ABSTRACT
Escherichia coli
has a ribonucleoprotein complex that is composed of a 114 nucleotide 4.5S RNA
and a 48 kDa polypeptide (P48) that has been demonstrated to function in
translation and in the secretion of periplasmic polypeptides. A small RNA of
~
220 nucleotides has been identified in maize mitochondria that includes sequence
identity with the highly conserved domain of the bacterial 4.5S RNA. The
transcript is mitochondrially encoded and maps to a region upstream of the gene for ATP synthase subunit I. The mitochondrial 4.5S-like RNA has 15 nucleotides of sequence identity with the highly conserved
region of the bacterial 4.5S RNA. Sucrose density gradient centrifugation of a
maize mitochondrial lysate demonstrated that the 4.5S RNA is a component of a
high molecular weight complex under native conditions, and could be disrupted
by phenol. Anti-P48 immune serum immunoprecipitated a mitochondrial protein of
~48 kDa, and RNA gel blot analysis of the immunoprecipitation reaction indicated
that the 4.5S-like RNA co-immunoprecipitated with the 48 kDa polypeptide. The mitochondrial
4.5S ribonucleoprotein complex could function in translation or protein
targeting.
The 4.5S RNA is a small bacterial RNA that has been identified from a number of
procaryotes (
1
,
2
). The 4.5S gene (
ffs
) is essential for growth of
Escherichia coli
(
3
), and other procaryotic 4.5S genes can complement
E.coli
strains depleted in the 4.5S RNA (
2
). The 4.5S RNA of
E.coli
is a 114 nucleotide (nt) transcript, and
ffs
genes from other procaryotes express small structural transcripts of 105-271 nt when expressed in
E.coli
(
2
). The procaryotic 4.5S RNAs have a highly conserved 22 nt domain and conserved
secondary structure, although the 4.5S RNAs differ in size and have relatively
little (29%) sequence identity outside of the conserved domain (
2
,
4
).
The function of the 4.5S RNA has been controversial and continues to be an
intriguing puzzle. Suppression analysis of a 4.5S RNA depletion strain of
E.coli
lead to the identification of suppression mutations that mapped to elongation
factor G (EF-G) and the EF-G binding site on the 23S rRNA (
5
), or resulted in increased concentrations of uncharged valyl-, glutamyl- or alanyl-tRNA (
6
). In addition, the 4.5S RNA is known to sediment with ribosomes or
polyribosomes in sucrose gradients (
5
), and is released by puromycin (
5
-
7
). Thus, genetic and biochemical evidence support a role for the 4.5S RNA in
translation.
The 4.5S RNA associates with a 48 kDa polypeptide (P48, product of the
ffh
gene) to form a ribonucleoprotein (RNP) complex that has considerable
similarity to the cytosolic signal recognition particle (SRP) of eucaryotic
cells. The SRP binds to the hydrophobic signal sequence of nascent polypeptide
chains and targets the synthesis of these polypeptides to the rough endoplasmic
reticulum. The SRP is composed of a ~300 nt RNA (SRP-RNA) and six polypeptides (
8
) and has secondary structure and sequence identity with the bacterial 4.5S/P48
RNP (
9
,
10
). The conserved 22 nt domain of the 4.5S RNA is also conserved in mammalian SRP-RNAs, as is the predicted secondary structure in this region of the SRP-RNA (
11
). P48 and SRP54, the signal sequence binding subunit of the SRP, have 31%
sequence identity over the entire length of the polypeptides, as well as a
highly conserved GTP-binding domain (
12
). In addition, SRP54 and P48 interact with the 4.5S or SRP-RNAs interchangeable: P48 binds both 4.5S RNA and human SRP-RNA (
10
); and SRP54 binds SRP-RNA or 4.5S RNA (
10
,
12
).
Similarities between the 4.5S/P48 RNP and the SRP have lead to speculation and
experimentation on the possible role of the RNP in protein targeting in
bacteria (
9
,
10
). Expression of 4.5S RNA levels through a repressible promoter have been
utilized to study the phenotype of 4.5S RNA limitation (
9
,
10
). Depletion of the 4.5S RNA lead to accumulation of unprocessed precursors of [beta]-lactamase; in addition, maltose binding protein was depleted,
although accumulation of precursors was not evident (
9
,
10
). Thus, processing of the [beta]-lactamase precursor was dependent on the 4.5S RNA. In addition, the
role of P48 has been analyzed through mutation of the
ffh
gene and expression through a regulated promoter (
13
,
14
). Depletion of P48 polypeptide results in the accumulation of precursors for
secretory polypeptides such as lambda B, maltose binding protein, alkaline
phosphatase and ribose binding protein. These results strongly indicate the
4.5S/P48 RNP must play some role in the bacterial secretory pathway.
Recently a chloroplast homologue of the P48 polypeptide, 54CP, has been
identified (
15
). The 54CP protein has 44% sequence identity with the
ffh
gene product, and acts as a molecular chaperone in the chloroplast stroma (
16
). 54CP binds the light harvesting chlorophyll a/b binding protein after import
into the chloroplast, and may be involved in targeting the protein to the
thylakoid.
The importance of the 4.5S/P48 RNP in bacterial gene expression is clear,
although the function would appear to include both roles in protein synthesis
and secretion. The presence of a procaryotic homologue of the SRP in bacteria
and chloroplasts prompted us to determine whether higher plant mitochondria may
have retained a structurally related RNP.
Mitochondria were isolated from 7-10 day old seedlings of
Zea
mays
(B37N) and purified by centrifugation on a discontinuous sucrose density
gradient and resuspended in homogenization buffer containing 0.4 M mannitol (
17
).
Total maize mitochondrial RNA (mtRNA) was extracted at 65oC with phenol equilibrated with 0.3 M sodium acetate (pH 5.0) and
subsequently with chloroform (
18
). Mitochondrial RNA was fractionated in an 8 M urea/6% polyacrylamide gel in 1* TBE buffer [TBE: 89 mM Tris-borate, 2 mM EDTA (pH 8.0)]. Gel temperature was maintained at 55oC during the electrophoresis. RNAs were electro-transferred to a nylon membrane (Hybond-N
+
) at 25-40 V (100 mA) at 4oC for 16 h. The RNAs were UV cross-linked and the RNA blot was pre-hybridized [prehybridization solution of 50% formamide, 5* SSPE (20 mM Na
2
EDTA, 3.6 M NaCl, 0.2 M Na
2
H
2
PO
4,
pH 7.0), 5* Denhardt's solution (0.1% Ficoll, 0.1% polyvinylprollidone, 0.1% bovine
serum albumin), 30 [mu]g/ml herring sperm DNA] for at least 1 h at 42oC. The membrane was probed with 5'end-labeled oligonucleotide probe for 24-36 h. The sequence of the oligonucleotide is
complementary to the conserved domain of the 4.5S RNA (5'-GCTGCTTCCTTCCGGACCTGAC-3';
2
). Blots were washed with 2* SSPE at room temperature for 10 min, 2* SSPE plus 0.1% SDS for 1 h at 42oC and 0.1% SSPE for 10 min at 42oC. The RNA was detected by autoradiography.
A 1.3 kb
Bam
HI-
Pst
I restriction fragment from maize mitochondrial DNA that encoded the 4.5S-like RNA sequence was cloned into pBluescript and identified as pBP1.3.
Maize mtRNA (5-10 [mu]g) and 1 * 10
6
c.p.m. T7 or T3 promoted transcripts of pBP1.3 were precipitated and the RNA was
collected by centrifugation. The RNA was resuspended in 10 [mu]l hybridization buffer (40 mM PIPES-NaOH pH 6.4, 400 mM NaCl, 1 mM EDTA, 80% formamide). After
denaturation at 85oC for 10 min, the RNA was hybridized at 45oC for 4-6 h. The annealed RNA was treated with 300 U RNase T1 and 100
ng RNase A in 300 [mu]l RNase buffer (10 mM Tris-HCl pH 7.7 and 300 mM NaCl) at 30oC for 1 h. The RNase digestion was terminated with 0.1% SDS, 100
[mu]g proteinase K. The protected RNA fragments were extracted with
phenol/chloroform and precipitated. The protected fragments were fractionated
in an 8 M urea/6% polyacrylamide sequencing gel and autoradiographed.
Maize mitochondrial protein (10 [mu]g) was separated on a 10% SDS-PAGE and electrotransferred to a nitrocellulose membrane. The
membrane was blocked with 10% (w/v) dried milk in the TTBS solution (0.3% Tween 20, 20 mM Tris-HCl pH 7.5, 500 mM NaCl) at 4oC overnight. The blot was washed three times with 50-100 ml TTBS at room temperature for 5 min. The blot was incubated with either rabbit anti-
E.coli
P48 immune or preimmune serum at a dilution of 1:1000 for 1 h at room temperature. The immunoblot was washed three times with TTBS for 10 min. The primary antibodies were detected by the alkaline phosphatase conjugated secondary body (goat anti-rabbit IgG alkaline phosphatase conjugate; Sigma) at a dilution of 1:3000. Immunoblot was then washed
three times with TTBS and incubated with 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium for 5-30 min.
Maize mitochondria were washed twice with 1 ml native buffer [50 mM
triethanolamine-HCl (pH 7.5), 100 mM potassium acetate, 16 mM magnesium
acetate, 1 mM dithiothreitol (DTT), 1 mM EDTA, 0.1% Nikkol (octaethylene glycol
dodecyl ether)] at 4oC and collected by centrifugation at 10 000
g
for 10 min (
9
). The mitochondrial pellet was resuspended in 1 ml native buffer containing 0.5
mM phenylmethylsulfonyl fluoride, and 1 mg/ml of each of leupeptin, pepstatin,
chymostatin and antipain. The mitochondrial suspension was sonicated four times with a Biosonicator at maximum setting for 10 s. The mitochondrial lysate was clarified by
centrifugation at 10 000
g
for 10 min; and the supernatant was saved and stored at -80oC.
The experiment was performed essentially as described (
9
). Anti-
E.coli
P48 antibody was bound to protein G-agarose beads for 1 h in phosphate buffered saline (PBS) containing 2%
Triton X-100 at room temperature. The antibody-protein G beads were washed once in PBS containing 1 M NaCl, and
twice with native buffer at room temperature. The antibody-protein G beads were then resuspended with 3 vol native buffer.
The mitochondrial lysate (~100 [mu]g protein) was mixed with 40 [mu]l of a 25% slurry of antibody-protein G beads containing in a microfuge tube. The reaction was incubated at room
temperature for 1 h with constant rotation. The beads were washed twice with 1 ml native buffer, transferred to a new microfuge tube, and washed twice with 1 ml PBS. Immunoprecipitated RNA was eluted with 1% SDS, 50 mM Tris-HCl (pH 7.5), 25 mM EDTA and 20 [mu]g glycogen. RNA was extracted with phenol/chloroform, precipitated with ethanol, and subjected to RNA gel blot hybridization with the
oligonucleotide probe for the 4.5S RNA.
KCl was added to the mitochondrial extract to a final concentration of 100 mM,
and the extract was clarified by centrifugation at 10 000 r.p.m. in the
microfuge as described (
10
). The supernatant was then layered on a 5-20% linear sucrose gradient containing 10 mM Tris-HCl pH 7.0, 5 mM MgCl
2
, 100 mM KCl, 1 mM dithiothreitol, 0.01% (w/v) Triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride. Gradients were centrifuged in a Beckman SW41TI rotor at 30 000 r.p.m. (154 000
g
) for 15 h. Fractions (500 [mu]l) were collected, RNA was extracted and analyzed by RNA gel blot
hybridization.
In order to determine whether a small mitochondrial transcript with sequence
similarity to the procaryotic 4.5S RNA was present in maize mitochondria, RNA
gel blots were hybridized with an oligonucleotide probe for the 22 nt conserved
domain of 4.5S RNA. The probe detected a transcript of ~220 nt (Fig.
1
), and indicated that a small transcript with sequence similarity to the
procaryotic 4.5S RNA was present in maize mitochondria.
The
E.coli
4.5S RNA has been shown to interact with a 48 kDa polypeptide (P48) to form a
ribonucleoprotein complex. The presence of a maize mitochondrial polypeptide
that was immunologically related to the P48 antigen of
E.coli
was tested by reaction with anti-P48 immune sera (Fig.
4
A). Immune sera specifically recognized a polypeptide from maize mitochondria
(lane 1), that was comparable in size with purified P48 from
E.coli
(lane 2). Preimmune sera reacted non-specifically with a small polypeptide present in the maize mitochondrial fractions (lanes 1 and 3).
An RNP in maize mitochondria has been characterized that bears similarity to the
bacterial 4.5S RNA/P48 RNP. A gene for a 4.5S-like RNA is mitochondrial encoded and expresses a small transcript of ~220 nt. The mitochondrial 4.5S-like RNA has 15 nt of sequence identity within the 22 nt
conserved domain of the prokaryotic 4.5S RNAs. The 4.5S-like RNA from maize mitochondria has sedimentation behavior indicated that
it is associated in a high molecular weight complex. In addition, maize
mitochondria have a ~50 kDa polypeptide recognized by P48 antibody that is capable of
immunoprecipitation of the 4.5S-like RNA. These results strongly support the presence of an RNP in maize
mitochondria that is comparable in composition and structure with the bacterial
4.5S RNA/P48 RNP and the cytosolic SRP.
The cytosolic SRP binds the hydrophobic signal sequence and subsequently to a
receptor on the endoplasmic reticulum, thereby directing translation of the
polypeptide with translocation into the lumen of the endoplasmic reticulum. A
comparable process has recently been elucidated for bacterial secretion. The
4.5S/P48 RNP has structural and functional similarity to the cytosolic SRP (
21
), and the 4.5S RNP has been shown to specifically interact with the signal
sequence of secretory proteins (
22
). A receptor for the 4.5S RNP, the
ftsY
gene product, has been identified and shows sequence identity with the [alpha]-subunit of the SRP receptor of the ER membrane, and both receptor
subunits exhibit GTPase activity (
23
). Disruption of the secretory pathway through depletion of the 4.5S RNA, the
P48 polypeptide, or the receptor protein results in accumulation of precursor
of several periplasmic polypeptides including [beta]-lactamase, OmpF and ribose binding protein (
13
,
14
,
24
). However, processing and translocation of other presecretory proteins remain
unaffected, presumably because these proteins are targeted by an independent
system utilizing the Sec proteins (
25
). Thus, there is compelling evidence that bacterial protein secretion and
cytosolic targeting to the ER utilize a homologous pathway.
The SRP-like targeting pathway has also been identified in chloroplasts. 54CP is a
chloroplast protein with substantial sequence identity with bacterial P48 (
15
,
16
); however no 4.5S-like RNA has yet been identified in the higher plant chloroplast system. 54CP apparently acts as a molecular chaperone by interacting with the chlorophyll binding protein after import, and
facilitating assembly of light harvesting chlorophyll complexes in the
thylakoids. Thus, 54CP may be involved in targeting proteins to the thylakoid
membrane through an ancestral pathway. Light harvesting complex and other
photosynthetic polypeptides in prokaryotic algae would need to be targeted to
the membrane by a secretory process. In the chloroplast, the photosynthetic
membranes are localized in the thylakoids, an internal membranous system with
no direct continuity to the inner envelope. Thus polypeptides of the thylakoid
must pass through the aqueous stroma before integration into or translocation
across the membrane. The SRP-like targeting system of the ancestral endosymbiont may have been
recruited to target nuclear encoded and imported polypeptides to the thylakoid
membrane.
The role of an SRP-like RNP in maize mitochondria is unknown. It is tempting to speculate a
role in protein targeting. Early work on protein targeting to the inner
membrane and intermembrane space in mitochondria suggested that imported
polypeptides were first translocated to the matrix, and subsequently directed
through an ancestral pathway to the inner membrane or translocated across the
inner membrane to the intermembrane space (
26
). Recent studies support a stop-transfer model for protein targeting to the inner membrane or
intermembrane space (
27
) such that proteins would never completely enter the matrix, but would stop
when associated with the inner membrane. In this case, matrix targeting
proteins would not be required for protein targeting.
Regardless of how the import and targeting controversy of nuclear-encoded mitochondrial polypeptides is resolved, many mitochondrial-encoded proteins are synthesized in the matrix and targeted to the
inner membrane. In several cases nuclear-encoded translation and assembly factors are known to be involved in the
production of membrane complexes (
28
). The topological relationship between the site of synthesis and assembly is
the same in mitochondria and prokaryotic progenitors, and the SRP-like system could be involved in this process. Both ATP6 and COX2
polypeptides have hydrophobic presequences which are cleaved during the
biogenesis of the mature complexes (
29
-
31
). The maize mitochondrial 4.5S RNP may play a role in targeting these
polypeptides to the inner mitochondrial membrane for subsequent assembly or
translocation.
This research was supported by USDA grant # 94-37301-0498. The purified P48 polypeptide and anti-P48 immune serum was a gift from Dr Peter Walter (University
of California, San Francisco). We wish to thank Dr Jodi Nunnari (University of
California, Davis) for helpful discussions. We wish to thank Dr Christiane
Fauron (University of Utah, Salt Lake City) for help identifying and providing
cosmid clones.
REFERENCES
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