From the Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703
Received for publication, October 27, 2000, and in revised form, November 17, 2000
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ABSTRACT |
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The protein specified by the Saccharomyces
cerevisiae nuclear gene PET111 specifically activates
translation of the mitochondrially coded mRNA for cytochrome
c oxidase subunit II (Cox2p). We found Pet111p specifically
in mitochondria of both wild-type cells and cells expressing a
chromosomal gene for a functional epitope-tagged form of Pet111p.
Pet111p was associated with mitochondrial membranes and was highly
resistant to extraction with alkaline carbonate. Pet111p was protected
from proteolytic digestion by the mitochondrial inner membrane. Thus,
it is exposed only on the matrix side, where it could participate
directly in organellar translation and localize Cox2p synthesis by
virtue of its functional interaction with the COX2 mRNA
5'-untranslated leader. We also found that Pet111p is present at levels
limiting the synthesis of Cox2p by examining the effect of altered
PET111 gene dosage in the nucleus on expression of a
reporter gene, cox2::ARG8m, that was
inserted into mitochondrial DNA. The level of the reporter protein,
Arg8p, was one-half that of wild type in a diploid strain heterozygous
for a pet111 deletion mutation, whereas it was increased 2.8-fold in a strain bearing extra copies of PET111 on a
high-copy plasmid. Thus, Pet111p could play dual roles in both membrane localization and regulation of Cox2p synthesis within mitochondria.
The key products of mitochondrial gene expression are integral
membrane proteins that are assembled with proteins encoded by nuclear
genes to form energy-transducing complexes in the inner membrane
(1-3). Thus, the activity of mitochondrial genetic systems must be
coordinated with cellular gene expression and adapted for efficient
delivery of hydrophobic proteins to the inner membrane.
Translation of at least five of the seven major Saccharomyces
cerevisiae mitochondrially coded mRNAs is under the control of
mRNA-specific activator proteins encoded by nuclear genes (4). Genetic analysis has revealed that the activator proteins functionally interact with the 5'-UTLs1 of
their target mRNAs and probably mediate productive interactions between those mRNAs and ribosomes (4, 5). As discussed below, several of the activator proteins have been found to be associated with
mitochondrial membranes, suggesting that they could function to
localize the synthesis of hydrophobic mitochondrial gene products to
their sites of insertion in the inner membrane (3, 4, 6, 7). This
hypothesis is strongly supported by the fact that the untranslated
regions of the COX2 and COX3 mRNAs contain information that facilitates correct targeting of the Cox2p and Cox3p
cytochrome oxidase subunits (8).
The translational activator proteins themselves are present at low
levels (9) and have therefore been difficult to study. In no case have
they been shown to be localized within mitochondria isolated from
wild-type (nonoverproducing) cells such that they could participate
directly in organellar protein synthesis. Cbs1p and Cbs2p, activators
required for translation of the cytochrome b mRNA (10),
were detectable at normal levels in wild-type cells. They were found
associated with the total mitochondrial membrane fraction. Cbs1p
behaved like an integral membrane protein, whereas Cbs2p was
peripherally associated, but they were not further localized within the
organelle (6). Pet309p, required for Cox1p translation (11), could only
be detected as an epitope-tagged species in cells overproducing the
protein (12). Under these conditions it fractionated as an integral
inner membrane protein, partially exposed on the outer surface
(intermembrane space side). Mss51p, also required for Cox1p synthesis,
was peripherally associated with mitoplast (mitochondria with ruptured
outer membranes) membranes when expressed from a low-copy plasmid (13).
Synthesis of Cox3p requires three interacting proteins, Pet54p,
Pet122p, and Pet494p (14, 15). Only Pet54p could be detected
immunologically in wild-type cells and was found to be peripherally
associated with the inner membrane (16). Pet122p and Pet494p, when
overproduced, fractionated as integral inner membrane proteins
(16).
The mRNA-specific activators could regulate expression of
mitochondrial genes in addition to targeting synthesis on the membrane if they were present at levels limiting translation. Alternatively, the
activators could be present in excess, with regulation of mitochondrial
gene expression controlled at some other step. This has been examined
to date only in the case of COX3 expression using the
synthetic reporter gene ARG8m inserted into
mtDNA at COX3 (17). Reduction of the gene dosage of
PET494 caused reduced expression of the mitochondrial
reporter, although increased dosage of PET494 caused only a
modest increase in reporter expression.
In this study we have focussed on Pet111p, the only known translational
activator for the COX2 mRNA (18, 19). Pet111p is known
to be mitochondrially located (20) and to interact functionally with
the COX2 mRNA 5'-UTL (21). Although the amino acid
sequence of Pet111p is not highly conserved among budding yeasts, its
function in specifically activating translation of the COX2
mRNA has been conserved, indicating an important role in the
production of cytochrome c oxidase (22). Here we study the
submitochondrial localization of Pet111p in cells expressing the
PET111 gene at normal levels and examine the effect of
altered PET111 gene dosage on expression of the
mitochondrial COX2 gene. Our results are consistent with a
direct role for Pet111p in localization of mitochondrial translation
and demonstrate that it is a rate-limiting factor in the expression of
the COX2 gene.
Yeast Strains and Genetic Methods--
S. cerevisiae
strains used in this study are listed in Table
I. All strains are congenic to the
wild-type strain D273-10B (ATCC 25657) except DFS160rho0, HMD122, and
NSG175, which were derived from DBY947 (23). Cells were grown in rich
medium (1% yeast extract, 2% bacto-peptone) containing 2% galactose
or 2% raffinose or in minimal medium (0.67% yeast nitrogen base)
containing 2% raffinose, as indicated in the figure legends. Standard
genetic methods were as described (24, 25). Strains deleted for
PET111 carry either the pet111-14 deletion (26)
or the pet111-9 deletion with LEU2 in its place
(27).
Epitope Tagging of Pet111p--
The PET111 reading
frame was modified to encode three copies of the influenza virus HA
epitope (28) at its 3' end using the plasmid pCS124 (obtained from C. Shamu and J. Nunnari), an integrative plasmid carrying three copies of
the HA sequence and the TRP1 gene. The downstream 556 base
pairs of PET111 were inserted as an
EcoRI-NcoI fragment into pCS124 such that the
epitope-coding sequence was in-frame with PET111. The
resulting plasmid, pNSG30, was cut at a unique HpaI site
within PET111 and used to transform strain PTY11 to
Trp+. In the resulting integrative transformant, NSG91, the
only complete copy of PET111 was the tagged allele,
PET111-HA. Correct integration was confirmed by Southern analysis.
Mitochondrial Isolation, Purification, and
Subfractionation--
Mitochondria were prepared from cells grown to
late exponential phase in complete medium (yeast
extract/peptone) containing 2% galactose as described (29),
except that spheroplasts were disrupted using a Parr-Bomb (Parr
Instrument Co., Moline, IL) as described previously (30). Crude
mitochondria were purified by equilibrium density gradient
centrifugation on 5-25% Nycodenz (5-(N-2,3-dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihydroxypropyl) isophthalimide; Sigma) step gradients as described (29). Mitochondria were separated into membrane and soluble fractions as described (31),
and alkaline carbonate extractions of membranes in 0.1 M
Na2CO3, pH 11.5, were as described (32).
Soluble fractions were precipitated in 10% trichloroacetic acid before
SDS-polyacrylamide gel electrophoresis. Mitochondria were converted to
mitoplasts by diluting 10-fold into 20 mM
K+-HEPES, pH 7.4, 1 mg/ml bovine serum albumin containing
either 1 mM phenylmethylsulfonyl fluoride or 100 µg/ml
proteinase K and incubated on ice for 30 min (31, 33). Control
mitochondria were diluted 10-fold into 0.6 M sorbitol, 20 mM K+-HEPES, pH 7.4, 1 mg/ml bovine serum
albumin containing either 1 mM phenylmethylsulfonyl
fluoride or 100 µg/ml proteinase K and similarly incubated. Fractions
were subjected to SDS-polyacrylamide gel electrophoresis on 10% gels
and Western-blotted as described (34).
Antisera and Immunological Methods--
The C-terminal 542-amino
acid fragment of Pet111p was isolated as a GST-Pet111p fusion protein
encoded by pJAM6 (35). Inclusion bodies were isolated after induction
in Escherichia coli (BL21) with 2 mM
isopropyl-1-thio-
For quantitative Western analysis, cells were harvested at mid-log
phase, and total protein was isolated as described (37). Samples were
subjected to SDS-gel electrophoresis in 12% polyacrylamide and then
transferred to Immobilon-P (Millipore). Membranes were blocked in 5%
ECF blocking compound (Vistra) in Tris-buffered saline, pH 7.6, containing 0.1% Tween 20, washed in Tris-buffered saline, and then
incubated with the primary antibody in Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 either at 25 °C for 1 h or overnight
at 4 °C. The filters were next washed in Tris-buffered saline and
then incubated with secondary antibody in Tris-buffered saline, pH 7.6, containing 0.1% Tween 20 for 1 h at 25 °C. The secondary
antibody was fluorescein-linked donkey anti-rabbit Ig (Vistra) diluted
1:250 for anti-Arg8p and 1:125 for anti-glucose-6-phosphate dehydrogenase. The membranes were then washed in Tris-buffered saline
and allowed to dry at room temperature for at least 20 min before
scanning on a Molecular Dynamics STORM 840 PhosphorImager (Blue
Fluorescence/Chemi-fluorescence screen, 100 µm, photomultiplier tube voltage 650). Membranes were probed first with anti-Arg8p antiserum (17) that had been purified on a DEAE Affi-Gel Blue column
(Bio-Rad) and treated with acetone powder (34) derived from an
arg8
Scanned images were analyzed using ImageQuant version 1.1 software from
Molecular Dynamics. Area, the summation of pixel intensity times the
number of pixels, was quantitated for the desired bands. Each Arg8p
signal was normalized to the corresponding G6PD signal.
Construction of the cox2::ARG8m Reporter
Gene--
The plasmid pHD6 was constructed in the vector pTZ18u
(Bio-Rad). It contains the ARG8m sequence (17)
(see GenBankTM accession U31093) flanked by 0.57 kilobases
of mitochondrial DNA upstream of the COX2 reading frame and
0.81 kilobases of mitochondrial DNA downstream of the COX2
reading frame. The sequence immediately downstream of the
ARG8m stop codon contains an artificial
BamHI site for cloning purposes. In addition, pHD6 contains
a 0.75-kilobase PacI-MboI COX3
fragment inserted between the BamHI and HincII
site of the polylinker to serve as an additional mitochondrial genetic
marker. The sequence of pHD6 is available upon request.
pHD6 was introduced into the Detection of Pet111p, Expressed at Normal Levels, in Purified
Mitochondria--
Pet111p is expressed at very low levels (9), and we
have previously not been able to detect the protein in wild-type cells (21). Yeast cells overproducing Pet111p accumulate it in mitochondria, associated with membranes.2
However, firm conclusions regarding localization of a protein cannot be
drawn from analysis of cells overproducing it. We therefore sought to
improve the sensitivity of Pet111p detection.
A new rabbit polyclonal antiserum was raised against a fusion protein
bearing the C-terminal 542 Pet111p amino acid residues and glutathione
S-transferase, expressed in E. coli
("Experimental Procedures"). This serum was used to probe
subcellular fractions from a wild-type strain (PTY11) on a Western
blot. It reacted with a protein of the size expected for Pet111p (94 kDa) that was highly enriched in gradient-purified mitochondria and
absent from post-mitochondrial supernatant (Fig.
1A). This protein was not
detected in fractions isolated from a pet111 deletion mutant (Fig. 1) nor in wild-type fractions probed with pre-immune serum (not
shown). Furthermore, the level of this protein was increased severalfold relative to total mitochondrial protein in the wild-type strain containing the multicopy plasmid pJM20, which carries a genomic
fragment including the PET111 promoter and transcription terminator (26) (Fig. 1). Cross-reacting species of ~56 kDa, which
may include Pet111p degradation products, were also detected (not
shown).
In addition, we were able to detect Pet111p as a tagged protein bearing
three copies of the HA epitope, expressed from its chromosomal locus.
Such strains were fully Pet+, demonstrating that Pet111p-HA
is a functional protein. Mitochondria were purified from a strain
expressing Pet111p-HA and from a wild-type strain and were probed with
an anti-HA monoclonal antibody and with anti-Pet111p (Fig.
1B). The anti-HA antibody reacted with a protein of the
appropriate size in the tagged mitochondria but not in the
corresponding wild-type mitochondria. The anti-Pet111p reacted with the
same protein in both samples, confirming that the species in question
is indeed Pet111p.
Pet111p Is Firmly Bound to the Inner Membrane, on the Matrix
Side--
To determine the location of Pet111p within the
mitochondria, submitochondrial fractions were prepared from a strain
expressing Pet111p-HA (NSG91). Purified mitochondria were disrupted by
osmotic shock and sonication, then fractionated by centrifugation to
separate soluble and membrane-associated proteins. The membrane
fraction was then extracted with alkaline sodium carbonate to separate peripheral membrane proteins from integral membrane proteins. The
resulting fractions were then analyzed by Western blots probed with
anti-HA to determine the distribution of Pet111p-HA. Most of the
Pet111p-HA was recovered in the membrane pellet after sonication (Fig.
2). Most of the membrane-associated
Pet111p-HA remained with the integral membrane protein fraction after
alkaline extraction, indicating that Pet111p is firmly membrane-bound.
The known integral membrane protein Cox2p was completely associated
with membranes in this experiment, whereas the soluble matrix protein
Arg8p was not.
Protease protection experiments were used to determine the
submitochondrial location of Pet111p-HA (Fig.
3). Proteinase K treatment of
mitochondria solubilized by detergent eliminated detectable Pet111p-HA,
showing that the epitope is not protected by a stable protein complex.
However, Pet111p-HA in both whole mitochondria and in mitoplasts
(osmotically shocked to rupture the outer membrane only) was protected
from digestion by proteinase K. Thus, the C-terminal epitope appears to
be on the inside of the inner membrane. Furthermore, since no shorter
species were detected, it appears that the entire Pet111p-HA protein is
protected from protease by the inner membrane. The same behavior was
observed for the soluble matrix marker mRNA-specific Translational Activation by Pet111p Is
Rate-limiting for Expression of the Mitochondrial Reporter Gene
cox2::ARG8m--
Expression of the mitochondrial
COX2 gene could be limited at the level of translation by
the activity of Pet111p. To examine this possibility, we constructed a
cox2::ARG8m reporter gene that had the
second codon of ARG8m fused to the
COX2 translation initiation codon and lacked all other
COX2 codons ("Experimental Procedures"). This reporter
was inserted into mitochondrial DNA, cleanly replacing the
COX2 open reading frame with ARG8m.
The resulting chimeric gene specifies an mRNA with the Pet111p target in the COX2 5'-UTL, upstream of the
ARG8m-coding sequence, followed by the
COX2 3'-untranslated region. Expression of this
mitochondrial reporter gene fully complemented the Arg
To ask whether the level of
cox2::ARG8m mRNA
translation was limited by the level of Pet111p, we constructed strains
containing the mitochondrial reporter with different dosages of the
PET111 nuclear gene. We then measured the relative
steady-state levels of Arg8p in whole cell extracts by quantitative
Western blotting ("Experimental Procedures"). Measurements of Arg8p
were internally controlled by normalization to the similarly measured
steady-state level of the cytoplasmic enzyme glucose-6-phosphate
dehydrogenase. Expression of the gene encoding glucose-6-phosphate
dehydrogenase, ZWF1, appears to be relatively constant under
different growth conditions (42, 43).
First, the dosage of PET111 was halved by constructing a
diploid strain heterozygous for a pet111 mutation. The level
of Arg8p reporter protein in this pet111/PET111 strain was
approximately half that of a control strain homozygous for wild-type
copies of PET111, as would be expected if Pet111p levels
limit COX2 mRNA translation (Fig.
4). As a control for mRNA
specificity, we also examined the effect on
cox2::ARG8m expression of lowering the
gene dosage of PET494, which encodes a subunit of the
COX3 mRNA-specific translational activator (15) and is
limiting for cox3::ARG8m expression
(17). Arg8p levels were not decreased in the
pet494
We next examined the effect of increasing PET111 gene dosage
on the expression of the cox2::ARG8m
reporter gene. The multicopy PET111 plasmid pJM20 (26) was transformed into a homozygous wild-type diploid strain. The
steady-state level of Arg8p present in this transformant was determined
relative to that of the same strain transformed with the empty vector
YEp352 (44). The presence of extra copies of PET111 caused
the steady-state level of Arg8p to increase by ~2.8-fold (Fig.
5), confirming that Pet111p activity is a
limiting factor in the expression of the mitochondrial COX2
gene.
S. cerevisiae Pet111p is synthesized in the cytoplasm
as an 800-amino acid protein, which may be processed upon entry into mitochondria. It is largely hydrophilic and has a net positive charge.
Previous genetic data have argued for a close functional interaction
between Pet111p and the COX2 mRNA in the mitochondrial matrix, where translation occurs. Here, we have detected Pet111p in
mitochondria of cells expressing it at normal levels and demonstrated that it is tightly associated with the inner membrane. Pet111p is
protected by the inner membrane from added protease, indicating that
its hydrophilic domains must be exposed on the matrix side where they
could interact with the COX2 mRNA and components of the
organellar translation system. Thus, Pet111p is very likely to be the
membrane component that recognizes targeting information in the
untranslated portions of the COX2 mRNA, promoting
efficient cytochrome c oxidase assembly (8).
Pet111p was largely resistant to removal from mitochondrial membranes
by extraction with alkaline carbonate, suggesting that it is an
integral membrane protein (32). However, it was not as firmly bound as
the known integral membrane protein Cox2p. Although analysis of the
S. cerevisiae Pet111p sequence using the algorithm TMAP
suggests two possible transmembrane helical domains (125 - 146 and
548-564), the more robust analysis, with the same program, of aligned
Pet111p homologs from three budding yeasts (22) does not suggest any
such domains (45, 46). The Pet122p subunit of the COX3
mRNA-specific activator behaves as an integral membrane protein
both when overproduced (16) and when expressed from a single
chromosomal gene.3 Yet
Pet122p also lacks apparent transmembrane helical domains and residues
exposed to proteolysis on the outer surface of the inner
membrane.3 This behavior contrasts with that of
overproduced Pet309p, the COX1 mRNA-specific activator,
that spans the inner membrane and is accessible to protease from the
outside (12).
The amount of Pet111p appears to limit expression of the mitochondrial
gene COX2 at the level of translation, since the level of
Arg8p encoded by a cox2::ARG8m
reporter gene varied with PET111 gene dosage. The reduction
of Arg8p was clearly proportional to PET111 dosage when we
reduced the number of PET111 genes by half. The Arg8p level
increased 2.8-fold when PET111 was introduced into cells on
a multicopy vector that caused an approximately similar increase in the
level of Pet111p. This result is consistent with a previous report that the COX2 mRNA level is not limiting for gene expression
since overexpression of that mRNA does not lead to increased
synthesis of Cox2p (47). We conclude that modulation of the level of
Pet111p and/or its translational activation activity could regulate the level of Cox2p synthesis within mitochondria.
Increased activity of a limiting translational activator will increase
translation of the target mitochondrial mRNA up to the point that
some other component (mRNA, ribosomes, etc.) becomes limiting. We
cannot be certain whether this point was reached in our overproduction
experiments. However, we did not greatly exceed it since the increase
in Pet111p levels in mitochondria from cells with increased
PET111 dosage was approximately similar to the 2.8-fold
increase in reporter gene expression. In a previous study of
cox3::ARG8m expression we found that
overproduction of the limiting activator subunit, Pet494p, increased
the mitochondrial reporter expression only by ~65% (17). The
possibility of competition between translational activators for low
levels of some general translation component is suggested by the
apparent slight increase in
cox2::ARG8m expression in a diploid
heterozygous for a pet494 deletion, but this effect may not
be significant.
The PET111 mRNA has an unusually long 5'-UTL of 470 nucleotides that contains four overlapping open reading frames upstream of the Pet111p-coding sequence (20), suggesting the possibility that
PET111 expression itself might be controlled at the level of
cytoplasmic translation (48). Such an mRNA structure could serve
either as a target for regulation that limits gene expression independently of mRNA levels via feedback, or it could simply reduce the translational efficiency of the PET111 mRNA.
The fact that increased copy number of the PET111 gene
causes an increase in Pet111p levels and activity demonstrates Pet111p
synthesis is not translationally limited independently of the mRNA
level. However, we have not established whether the protein and
mRNA levels vary proportionately.
Translational feedback regulatory loops that couple synthesis of
specific components to the assembly of a chloroplast membrane complex
in Chlamydomonas (49, 50) and of the basal body-hook structure of Caulobacter (51) have been described. Our
results show that Pet111p is very likely to play roles in both
regulating the rate of Cox2p synthesis and localizing that synthesis on
the surface of the inner membrane. Thus, Pet111p could participate in a
feedback mechanism coupling Cox2p translation with its assembly into
the cytochrome c oxidase complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
-D-galactopyranoside, and the
GST-Pet111p fusion protein was purified by polyacrylamide gel
electrophoresis. Rabbits were immunized and serum-prepared as described
previously (34). Immune serum (5-165) was treated with acetone powder
from a pet111
strain (34), purified on a DEAE Affi-Gel
blue column (Bio-Rad), and used at a 1:1000 dilution. The mouse
monoclonal antibody 12CA5 against the HA epitope was purchased from
Berkeley Antibody Corp. (Berkeley, CA) and was used at 1:30,000.
Monoclonal anti-Cox2p (CCO6) was a gift from T. L. Mason.
Polyclonal anti-cytochrome b2 and
anti-
-ketoglutarate dehydrogenase were gifts from B. Glick and G. Schatz. Preparation of antisera against Arg8p (17) and Yme1p (I-473)
(36) was described previously. For the experiments of Figs. 1, 2, and
3, antigen-antibody complexes were visualized on Western blots using
horseradish peroxidase-conjugated goat anti-mouse IgG or goat
anti-rabbit (Life Technologies, Inc.) secondary antibody and the
enhanced chemiluminescence system (Amersham Pharmacia Biotech).
yeast strain. After scanning, blots were stripped with 100 mM 2-mercaptoethanol, 2% (w/v) SDS, 62.4 mM Tris-HCl pH 6.7 at 50 °C for 30 min, washed
extensively with Tris-buffered saline, dried at room temperature, and
scanned to check for removal of the Arg8p antibody. Blots were then
reprobed with anti-glucose-6-phosphate dehydrogenase polyclonal
antiserum (Sigma) diluted 1:125.
0 mitochondria of strain
DFS160rho0 (17) by microprojectile bombardment as described (38, 39),
and mitochondrial transformants were identified by their ability to
marker rescue the cox3-10 mutation (40). One such transformant, HMD122, was isolated and used to transfer pHD6 into
+ mitochondria by mating to appropriate strains and
selecting Arg+ haploid cytoductants.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Detection of Pet111p in purified
mitochondria. A, mitochondria were prepared from cells
of PET111 wild-type (PTY11), pet111 (NB39-16D),
and wild-type containing PET111 on a high-copy
(H-C) plasmid, PET111 (PTY11 [pJM20]), grown to
late logarithmic phase in complete medium containing galactose, and
purified by equilibrium density gradient centrifugation
("Experimental Procedures"). Approximately 50 µg of protein from
total cell extracts (T), post-mitochondrial supernatant
(S), and purified mitochondria (M) were analyzed
by Western blot, probed with the polyclonal anti-Pet111p antiserum. The
arrow indicates the Pet111p-specific band of ~94 kDa. A
cross-reacting band of ~55 kDa was also evident (not shown).
B, mitochondria were prepared as described above from
PET111 wild-type (PTY11) and a PET111-HA strain,
whose chromosomal gene encodes HA epitope tags at the 3' end (NSG91).
Approximately 50 µg of purified mitochondria from each strain were
subjected to Western blotting in duplicate. One blot was probed with a
monoclonal anti-HA antibody, the other with polyclonal anti-Pet111p
antibody, as indicated.
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Fig. 2.
Pet111p is firmly bound to mitochondrial
membranes. Mitochondria were purified from cells with the
chromosomally integrated PET111-HA gene (NSG91), grown to
late logarithmic phase in complete galactose medium ("Experimental
Procedures") and 50-µg aliquots of total mitochondrial protein
(T), and submitochondrial fractions were analyzed by Western
blotting. Mitochondria were separated into soluble (S) and
membrane (M) fractions ("Experimental Procedures").
Membranes were further extracted with alkaline carbonate
("Experimental Procedures") to separate peripheral membrane
proteins (PM) from integral membrane proteins
(IM). The Western blots were probed with anti-HA to detect
Pet111p-HA as well as antisera against inner membrane-bound Cox2p and
the soluble matrix enzyme Arg8p (encoded by the wild-type nuclear
ARG8 gene in this strain).
-ketoglutarate dehydrogenase
(Fig. 3). Yme1p is an integral inner membrane protein exposed on the outer surface of the inner membrane (41) and is not protected from
protease by mitoplasts, as expected (Fig. 3). Taken together, these
data indicate that Pet111p is tightly associated with the inner surface
of the inner mitochondrial membrane.
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Fig. 3.
Pet111p is an inner membrane protein facing
the matrix. Mitochondria (M) were purified from the
PET111-HA strain NSG91 and converted to mitoplasts
(MP) by osmotic shock in the absence or presence of
proteinase K, as indicated for each lane ("Experiment Procedures").
Mitochondria were also treated with proteinase K in the presence of the
detergent octyl glucoside (1%) to solubilize membranes. Treated
samples were analyzed by Western blots probed with anti-HA to detect
Pet111p-HA as well as antisera against the soluble matrix enzyme
-ketoglutarate dehydrogenase (
KDH) , the inner
membrane-bound protein Yme1p exposed on the outer surface, and the
soluble intermembrane space protein cytochrome (cyt)
b2.
growth
phenotype caused by a nuclear arg8 mutation. This
complementation was abolished by a pet111 nuclear mutation,
demonstrating that cox2::ARG8m expression
is Pet111p-dependent. As expected, the pet111
mutation prevented accumulation of immunologically detectable Arg8p
encoded by the mitochondrial reporter (not shown).
/PET494 heterozygous diploid relative to
the homozygous wild-type strain but instead appeared to be slightly
increased (Fig. 4).
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Fig. 4.
Expression of the mitochondrial reporter gene
cox2::ARG8m is reduced in a
diploid heterozygous for a pet111 mutation.
A, total protein was extracted from cells grown in complete
medium containing raffinose and analyzed by quantitative Western
blotting ("Experimental Procedures"). A representative Western blot
of the indicated amounts of total protein probed with antisera against
Arg8p and glucose-6-phosphate dehydrogenase (G6PD) is shown.
The Arg8p detected here is encoded by the mitochondrial
cox2::ARG8m reporter gene in diploids
whose relevant nuclear genotypes are wild type,
pet494/PET494, and pet111 /PET111. A
diploid lacking any active ARG8 gene (arg8) is
included as a control. B, four such blots were analyzed
quantitatively ("Experimental Procedures"), normalizing Arg8p
levels to glucose-6-phosphate dehydrogenase, and averaged. Error
bars indicate the S.D. The diploids used in this experiment were
constructed by mating the following haploids (Table I):
arg8, GW241rho0 and NB40-20D; wild-type, NSG176 and
NSG170rho0; pet111/PET111, NSG176 and
NSG165rho0; pet494/PET494, NSG176 and NSG164rho0.
The heterozygous diploids grew as well as the homozygous wild-type
diploid on medium lacking arginine (not shown).
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Fig. 5.
Expression of the mitochondrial reporter gene
cox2::ARG8m is increased in a
wild-type diploid containing extra plasmid-borne copies of
PET111. A, total protein was extracted from
homozygous wild-type cells transformed with either the empty vector
YEp352 (44) or the PET111 plasmid pJM20 (26) and grown in
minimal medium containing raffinose. A diploid lacking any active
ARG8 gene (arg8) is included as a control.
Proteins were analyzed as described in the legend to Fig. 4.
B, three such blots were quantitatively analyzed as
described in the legend to Fig. 4. Bars indicate S.D. The
strains used in this experiment were constructed by mating the
following haploids (Table I): arg8, GW241rho0 and NB40-20D;
wild-type, NSG176 and NSG170rho0.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We gratefully thank T. L. Mason, B. Glick, and G. Schatz for gifts of antisera.
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FOOTNOTES |
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* This investigation was supported by National Institutes of Health Research Grant GM29362 and Training Grant GM07617.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: Invitrogen Corp., 1600 Faraday Ave., Carlsbad, CA 92008.
§ To whom correspondence should be addressed: Dept. of Molecular Biology and Genetics, Biotechnology Bldg., Cornell University, Ithaca, New York 14853-2703. Tel.: 607-254-4835; Fax: 607-255-6249; E-mail: tdf1@cornell.edu.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M009856200
2 C. A. Strick and T. D. Fox, unpublished data.
3 C. A. Butler and T. D. Fox, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: 5'-UTL, 5'-untranslated leader; HA, hemagglutinin.
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