Addition of growth factors to a resting cell culture elicits a
wide variety of biochemical and genetic responses. The early
biochemical events trigger the cytoplasmic accumulation of newly
transcribed RNAs (primary or immediate-early response RNAs), which does
not require de novo protein synthesis. In fibroblastic cells,
a large number of genes have been characterized as being transiently
induced within 4 h of growth stimulation(1, 2) . They
encode various types of products, such as cytokines, structural
proteins, metabolic enzymes, protein phosphatases, and transcription
factors(3, 4, 5, 6) . However,
activation of immediate-early response genes is not sufficient per
se for eliciting DNA synthesis, which requires long-term growth
factor exposure and active protein synthesis for at least 10
h(7, 8) . Stimulated cells may also be blocked in late
G
upon transforming growth factor-
or glucocorticoid
treatment without affecting the early biochemical and genetic events (9, 10, 11) . A limited number of genes
activated during late G
have been characterized as (i)
early-early response mRNAs, the accumulation of which, like
immediate-early response mRNAs, does not require de novo protein synthesis. They encode products such as p53(12) ,
cyclin genes(13) , Ras-like proteins(14) , and
biosynthetic enzymes or secreted
proteases(4, 15, 16, 17) . A limited
number of genes have also been characterized as (ii) delayed-early
response RNAs, which accumulate upon growth stimulation only when
protein synthesis is active. They were found to encode various types of
products, such as structural proteins, non-histone chromosomal
proteins, extracellular matrix proteins, and transcription
factors(18, 19) .
Although mitochondrial activity
is not necessary for cell growth in defined cultured
conditions(20) , several nuclear genes encoding mitochondrial
proteins have also been described as being growth-regulated. These
include the mitochondrial chaperonin hsp60 (21) , the Fos
transformation effector(22) , an ADP/ATP carrier(23) ,
and a proton/phosphate symporter(19) .
We report here the
characterization of the mammalian mitochondrial ribosomal (mitosomal)
MRPL12 protein as being encoded by a delayed-early response mRNA.
MRPL12 mRNA accumulation in growth-stimulated cells occurs in
proportion to the mitogenic strength and is paralleled by the
accumulation of the corresponding protein. MRPL12 protein is
phylogenetically related to the chloroplastic and bacterial L12
ribosomal proteins, which control mRNA translation. Immunofluorescence
microscopy and cell fractionation indicate a predominant mitochondrial
localization of the protein in various mammalian cell lines. The
amino-terminal region is necessary and sufficient to promote a
mitochondrial targeting of exogenous proteins. MRPL12 proteins
associate in vitro as dimers and cofractionate with ribosomal
structures from mitochondrial extracts. Expression of a truncated
MRPL12 protein acting as a dominant inhibitor affects cell growth and
mitochondrial ATP synthesis.
MATERIALS AND METHODS
Cell Culture
CCL39, COS-7, NIH3T3, and HeLa
cells were grown, respectively, in Dulbecco's modified
Eagle's medium and RPMI 1640 medium supplemented with 10% calf
serum (FCS). (
)For serum starvation, confluent cells were
incubated for 20 h with plain Dulbecco's modified Eagle's
medium. Renewed growth was stimulated by addition of 10% FCS,
-thrombin (1 unit ml
), or epidermal growth
factor (EGF; 35 ng ml
) in combination with insulin
(10 µg ml
). Concentrations were 1 mM for 8-bromo-cAMP and 10 µg ml
for
cycloheximide.
RNA and Protein Analysis
RNA extraction and
analysis were carried out as described(24) . The hybridization
mixture contained 50% formamide, 5
Denhardt's solution,
10 mM phosphate buffer (pH 7.0), 0.75 M NaCl, 0.1%
SDS, 10% dextran sulfate, and 100 µg ml
denatured salmon sperm. Hybridization was at 42 °C for
12-24 h using a probe concentration of 2
10
cpm ml
. Filters were washed twice in 2
SSC, 0.1% SDS at room temperature and once in 0.2
SSC, 0.1% SDS
at 65 °C. Signals were quantified using either an image processing
workstation (BioImage, Millipore Corp.) or a PhosphorImager (Molecular
Dynamics, Inc.). Proteins were prepared from cells lysed for 1 h at 4
°C in radioimmune precipitation assay buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% deoxycholate, 0.1%
SDS, and 1% Triton X-100). Cells extracts were cleared by
centrifugation (15 min at 10,000
g). Protein detection
on Western blots was performed using the ECL detection kit (Amersham
Corp.).
Isolation of Full-length cDNA Clones
The
construction of cDNA libraries was as described(14) . Libraries
from hamster CCL39 cells growth-stimulated for 5 h and exponentially
growing HeLa cells were screened at low density on nylon membranes
(Hybond N, Amersham Corp.). DNA was extracted from pools of colonies
isolated from positive areas and restricted with BamHI and HindIII enzymes, and insert size was monitored by Southern
blotting. Pools harboring inserts larger than 1 kilobase pair (i.e. corresponding to the size of mRNA) were plated at a lower density,
and individual positive clones were selected after a second round of
screening.
cDNA Sequencing and Analysis
DNA sequence was
determined on crude double-stranded plasmid DNA (T7 sequencing kit,
Pharmacia Biotech Inc.). The complete hamster and human sequences were
obtained on both strands by subclones generated by restriction
cleavage. Due to their high G + C content, the sequence
corresponding to the 5`-end of both RNAs was determined using M13
subcloning and manual and automatic sequencing using an Applied
Biosystems 373A sequencer. Data base searches and sequence analysis
were worked out by using the facilities of the Centre
Inter-universitaire de Traitement de l'Information
(Paris)(25) .
Construction of Expression Vectors
For eukaryotic
expression, pcDNAI (Invitrogen) was modified as follows. A
double-stranded oligonucleotide containing a canonical ribosome-binding
sequence and a translation initiator
(5`-(HindIII)-CCCCCGCCTCGGGAGCCGCCACCATGCC-(BamHI)-3`)
was inserted in the HindIII and BamHI sites of
pT7T318U (Pharmacia Biotech Inc.). Oligonucleotides containing the
hemagglutinin (HA) epitope (5`-TACCCATACGATGTTCCGGATTACGCTAGCCTC-3`)
were then inserted in the NcoI and EcoRI sites. A HindIII/ScaI fragment containing the ribosome-binding
sequence with the HA epitope was inserted in the HindIII and EcoRV sites of the pcDNAI vector, yielding pcDNAFT. p(FT)L12
is pcDNAFT containing the PCR-amplified human MRPL12 ORF.
p
L(FT)L12 contains a DNA fragment encoding amino acids
51-191 that was PCR-amplified and subcloned in the EcoRI
and XhoI sites of pcDNAFT (see above). A PCR-amplified DNA
fragment corresponding to the amino-terminal region of human MRPL12
(amino acids 1-48) was subcloned in the HindIII and EcoRI sites of pcDNA3 (Invitrogen). A fragment encoding a
HA-tagged version of RhoG (14) was then inserted 3` of the
MRPL12 fragment, yielding pL(FT)RhoG. pL12
(FT) was obtained by
cloning the human MRPL12 ORF in pcDNAI and replacing the EcoRV/XbaI fragment corresponding to amino acids
123-199 with a double-stranded oligonucleotide encoding the HA
epitope followed by a stop codon.
In Vitro Translation and Assay for
Interaction
RNAs were in vitro transcribed from the T3
promoter and translated in rabbit reticulocyte extracts according to
the suppliers' recommendations (Pharmacia Biotech Inc. and
Amersham Corp.). For interaction assays, wild-type MRPL12 and HA-tagged
L12
(FT) proteins were cotranslated, immunoprecipitated with 12CA5
antibodies, and separated by SDS-PAGE. Signal for MRPL12 (A)
was quantified on PhosphorImager screens. The efficiency of interaction
was monitored as follows. Assuming an interaction rate
and p and (1 - p) representing the proportion of
wild-type L12 and L12
(FT) proteins after translation,
respectively, the relative amount of interacting molecules is 2
p(1 - p). This value was
corrected by the efficiency of immunoprecipitation (
) and the
unspecific background (B), estimated by immunoprecipitation of
HA-tagged L12
(FT) products and of wild-type L12, respectively.
Estimation of
is given by
= (A - B)/2
p(1 - p).
Preparation of Glutathione S-Transferase-MRPL12 and
Maltose-binding Protein-MRPL12 Fusion Proteins
The human ORF
(positions +138 to +735) was amplified by PCR, subcloned in
pUC18, and sequenced. It was then cloned in pMalcR1 (New England
Biolabs, Inc.) and pGEX2T (Pharmacia Biotech Inc.) vectors.
Maltose-binding protein and glutathione S-transferase fusion
proteins were expressed in TG1 bacteria upon addition of 0.3 mM isopropyl-
-D-thiogalactopyranoside for 4 h or 0.1
mM isopropyl-
-D-thiogalactopyranoside for 4.5 h,
respectively. Recombinant maltose-binding proteins were purified on
amylose columns as described by the supplier (New England Biolabs,
Inc.).
Preparation of Specific Antibodies
Maltose-binding
protein-P2A1 fusion protein (100 µg) was used for subcutaneous
injection in rabbits. Five rounds of boost were performed, and the
immune response was monitored by Western blotting of the bacterial
protein. Two independent antisera were raised, termed a179 and a180.
Specific antibodies were affinity-purified against 50 µg of
glutathione S-transferase-P2A1 fusion protein immobilized by
Western blotting on a nitrocellulose membrane. A nitrocellulose slice
(3
0.2 cm) containing the fusion protein was incubated
overnight at 4 °C in a 5-ml test tube with 500 µl of PBS
containing 0.2% gelatin and 50 µl of a180 antiserum. The membrane
was washed for 30 min with cold PBS/Tween (0.2%) and then with cold
PBS, and antibodies were eluted by adding 400 µl of 100 mM glycine (pH 2.8). The solution was immediately neutralized in 3 M Tris-HCl (pH 8.8). Antibodies to the HA epitope were
prepared from the supernatant medium of the 12CA5 hybridoma cell
culture and purified on a protein A-Sepharose affinity column.
Immunolocalization
Hamster CCL39, human HeLa, and
monkey COS-7 cells were grown in coated 35-mm plastic tissue culture
dishes. Cell cultures were fixed with 3% formaldehyde in PBS for 30 min
at 4 °C, washed twice for 10 min with 50 mM NH
Cl in PBS and once for 10 min with PBS, and
permeabilized for 20 min at 4 °C with 0.1% Triton X-100 in PBS.
Cells were then washed for 10 min at 4 °C with PBS containing 0.2%
gelatin and incubated with affinity-purified rabbit anti-MalE/P2A1
(a180, at a 1:100 dilution in PBS/gelatin; see ``Preparation of
Specific Antibodies'') in PBS for 1 h at 22 °C.
Affinity-purified goat anti-rabbit IgG conjugated to fluorescein
isothiocyanate (1:200; Sigma) or biotinylated anti-rabbit IgG (1:200;
Amersham Corp.) was detected by incubating cells for 15 min with Texas
Red-streptavidin (1:400; Amersham Corp.). For mitochondrial labeling of
living cells, cells were incubated for 30 min at 37 °C in 5%
CO
in the presence of 10 µg ml
rhodamine 123, followed by four rounds of washings (10 min at 37
°C) with fresh Dulbecco's modified Eagle's medium. Cell
cultures were visualized with a Zeiss Axiophot epifluorescence or
confocal microscope.
Mitochondrial Extract Analysis
Cells were washed
in TTM buffer (10 mM Tris-HCl (pH 7.5), 100 mM NaCl,
and 15 mM MgCl
) and incubated for 20 min at 4
°C. Cells were broken with a Dounce homogenizer, and the lysate was
centrifuged for 5 min at 900
g. The pellet was
resuspended in TTM buffer containing 0.25 M sucrose and 1
mM EDTA and centrifuged for 15 min at 13,000
g. Pelleted mitochondria were resuspended in TTM buffer
containing 0.25 M sucrose and centrifuged on sucrose cushions
(1.5 M:1 M) for 20 min at 20,000
g.
Mitochondria were collected from the interphase, diluted in TTM buffer,
and centrifuged for 20 min at 20,000
g. For
mitochondrial extract fractionation, purified mitochondria were
resuspended in 10 mM Tris-HCl (pH 7.4), 100 mM KCl,
30 mM MgCl
, and 6 mM
-mercaptoethanol and then incubated for 15 min at 4 °C
after addition of 1% Triton X-100. Mitochondrial lysates were cleared
by centrifugation at 60,000
g for 10 min and then
fractionated by centrifugation on a 15-30% sucrose gradient
(60,000-140,000
g for 4 h at 3 °C). 16 S
rRNA distribution was determined by Northern blot analysis (see above).
The probe was a 2-kilobase pair-long ApaI/SacI
fragment isolated from Camelus bactrianus (kindly supplied by
Emmanuel Douzery, Institut des Sciences de L'Evolution de
Montpellier, Montpellier, France). Washing conditions were 0.1
SSC, 0.1% SDS at 65 °C for 30 min.
RESULTS
Mitogen Induction of P2A1 Expression
We
previously reported the isolation of a set of cDNA clones corresponding
to mRNAs that accumulate in mid-G
of resting CCL39
fibroblastic cells stimulated to grow(19) . One of these,
termed P2A1, revealed on Northern blotting a 1.1-kilobase pair mRNA
species, whose induction was strictly dependent on active protein
synthesis. As shown Fig. 1A, the P2A1 mRNA level
decreased when exponentially growing cells (lane E) were
serum-starved for 24 h (lane Q) or 48 h (lane C).
Following serum stimulation of cells (lane Q) for the
indicated times, the P2A1 mRNA level was found to increase up to 12 h (FCS lanes). When cycloheximide was added in combination with
FCS, mRNA accumulation was impaired (FCS+CHX lanes). This
inhibition does not reflect a long-range side effect of cycloheximide,
as a lag period of 1.5-2 h between serum stimulation and
cycloheximide treatment was sufficient for P2A1 mRNA accumulation to
resume (FCS/FCS+CHX lanes). This indicates that protein
synthesis is required during the first 2 h of stimulation for P2A1 mRNA
accumulation. To obtain information on the biochemical pathways
involved in increased P2A1 mRNA expression, we then examined resting
cells for their response to various purified growth factors, following
distinct biochemical pathways for transducing their mitogenic trigger,
either through receptor tyrosine kinase (EGF) or G-protein-coupled
receptor (
-thrombin)(26, 27) . Insulin is not
mitogenic on CCL39 cells, but enhances the effect of EGF. Total RNA
prepared from resting CCL39 cells stimulated for 1, 6, and 12 h was
analyzed by Northern blotting. Signal quantification is shown Fig. 1B.
-Thrombin led to a 3.5-fold increase in
P2A1 mRNA 9 h after stimulation, whereas a combination of EGF and
insulin led to a 2.5-fold induction. To ensure that P2A1 mRNA
accumulation correlated with the mitogenic response induced by growth
factors, we tested the antimitogenic effect of 8-bromo-cAMP on
-thrombin stimulation. The accumulation of P2A1 mRNA was inhibited
>80% when 8-bromo-cAMP was added in conjunction with
-thrombin,
indicating that biochemical pathways associated with DNA synthesis are
responsible for the increase in the P2A1 RNA level. Identical
cAMP-mediated inhibition was observed in cells treated with EGF or EGF
+ insulin (data not shown).
Figure 1:
A, RNA
was extracted from CCL39 cells serum-starved for 48 h (lane
C), exponentially growing cells (lane E), or cells
serum-starved for 24 h (lane Q) and then stimulated for 1, 3,
6, 9, and 12 h with 10% serum alone (FCS), for 1, 3, 6, 9, and
12 h with FCS in combination with 10 µg ml
cycloheximide (FCS+CHX), or for 0, 0.5, 1, 1.5, and
2 h with FCS and then for 6, 5.5, 4, 4.5, and 4 h with FCS +
cycloheximide (FCS/FCS+CHX). 10 µg of total RNA was
electrophoresed and transferred to nylon membranes. Filters were
sequentially probed with
P-labeled P2A1 and S26 cDNA (65) inserts. B, shown is the effect of purified
growth factors on P2A1 mRNA accumulation. Total RNA was extracted from
cells stimulated for the indicated times (in hours) with 1 unit
ml
-thrombin (
-thr),
-thrombin plus 1 mM 8-bromo-cAMP (
-thr+cAMP), or 35 ng ml
EGF plus
10 µg ml
insulin (EGF+Ins). 10
µg of total RNA was analyzed by Northern blotting, and
autoradiographic signals were quantified by using a Millipore BioImage
workstation. C, total proteins were extracted from
exponentially growing cells (lane E), serum-starved cells (lane Q), or cells stimulated for the indicated times (in
hours) with 10% fetal calf serum. Proteins (500 µg) were analyzed
by Western blotting, and P2A1 peptide was revealed using polyclonal
a180 antibodies. D, shown is a sequence comparison of human
and hamster MRPL12 proteins with chloroplastic proteins (ear cress (28) and spinach (29) ) and prokaryotic proteins (B. stearothermophilus(32) and E.
coli(33) ). Protein sequences were aligned using TREEALIGN
software(36) . Identical amino acids(-) and deletions (*) are
indicated. E, shown is the evolutionary tree of L12 proteins.
Distances using the TREEALIGN package (36) were used to trace
an evolutionary tree. Branch length is proportional to the computed
distances. The tree is unrooted.
To demonstrate that P2A1 activation
also occurs at the protein level, Western blots of proteins prepared
from cells in various physiological states were incubated with
affinity-purified rabbit polyclonal antibodies (a183) to the
bacterially expressed human protein. As observed in Fig. 1C, a single 21-kDa peptide was revealed, whose
level decreased in resting versus exponentially growing cells
(compare lanes E and Q) and increased when cells were
stimulated to grow.
P2A1 Encodes a Protein Homologous to L12 Ribosomal
Protein
The size of the insert in the original clone was 0.5
kilobase pair. Full-length cDNAs were then isolated from hamster and
human libraries, and complete sequences were determined (data not
shown). Searching for homologous sequences in the GenBank
Data Bank revealed no match with known DNA sequences except for
several randomly sequenced cDNAs (GenBank
accession
numbers H51431, H69197, H51739, R10227, and R10550). The largest ORFs
contained in hamster and human cDNAs encode proteins of 203 and 198
amino acids, respectively. Two additional AUG initiator codons were
found upstream of the human ORF, at positions 19 and 63 (Table 1). The codon at position 63 is in frame with the main ORF
(starting at position 138). Both codons are included within a
34-nucleotide-long tandem repeat (positions 13-47 and
57-91) and are followed by stop codons (positions 40 and 84).
Protein sequences derived from hamster and human main ORFs were
compared against NBRF, GenPro, and SwissProt data bases using the BLAST
program, which revealed that their carboxyl-terminal ends displayed a
significant similarity to chloroplastic and bacterial L7/L12 proteins
(smallest Poisson probability ranging from 10
to
10
) (Fig. 1D). We investigated more
precisely the relationships between P2A1 and L7/L12 proteins from
different sources: chloroplasts from Arabidopsis thaliana (ear
cress) (28) , Spinacia oleracea (spinach)(29) , Nicotiana tabacum (common
tobacco) and Nicotiana sylvestris (wood
tobacco)(30, 31) , and prokaryotes (Bacillus
stearothermophilus and Bacillus subtilis(32) , Escherichia coli(33) , Salmonella typhimurium(34) , and Synechocystis sp.(35) ).
Sequences were first aligned with the TREEALIGN package(36) .
Multiple sequence alignments were then used to compute identity and
distance scores between members of each pair and to trace the evolution
of RL7/RL12 proteins, presented in Fig. 1E. Proteins
from Gram-positive and Gram-negative bacteria are correctly clustered,
as are chloroplastic proteins. The tree consists of three main distinct
stems, corresponding to prokaryotic, chloroplastic, and P2A1 proteins,
the latter having diverged earlier from chloroplastic proteins than
from prokaryotic proteins. In addition to primary sequence similarity,
secondary structures (37) of human and hamster putative
proteins exhibit features very close to those of prokaryotic L7/L12
proteins(38) : a helical region spanning amino acids
45-100 and a highly flexible glycine/alanine-rich hinge region
(amino acids 100-113), followed by two large helical
- and
-segments separated by a ``turn'' region, constituting a
globular domain. The amino-terminal regions (amino acids 1-45) of
mammalian and chloroplastic proteins also share common features, i.e. two short helical segments separated by coiled
structures.
P2A1 Protein Colocalizes with the Mitochondria
The
topology of the evolutionary tree suggested that P2A1 might be the
mitochondrial homologue of L12 proteins. To address this hypothesis,
a180 antibodies were used in Western blotting of subcellular extracts (Fig. 2A) and immunolocalization (Fig. 2B). Exponentially growing HeLa cells were lysed,
and cytoplasmic extracts (Fig. 2A, lane C)
were separated from nuclei and membranes (lane N.E.) by low
speed centrifugation. Mitochondria (lane M) were then
fractionated from the cytosol (lane C-M) by centrifugation on
sucrose cushions. A single specific 21-kDa protein was detected in the
mitochondrial fraction, whereas no signal remained associated with the
cytosolic fraction. A significant amount of protein was also detected
in the nuclei and membranes, which probably results from mitochondria
trapped within the perinuclear microtubule network when cells are
broken in the absence of detergent. To confirm these observations,
immunofluorescence studies using a180 antibodies (Fig. 2B, row a) were carried out in hamster
CCL39 cells (panel 1), monkey COS-7 cells (panel 2),
human HeLa cells (panel 3), and murine NIH3T3 cells (panel
4). In all cell lines studied, most of the fluorescence appeared
as cytoplasmic punctations or short filaments, reminiscent of
mitochondrial distribution. This was further confirmed by specific
mitochondrial staining of living cells with rhodamine 123 (Fig. 2B, row b). No signal was obtained with
the preimmune serum (data not shown). Taking into account its
similarity to the bacterial and chloroplastic L12 proteins and its
mitochondrial localization, this protein will be termed MRPL12 for
mitochondrial ribosomal protein L12.
Figure 2:
A, subcellular fractionation of MRPL12.
Nuclei and membranes (lane N.E.) were separated from
cytoplasmic extracts (lane C) by low speed centrifugation of
mechanically broken CCL39 cells. The cytosolic extracts (lane
C-M) were depleted of mitochondria (lane M) by high speed
centrifugation. Aliquots of either fraction were separated by SDS-PAGE,
and the amount of MRPL12 protein was detected by immunostaining of
Western blots using the affinity-purified a180 antibody. B,
immunolocalization of MRPL12. Exponentially growing CCL39 cells (panel 1), COS-7 cells (panel 2), HeLa cells (panel 3), and NIH3T3 cells (panel 4) were incubated
for 30 min with rhodamine 123, and mitochondria were observed under
confocal microscopy (row b). Alternately, cells were fixed and
incubated in the presence of the affinity-purified a180 antibodies (row a).
Characterization of MRPL12 Mitochondrial Targeting
Sequences
To confirm the efficient use of the third AUG codon,
the human ORF was selectively PCR-amplified, and in vitro translated products were compared with those synthesized from the
complete cDNA by SDS-PAGE (Fig. 3A). Proteins
translated from both substrates displayed an identical apparent
molecular mass of 29 kDa, indicating that the third AUG codon is
efficiently used. However, the in vitro translated peptide
exhibited a SDS-PAGE mobility 7-8 kDa higher than that of the
MRPL12 protein (21 kDa) extracted from hamster or human cells. To rule
out any artifactual modifications during in vitro translation,
we first transiently transfected human 293 cells with the p(FT)L12
plasmid (Fig. 4A), a construct expressing the complete
human MRPL12 protein fused to the HA epitope. A single product was
specifically detected in transfected cells (Fig. 3B),
whose apparent mobility (29 kDa) was identical to that of the in
vitro translated peptide. We next examined the amino-terminal
sequence of both hamster and human proteins, which revealed classical
features of a mitochondrial sequence leader(39, 40) :
(i) absence of acidic residues; (ii) a high content of arginine,
leucine, and serine residues (34% (man) and 44% (hamster)); (iii) a
high content of positively charged amino acids (23% (man) and 18%
(hamster)); and (iv) a high hydrophobic helical moment. A potential
cleavage site
(RXZ(X)
(S/T/G)(X)
R,
where Z is any hydrophobic residue) for mitochondrial
processing peptidase (39, 40) was found at positions
45 (man) and 47 (hamster), as shown in Fig. 1B. To
address the role of the amino-terminal sequence, we transiently
transfected human HeLa cells with the p
L(FT)L12 construct (Fig. 4B), expressing a HA-tagged truncated version of
human MRPL12 lacking 48 amino acids in its amino-terminal part. In
vitro translation yielded a product of 21 kDa, in agreement with
the size of the endogenous protein (Fig. 3D).
Immunolocalization of the
L(FT)L12 protein with both anti-HA
monoclonal 12CA5 antibodies and anti-MRPL12 polyclonal a180 antibodies
revealed a distribution throughout the cytoplasm (Fig. 3C), indicating that at least part of the
targeting signal has been deleted from the protein. We next transiently
transfected the pL(FT)RhoG construct (Fig. 4C), which
drives the expression of a HA-tagged mutated version of RhoG protein,
normally localized in the perinuclear endoplasmic
reticulum(41) , fused to the first 49 amino acids of MRPL12
leader sequence. At variance with the p
L(FT)L12 construct,
staining of cells transfected with the pL(FT)RhoG construct with 12CA5
antibodies showed a typical mitochondrial distribution. These data
establish that the NH
-terminal 49 amino acids are necessary
and sufficient for mitochondrial targeting and suggest that the leader
peptide is cleaved off during MRPL12 mitochondrial import.
Figure 3:
A, comparison of electrophoretic
mobilities of in vitro translated human products on SDS-PAGE.
Proteins were translated in vitro from the full-length cRNA or
from RNA corresponding to the largest ORF and separated by SDS-PAGE.
Size markers are indicated on the right (lane M). B,
detection of the unprocessed MRPL12 product. Extracts from HeLa cells
transfected with the p(FT)L12 construct (lane T) or from
control cells (lane C) were analyzed by Western blotting using
the a180 antibodies. C, immunolocalization of
L(FT)L12
and L(FT)RhoG proteins. HeLa cells were transiently transfected with
p
L(FT)L12 and pL(FT)RhoG (see ``Materials and Methods''
and Fig. 4). 24 h after transfection, cells were fixed and
incubated in the presence of immunopurified 12CA5 antibodies or
affinity-purified a180 antibodies. D, in vitro translation of
L(FT)L12 protein. RNA was synthesized and
translated in vitro in the presence of
[
S]methionine. Labeled products were separated
by SDS-PAGE. Lane wt, (FT)L12 protein; lane
,
L(FT)L12 protein.
Figure 4:
Constructs expressing various modified
MRPL12 proteins. The corresponding amino acid sequences are indicated
below each map. The HA epitope sequence is indicated in boldface, and the mitochondrial targeting peptide is underlined. A, p(FT)L12, derived from pcDNAI,
contains the complete ORF (MRPL12) fused to the HA epitope. B,
p
L(FT)L12, derived from pcDNAI, contains the ORF from which a
portion coding for the NH
-terminal 48 amino acids was
deleted. C, pL(FT)RhoG, derived from pcDNA3, contains a
HA-tagged version of the human RhoG ORF fused to a fragment coding for
the NH
-terminal 49 amino acids of MRPL12. D,
pL12
(FT), derived from pcDNAI, contains the MRPL12 ORF from which
a fragment coding for the carboxyl-terminal 77 amino acids was replaced
with the HA epitope. CMV, cytomegalovirus; H, HindIII; E, EcoRI; Xb, XbaI; Xh, XhoI; R, EcoRV.
Expression of MRPL12
, a Truncated Version of MRPL12
Protein
Prokaryotic L7/L12 proteins are associated with
ribosomes as structures made of two homodimeric complexes bound to a
single molecule of L10 protein(42) . The protein segments
involved in homodimer formation span the amino-terminal helix,
corresponding to amino acids 46-91 in the human sequence, while
the region spanning amino acids 119-198 displays a basic
helix-turn-helix structure, required for binding to ribosomal
RNA(43, 44, 45) . To inhibit the activity of
endogenous MRPL12, we expressed a MRPL12 protein lacking its RNA
binding activity upon deletion of 76 amino acids in its
carboxyl-terminal tail (pL12
(FT) construct) (Fig. 4D), but retaining its ability to be targeted to
the mitochondria and to interact with the endogenous protein. The
correct processing and targeting of the exogenous protein were
monitored by analyzing the apparent mobility and the localization of
the truncated protein in transiently or stably transfected HeLa cells (Fig. 5A). 18-kDa (i.e. the same size as the in vitro translated product (lane C)) and 15-kDa
peptides were detected with 12CA5 antibodies in cellular extracts from
transiently transfected cells (lane T), while a single band of
15 kDa was observed in extracts from stably transfected cells (lane
S). Although the mobility shift approximates 3 kDa, this indicates
that stably transfected cells express prominently the processed
protein. We next isolated six independent stably transfected clones and
compared the subcellular distribution of the truncated protein with
that of endogenous MRPL12 by immunofluorescence analysis. All cell
clones elicited a strong signal for the truncated construct,
corresponding to a mitochondrial distribution, as illustrated in Fig. 5B.
Figure 5:
Characterization of clones expressing
MRPL12
protein. A, HeLa cells were transfected with
pL12
(FT), and total protein extracts were analyzed by Western
blotting using 12CA5 antibodies. Lane S, stably transfected
cells; lane T, transiently transfected cells; lane C, in vitro translated peptide. B, cells from stably
transfected independent HeLa clones (1, 2, and 6) were fixed and incubated in the presence of immunopurified
12CA5 antibodies (row a) or affinity-purified a180 antibodies (row b) or were stained in the presence of rhodamine 123 (row c). Cells were visualized under confocal microscopy.
Signals corresponding to full cell sections are
shown.
In Vitro Interaction between MRLP12 and
MRPL12
We then examined whether wild-type MRPL12 and
MRPL12
proteins were able to associate. mRNAs transcribed from a
wild-type MRPL12 cDNA and the pL12
(FT) construct were cotranslated in vitro in the presence of
[
S]methionine, and translation products were
immunoprecipitated using 12CA5 antibodies (Fig. 6A).
Equal amounts of nonimmunoprecipitated (lanes C) and
immunoprecipitated (lanes IP) reactions were analyzed by
SDS-PAGE. Under these conditions,
19% of the 18-kDa MRPL12
protein immunoprecipitated (MRPL12
, lane IP versus
lane C), while 12CA5 antibodies gave a background of 0.05% with
the untagged 29-kDa complete protein (MRPL12, lane IP
versus lane C). After cotranslation, addition of 12CA5 antibodies
led to the immunoprecipitation of 5% of the untagged 29-kDa protein (MRPL12+MRPL12
, lane IP versus lane C).
According to the relation described under ``Materials and
Methods'' and given that the relative amounts of the proteins
synthesized during cotranslation were 68% (29 kDa) and 32% (18 kDa) (MRPL12+MRPL12
, lane C), this indicates
that
60% of the translated proteins have interacted, probably as
heterodimers, as shown for the bacterial L7/L12 proteins(42) .
Figure 6:
Dimerization and submitochondrial
distribution of MRPL12 and MRPL12
proteins. A, MRPL12 (29
kDa) and HA-tagged MRPL12
(21 kDa) proteins were synthesized alone
or in combination from in vitro translation mixtures in the
presence of
S-labeled methionine and immunoprecipitated
using anti-HA 12CA5 antibodies. Aliquots from immunoprecipitated
proteins (lanes IP) or from the translation mixture (lanes
C) were separated by SDS-PAGE and exposed to autoradiography. B, mitochondria were purified from stably transfected cells
(clone 6). After lysis and membrane removal, mitochondrial matrix
extracts were fractionated on a 15-20% sucrose gradient.
Fractions were collected from the top (fraction 1) to the bottom
(fraction 32) of the gradient. Upper panel, nucleic acid
content was estimated by A
spectrophotometry (empty circles), and assay for 16 S rRNA distribution (filled squares) was performed by Northern analysis of an
aliquot of each fractions. Lower panel, fractions were pooled,
and proteins were extracted and analyzed on Western blots. Membranes
were incubated with a180 antibodies or with 12CA5 antibodies to detect
the endogenous protein (21 kDa) or the exogenous protein (15 kDa).
Molecular mass markers are indicated on the right. C, extracts
from the mitochondrial matrix were immunoprecipitated with 12CA5
antibodies. Immunoprecipitates were analyzed by Western blotting, and
proteins were revealed using a180 antibodies. The strong signal at the
top of the gel corresponds to mouse IgG heavy chain detected by goat
antibodies to rabbit IgG.
Cofractionation of Wild-type and Mutant MRPL12
Proteins
The previous experiments showed that the truncated
version of MRPL12 protein was processed and targeted to the
mitochondrion and could associate in vitro with the wild-type
protein. As bacterial L12 proteins were shown to associate with
ribosomes, we next examined the submitochondrial localization of both
endogenous and truncated proteins in stably transfected HeLa cells.
Lysates from purified mitochondria were fractionated on a 15-30%
sucrose gradient, and aliquots of the collected fractions were analyzed
by Western blotting for their content in MRPL12 and MRPL12
proteins, by Northern blotting for their content in 16 S ribosomal
RNAs, and by spectrophotometry for their content in nucleic acids (Fig. 6B). The overall distribution of nucleic acids (upper panel, dashed line) is in agreement with
published data (46) , exhibiting three peaks corresponding to
35-45 S (ribosomal subunits), 60 S (monomeric ribosome), and 74 S
(polysomal structures). 16 S rRNA is mainly distributed within all
ribosomal structures (filled squares). Western analysis of
MRPL12 proteins shows a loose distribution of the endogenous protein
centered at 35-45 S (Fig. 6B), while the
truncated protein exhibits a much tighter distribution on the same
ribosomal structure (Fig. 6B). This establishes that
both endogenous and truncated MRPL12 proteins cofractionate with
ribosomal subunits. As the truncated protein has lost its basic
helix-turn-helix RNA-binding domain, this also suggests that both
wild-type and truncated proteins are present in the same protein
complex. To address this latter issue, mitochondrial extracts were
immunoprecipitated using anti-HA 12CA5 antibodies. Immunoprecipitated
products were analyzed by Western blotting using anti-MRPL12 a180
antibodies (Fig. 6C). Two peptides of 21 and 15 kDa
were detected, indicating that both proteins are associated with the
same structures.
Expression of MRPL12
Inhibits Cell Growth and
Mitochondrial ATP Production
We next examined transfected HeLa
clones for their physiological properties. We first compared the growth
kinetics of six independent transfected clones. Cells transfected with
wild-type pcDNAI/Neo were included as a control. Cells were seeded at
20
10
cells ml
in RPMI 1640
medium supplemented with 10% FCS, and cell populations were estimated
up to 172 h after seeding. As observed in Fig. 7, all
transfected clones exhibit a reduced growth rate (average doubling
times ranging from 26 to 28 h for clone 2 to 34 to 36 h for clones 1
and 5) compared with the control cells (average doubling time of
18-20 h). As the mitochondrial genome codes exclusively for
proteins involved in oxidative phosphorylation, we analyzed the
transfected clones for their mitochondrial ATP production. Three clones
exhibiting different ranges of growth inhibition were compared for
their behavior with regard to the absence of glycolysis by incubating
cells in glucose-free medium and with regard to mitochondrial ATPase
inhibition upon addition of 10 µg ml
oligomycin (Fig. 7B). In the presence of oligomycin, the doubling
time of control cells increased moderately (20-22 h), while the
doubling times of MRPL12-transfected cells remained similar to those
observed in Fig. 7A, indicating that inhibition of
mitochondrial ATPase in the transfected clones has no effect on their
growth properties. At variance, glucose starvation led to an expected
reduction in the growth rate of control cells (doubling time of
28-30 h), but to a rapid death of the transfected clones,
confirmed by trypan blue staining (data not shown). Identical cell
death was observed in control cells upon inhibition of both pathways.
These data demonstrate that mitochondrial ATPase activity is inhibited
or severely reduced in the transfected clones and that their ATP supply
derives exclusively from glycolysis.
Figure 7:
Growth properties of HeLa clones
expressing MRPL12
. A, cells transfected with pcDNAI (C) or cells from six independent clones (Cl1, Cl2, Cl4, Cl5, Cl6, and Cl7) were seeded at a density of 2
10
ml
and grown for the indicated times in RPMI
1640 medium supplemented with 10% FCS. Asterisks indicate
times at which cells were refed with fresh medium. B, cells
transfected with pcDNAI (Control) or cells from three
independent clones (Cl1, Cl2, and Cl6) were
seeded at 10
cells ml
in minimum RPMI
1640 medium supplemented with 10% FCS and 2 g liter
glucose. 24 h later, cells were cultured for the indicated times
in the absence (empty symbols) or presence (filled
symbols) of 10 µg ml
oligomycin, in the
absence (squares) or presence (circles) of 2 g
liter
glucose.
DISCUSSION
To get a better understanding of the molecular events
associated with cell proliferation, several groups have initiated an
overall survey of mRNAs that accumulate in late G
of
serum-stimulated cells. We previously characterized a subset of cDNA
clones complementary to serum-inducible RNAs, derived from hamster
CCL39 fibroblastic resting cells stimulated with serum for 5 h. Three
of them were shown to participate in the delayed-early response (19) . Sequence analysis showed that two delayed-early response
mRNAs encoded the plasminogen activator inhibitor (type I) (19) and the immunophilin-binding FKBP59 protein(41) .
We report here the characterization of the third delayed-early response
mRNA, originally termed P2A1(19) , as well as its human
counterpart.
This represents the first cloning and characterization
of a mammalian mitochondrial ribosomal protein encoded by the nucleus.
Previous reports led to the identification of various mammalian L12
ribosomal proteins related to prokaryotic L11 and yeast L15
proteins(47, 48, 49) that were shown to bind
28 S rRNA and to be cross-linked to elongation factors 1
and
2(50, 51, 52) . Although attempts were made
to relate these proteins to prokaryotic L12(30) , our data
unambiguously demonstrate that P2A1 is the mitochondrial homologue of
prokaryotic L7/L12 ribosomal protein, which will now be termed MRPL12.
Although sharing a low level of identity in their primary structures,
MRPL12 and bacterial L12 proteins exhibit a conserved overall secondary
structure (two helical structures separated by a flexible hinge
region). In bacteria, the NH
-terminal helix is involved in
dimerization, while the COOH-terminal helix displays a helix-turn-helix
structure, involved in binding to ribosomal RNA. Assuming similar
properties for MRPL12 protein, we showed that the expression of a
protein potentially devoid of RNA binding activity impairs the normal
function of the protein complex, probably due to reduced translation,
leading to inhibition of oxidative phosphorylation.
Mitochondria are
organelles whose main function is ATP production, but that also
contribute to the biosynthesis of many metabolites such as pyrimidines,
amino acids, and phospholipids. Although not essential for in vitro cell cultures(20) , the mitochondrial activity is
regulated at developmental and physiological
levels(53, 54) , and its failure is probably involved
in numerous degenerative diseases (55, 56, 57) . Several nuclear genes
activated by growth factors or hormones were shown to encode proteins
controlling various aspects of mitochondrial metabolism: energy
production (the adenine nucleotide translocator ANT2(23) , a
proton/phosphate symporter(19) , or cytochrome c(58) ), lipid biosynthesis (the p90
glycerol-3-phosphate acyltransferase (59) or the uncoupling
protein (60) ), and protein import (the chaperonin hsp60 (61) or Fos effector protein-1, similar to a yeast
mitochondrial protein import(22) ).
In mammalian cells, the
mitochondrial genome encodes a limited number of proteins involved in
oxidative phosphorylation. Mitochondrial RNAs are transcribed as
polycistronic primary transcripts from a unique bidirectional promoter,
implying that regulation of gene expression mainly relies on
post-transcriptional mechanisms such as RNA or protein turnover and
translational control(62, 63, 64) . As L7/L12
proteins have been shown to control the efficiency of mRNA translation
in E. coli, it is tempting to speculate a similar function for
MRPL12 in the mitochondria. Our data establish that MRPL12 mRNA and
protein accumulate in response to growth factor stimulation and that
mRNA induction is inhibited when cells are treated with antimitogenic
agents such as cAMP. Growth regulation of MRPL12 protein could then
represent a direct regulatory mechanism for increasing the amount of
proteins involved in mitochondrial metabolic pathways in response to
external stimulation.