable strain) was grown for 24 h in rich
media with 2% sucrose and the indicated concentrations of doxycycline
([Doxy], µg/ml). The cells were subsequently grown for
eight generations in synthetic complete sucrose medium with
[32P]Pi to the mid-exponential phase of
growth. Lipids were extracted and separated by thin layer
chromatography. A shows one-dimensional analysis, although
the data in B are a representation of both one- and
two-dimensional analyses. The phospholipid species were identified with
standards and quantified by both PhosphorImager analysis and
scintillation counting of the isolated spots. PG and PS, which are not
visible in the autoradiogram reproduction, were nevertheless identified
by phosphorimaging and can be visualized by overexposure of the TLC
plate to film. The results in B are expressed as a
percentage of total phospholipid counts. The experiment was performed
using four distinct phospholipid preparations. The results are
indistinguishable from those obtained with strain YCD4/pDO292
(pgs1-regulatable strain) and represent averages of data
obtained with both strains. The data have a standard deviation of less
than 8%. PA, phosphatidic acid; PC,
phosphatidylcholine; PE, phosphatidylethanolamine;
PI, phosphatidylinositol; PS, phosphatidylserine;
SL, sphingolipids.
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Fig. 4.
Nonyl acridine orange staining.
SDO225/pDO292 (pgs1-regulatable strain) was grown in rich
sucrose media with no ( ) or 10 µg/ml (+) doxycycline to the
mid-exponential phase of growth. Cells were stained for 30 min with 50 nM 10-N-nonyl acridine orange and visualized
with a 100× oil immersion phase contrast objective using a FITC
filter. Exposure times were 1/60th second for phase contrast, 1 s
for no doxycycline, and an 8-s overexposure for doxycycline fluorescent
images.
yeast cells cannot grow on non-fermentable carbon
sources and are temperature-sensitive at 37 °C (32). In order to observe the effect of doxycycline on these phenotypes, the optical density of a culture using non-fermentable carbon sources was measured
in different doxycycline concentrations. Increased doxycycline caused
an increased doubling time of the strain to the point of senescence
(Fig. 5). This lack of growth corresponds
to the concentration of doxycycline that showed no expression of the
gene, no detectable enzymatic activity, and no detectable PG or CL. The
same results were observed with growth at 37 °C in rich medium with
2% sucrose with increasing concentrations of doxycycline.
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Fig. 5.
Effect of doxycycline on growth rate in
non-fermentable carbon sources. A late logarithmic culture of
SDO225/pDO292 (pgs1-regulatable strain) grown in rich media
containing ethanol and glycerol was used to inoculate cultures at an
A540 of 0.025 with the same composition but with
the indicated concentrations of doxycycline ([Doxy],
µg/ml). The optical density of the culture was determined at the
times indicated. The experiment was performed in duplicate on four
occasions. The results are indistinguishable from those obtained with
strain YCD4/pDO292 (pgs1-regulatable strain) and represent
averages and standard deviations of data obtained with both
strains.
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Fig. 6.
DASPMI staining. SDO224 (wild type) and
SDO225/pDO292 (pgs1-regulatable strain) were grown in rich
sucrose media with no or 10 µg/ml doxycycline (Doxy).
Cells were stained with 20 µM DASPMI and visualized with
a 100× objective using a FITC filter. 50 µM FCCP was
also added to the no doxycycline strain as an uncoupling agent (+ Unc). Exposure times were 1/60th second for phase contrast, 1 s for parental and no doxycycline fluorescent, and an 8-s overexposure
for doxycycline and uncoupler fluorescent images.
-D-mannosyltransferase (an
endoplasmic reticulum lumenal enzyme) was employed (Fig.
7B). Significant amounts of this protein were observed in
the MAM fraction. However, no detectable amounts were observed in the
purified mitochondrial fraction.
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Fig. 7.
Mitochondrial isolation. A
and B, samples of identical protein amounts (25 µg) from
various steps in the isolation of purified mitochondria from SDO224
(wild type) were analyzed by immunoblotting. A is an
immunoblot with antibodies to porin, a mitochondrial outer membrane
protein. Lane 1, cell lysate; lane 2, crude
mitochondrial fraction; lane 3, purified mitochondria;
lane 4, MAM. B is an immunoblot with antibodies
to dolichyl-phosphate- -D-mannosyltransferase
(DPMT), an endoplasmic reticulum protein. Lane 1,
cell lysate; lane 2, microsomal fraction; lane 3,
crude mitochondria; lane 4, purified mitochondria;
lane 5, MAM. C and D, purified
mitochondria containing 12 µg of protein from SDO225/pDO292
(pgs1-regulatable strain) grown without (lane 1)
or with 10 µg/ml (lane 3) doxycycline were separated by
SDS-PAGE. Lanes 2 and 4 contain the MAM fraction
from SDO225/pDO292 (pgs1-regulatable strain) grown without
or with doxycycline, respectively. C is an anti-Cox2p (inner
mitochondrial membrane) immunoblot, and D is an anti-porin
immunoblot as a loading control for the results in C.
strain and the wild type parental
strain (not shown) to rule out any effect of doxycycline. The amount of
each protein in the doxycycline-grown psg1-regulatable
strain was indistinguishable from that of the psg1
strain, and the amount of each protein in non-doxycycline-grown cells
was indistinguishable from that of wild type cells.
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Fig. 8.
Immunoblot analyses of anionic
phospholipid-depleted mitochondria. Purified mitochondria were
analyzed by immunoblotting with the indicated antibodies.
A-F, purified mitochondria containing 12 µg of protein
from either no (lanes 1) or 10 µg/ml (lanes 2)
doxycycline-grown SDO225/pDO292 (pgs1-regulatable strain)
were separated by SDS-PAGE. Immunoblots used the antibodies indicated.
Protein quantification was accomplished by densitometry in duplicate at
least three times using three different highly purified mitochondria
preparations. F is an anti-porin immunoblot as a loading
control. G is a Cox4p immunoblot. Lane 1 is
mitochondria isolated from the parental wild type strain, and
lanes 2-5 are from SDO225/pDO292
(pgs1-regulatable strain) grown with different
concentrations of doxycycline (µg/ml): lane 2, 0;
lane 3, 0.1; lane 4, 1.0; and lane 5,
10. H is a Cox2p immunoblot of SDO225/pDO292
(pgs1-regulatable strain) grown with different
concentrations of doxycycline (µg/ml): lane 1, 0;
lane 2, 0.1; lane 3, 1.0; and lane 4, 10. In blots with multiple bands, the arrows indicate the
protein of interest.
DNA primers (5' to 3') used to amplify RNA in RT-PCR experiments
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Fig. 9.
Quantitative RT-PCR. SDO225/pDO292
(pgs1-regulatable strain) was grown for eight generations in
rich sucrose media with different concentrations of doxycycline to the
mid-exponential phase of growth. Total RNA was isolated and subjected
to quantitative RT-PCR with primers specific for the messages indicated
(see Table I). Lanes 1-4 of A and B
are PGS1. Note that a low molecular weight nonspecific
amplicon is amplified when PGS1 message is in low abundance.
Lanes 5-8 of A are COX4; lanes
5-8 of B are COB (cytochrome b).
The cells were grown in the following concentrations of doxycycline
(µg/ml): lanes 1 and 5, 0; lanes 2 and 6, 0.1; lanes 3 and 7, 1.0;
lanes 4 and 8, 10. C, RT-PCR was
performed alternatively with RNA obtained from SDO224 (wild type,
odd lanes) and SDO225 (pgs1 , even
lanes). Lanes 1 and 2, PGS1;
lanes 3 and 4, COX2; lanes
5 and 6, COB; lanes 7 and
8, ATP6; lanes 9 and 10,
21 S rRNA. The markers shown in A and C are a
100-base pair ladder, and the markers in B are a 1-kilobase
pair ladder (New England Biolabs). The experiments were performed in
duplicate on at least two occasions with two distinct RNA preparations.
The reason for the apparent difference in sizes of some of the
amplicons from the different strains is unknown. Sequencing was
performed to ensure that the amplicons were the same.
strain. No antibodies are available to the three ATP synthase subunits (F0) encoded
by mtDNA. All three proteins are myristoylated and, therefore, are the
only mitochondrial proteins soluble in chloroform (56). Therefore, an
organic extraction was prepared of highly purified mitochondria from
both the wild type and the psg1
strains. Proteins from
these preparations were identified by silver staining of SDS-PAGE gels. All three subunits are present in the psg1
strain, and
densitometry demonstrated that the proteins are found in the same
amount as in wild type cells (Fig. 10).
This analysis also reveals that the eighth mitochondrial encoded
protein, Var1p, is also present, as the subunits of F0
could not be produced without this ribosomal protein (57).
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Fig. 10.
Isolation of F0 subunits of
ATPase. Highly purified mitochondria with 2 mg of protein from
SDO224 (wild type, lane 1) and SDO225 (psg1 ,
lane 2) were homogenized by sonication and lipids isolated
by organic extraction. Proteolipids were separated by SDS-PAGE and
visualized by silver staining. Proteolipid isolation and quantification
were accomplished twice in duplicate.
strain and the wild type
strain. Experiments with in vitro transcribed and translated
Cox4p (Fig. 11) and Cox6p (data not
shown) showed that protein import into mitochondria is unaffected by
the absence of the major anionic phospholipids. Cox4p shows proper
cleavage of signal sequences and is imported and retained in a
time-dependent manner in mitochondria isolated from
pgs1
cells (Fig. 11B). This is true although
Cox4p is not detectable in cells grown with high concentrations of
doxycycline. When the ionophore valinomycin is added to the reaction to
dissipate the membrane potential, no protein import is observed.
Therefore, membrane potential, although low as indicated by DASPMI
staining, can be induced to a level sufficient to support normal
protein import even of proteins not present in pgs1
cells.
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Fig. 11.
In vitro protein import
assays. COX4 was subcloned behind a T7 CAP-independent
translation enhancer and transcribed in vitro. The message
was translated with biotinylated amino acids in vitro, and
protein denatured with urea. The resulting biotinylated Cox4p (A,
lane 1, no mitochondria) was added to ATP-activated highly
purified mitochondria from SDO224 (wild type, A) or SDO225
(psg1 , B). A 60 min assay was performed in the
presence of 1 µg/ml valinomycin as a negative control in lane
2 of A and lane 1 of B. Aliquots
of the reactions were stopped with valinomycin at 20 min. intervals up
to an hour. The first time point (lane 3 of A,
and lane 2 of B) was approximately five min. The
mitochondria were treated with proteinase K, extensively washed, and
isolated by centrifugation. Protein was separated by SDS-PAGE, blotted
to membrane, and detected with streptavidin conjugated
chemiluminescence reagents. The in vitro
transcription/translation and mitochondrial protein import assays were
performed in duplicate on three occasions with the same results.
cells are translated and subsequently degraded,
pulse-chase labeling of spheroplasts was performed. No
immunoprecipitable translation products of any of the proteins found to
be absent in the doxycycline-grown psg1-regulatable cells
could be detected (Fig. 12,
A and B). This includes mitochondrial encoded
proteins, such as Cox2p, as well as the single nuclear encoded protein
found absent, Cox4p. The translation and proper processing of these proteins upon import into mitochondria were found normal in wild type
cells. Other nuclear encoded mitochondrial proteins, such as aconitase,
were translated, imported, and processed as expected in
psg1
cells (Fig. 12C).
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Fig. 12.
Pulse-chase labeling. Spheroplasted
cells from SDO224 (wild type, lanes 1-4) and SDO225
(psg1 , lanes 5-8) were labeled for 10 min
with [35S]methionine and [35S]cysteine. The
cells were then chased with a high concentration of cold amino acids.
Aliquots were stopped with cold TCA at 20 min intervals up to one hr.
The first time point was taken at ~5 min. The above samples were
divided into multiple aliquots for immunoprecipitation to isolate the
respective labeled proteins that were separated by SDS-PAGE and
detected by autoradiography. A, Cox2p; B, Cox4p;
C, Aconitase. The upper arrow in each panel
denotes the precursor, while the lower denotes the mature form of the
protein. Immunoprecipitations were performed in duplicate at least
twice from at least three different pulse-chase labelings with the same
results.
strain than in the wild type strain (Fig.
13A). When the actual
differences in message levels were quantified, only five of the eight
NEMTAFs were shown to be significantly repressed in the
psg1
strain as follows: MSS51 and
PET309, which activate the translation of COX1
message; PET111 for COX2; PET54 for
COX3; and CBS2 for COB (Fig.
13B). No statistical difference could be discerned in
message levels for PET122 and PET494, which also
activate the translation of COX3 message, and for
CBS1 for COB. However, Western blot analysis of
mitochondrial proteins using antibody specific for PET111p showed no
difference in the level of this NEMTAF between wild type and
psg1
cells (Table II).
Therefore, it is unlikely that the levels of the NEMTAFs are limiting
in the absence of PG and CL.
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Fig. 13.
NEMTAF message levels. A,
total RNA was isolated from SDO224 (wild type, odd lanes) or
SDO225 (psg1 , even lanes). RT-PCR was
performed with primers (see Table I) specific for the messages
indicated. Lanes 1 and 2, MSS51;
lanes 3 and 4, PET309; lanes
5 and 6, PET111; lanes 7 and
8, PET54; lanes 9 and 10, PET122; lanes 11 and 12,
PET494; lanes 13 and 14,
CBS1; lanes 15 and 16,
CBS2. The experiments were performed in duplicate on at
least two occasions with two distinct RNA preparations. The markers
shown are a 100-base pair ladder (New England Biolabs). The reason for
the apparent difference in sizes of some of the amplicons from the
different strains is unknown. Sequencing was performed to ensure that
the amplicons were the same. B, the image in A as
well as duplicate experiments were quantified by densitometry and the
changes in mRNA concentration calculated. The message levels in
SDO225 (psg1
) are plotted as a percentage of SDO224 (wild
type) message for each gene.
Mitochondrial proteins screened for dependence on anionic lipids
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells on a fermentable carbon source for growth. By introducing into a
pgs1
strain a plasmid carrying a copy of PGS1
under "tet-off" regulation, a dose-response relationship
between PGS1 message, PGPS activity, level of PG plus CL,
and mitochondrial properties dependent on PG plus CL was demonstrated.
strains, all other subunits of Cox are easily detected although at reduced levels
due to failure of assembly resulting in some degradation (60) as we
observed for Cox5, Cox6, and Cox7. Cytochromes c, c1, and b are all also present in
normal amounts in cox4
strains. Lack of cytochrome
c (not determined in our studies) results in low levels of
the mitochondrial encoded subunits of Cox that can be readily detected
in pulse-chase experiments (61). Lack of translation in
pgs1
cells rather than rapid degradation of these ETC
components is further supported by the lack of their detection in
pulse-chase experiments reported here.
strain
(lacks CL synthase) was grown on glucose resulting in depletion of both
PG and CL in this particular background (25). However, mitochondrial
assembly and function are themselves significantly reduced in glucose
media (66) compromising any conclusions implicating the anionic
phospholipids. Nevertheless, the effect of altering PG levels in a
crd1 null strain by carbon source cannot be reproduced in
our laboratory. In all carbon sources tested in three genetic backgrounds, PG levels remain elevated at least 5-fold in
crd1
strains.2
cells for protein import by ATP indicates that both of these mitochondrial components are present and at least partially functional in psg1
cells. Studies in crd1
cells indicated that the ADP/ATP translocase is compromised in
vivo due to the lack of CL and that the presence of PG does not
substitute (25). In vitro reconstitution studies showed a
requirement by the translocase for CL. Therefore, the translocase may
only be marginally active in the absence of PG and CL which may be the
basis for both the petite lethal and temperature-sensitive phenotypes
of pgs1
cells. Cells lacking the ADP/ATP translocase are
also petite lethal (70). It has been postulated that in the absence of
both the ETC and the F0 portion of the ATP synthase, the
only means of generating a membrane potential across the mitochondrial
membrane is by continuous exchange of cytoplasmic ATP4
for mitochondrial ADP3
mediated by the
translocase and driven by F1-ATPase activity in the
mitochondrial matrix (for review see Ref. 71). In pgs1
cells, F0F1-ATPase activity coupled to proton
export may be sufficient to compensate for a poorly functional
translocase (still necessary for import of ATP) in maintaining
mitochondrial membrane potential. However, loss of F0 by
eliminating mtDNA in a pgs1
strain may make a marginally
functional translocase coupled to remaining F1-ATPase
activity insufficient to maintain membrane potential. Above 37 °C
the function of either the translocase or the ATPase in a
pgs1
strain may be further compromised. For example, the nuclear encoded FMC1 gene product is required only at
elevated growth temperatures for proper assembly of the F1
component of the ATP synthase (72). It is not known whether FMC1p is
expressed or functional in pgs1
cells above 37 °C.
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ACKNOWLEDGEMENTS |
---|
We thank Oksana Takh and Rachel Israel for technical assistance. We are indebted to Drs. Schatz, Fox, and Koehler for providing us with the antibodies used in this work.
![]() |
FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grant GM56389 (to W. D.) and NHLBI Grant HL10304 from the National Institutes of Health (to D. O.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Present address: ICOS Pharmaceutical Corp., 22021 20th Ave. S.E.,
Bothell, WA 98021.
§ Present address: Catholic Research Institute of Medical Science, Research Institute of Cancer, Catholic University, 505 Banpo-dong, Seocho-ku, Seoul 137-701, Korea.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of Texas Medical School, P. O. Box 20708, Houston, TX 77225. Tel.: 713-500-6051; Fax: 713-500-0652; E-mail: William.Dowhan@uth.tmc.edu.
Published, JBC Papers in Press, May 2, 2001, DOI 10.1074/jbc.M103689200
2 M. Zhang, D. Ostrander, E. Mileykovskaya, and W. Dowhan, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
CL, cardiolipin;
Cox, cytochrome c oxidase;
DASPMI, 2-(4-dimethylaminostyryl)-1-methylpyridinium iodide;
DPMT, dolichyl-phosphate -D-mannosyltransferase;
ETC, electron
transport chain;
FCCP, carbonyl cyanide
p-trifluoromethoxyphenylhydrazone;
MAM, mitochondrial
associated microsomal membrane;
NAO, 10-N-nonyl acridine
orange;
NEMTAF, nuclear encoded mitochondrial translation activation
factor;
PG, phosphatidylglycerol;
PGPS, phosphatidylglycerol phosphate
synthase;
RT, reverse transcriptase;
PCR, polymerase chain reaction;
PAGE, polyacrylamide gel electrophoresis;
FITC, fluorescein
isothiocyanate.
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REFERENCES |
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