From the Department of Integrated Biosciences,
Graduate School of Frontier Sciences, University of Tokyo, 7-3-1 Hongo,
Bunkyo-ku, Tokyo 113-8656, § Department of Chemistry and
Biotechnology, Graduate School of Engineering, University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656,
Protein Research Group,
Genomic Sciences Center, RIKEN Yokohama Institute, Tsurumi-ku,
Yokohama, Kanagawa 230-0045, and ** Department of Physics, Osaka Medical
College, 2-41 Sawaragi-cho, Takatsuki, Osaka 569-0084, Japan
Received for publication, January 17, 2001
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ABSTRACT |
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The mammalian mitochondrial ribosome
(mitoribosome) is a highly protein-rich particle in which almost half
of the rRNA contained in the bacterial ribosome is replaced with
proteins. It is known that mitochondrial translation factors can
function on both mitochondrial and Escherichia coli
ribosomes, indicating that protein components in the mitoribosome
compensate the reduced rRNA chain to make a bacteria-type ribosome. To
elucidate the molecular basis of this compensation, we analyzed bovine
mitoribosomal large subunit proteins; 31 proteins were identified
including 15 newly identified proteins with their cDNA
sequences from human and mouse. The results showed that the proteins
with binding sites on rRNA shortened or lost in the mitoribosome were
enlarged when compared with the E. coli counterparts; this
suggests the structural compensation of the rRNA deficit by the
enlarged proteins in the mitoribosome.
Recent advances in structural biology has enabled the observation
of the detailed structure of bacterial ribosome at atomic resolution.
Electron microscopy has produced high resolution images of
Escherichia coli ribosome (1, 2). Topological orientation of
RNA helices and protein components has been determined with the help of
much biochemical data (3). In past few years, crystallographers have
reported primary images of crystal structures for 30 S, 50 S, and 70 S
ribosome subunits (4-7), where some proteins and rRNA helices were
located. Most recently, the crystallographic resolution was improved
greatly, allowing the identification of the complete structures of 50 S
(8, 9) and 30 S (10, 11) at 2.4 Å and 3.0-3.3 Å resolutions,
respectively. Because almost all nucleotide residues in rRNA and many
protein components were unambiguously assigned in the electron density
maps, we have a better understanding of how rRNA and proteins assemble
to make a functional ribosome. The most notable finding was that the
functional regions for peptide bond formation in the large subunit (9, 12) and the decoding center in the small subunit (13) consist entirely
of rRNA. The ribosome is thus a ribozyme (9, 12).
As compared with the bacterial ribosome, the mammalian mitochondrial
ribosome (mitoribosome) provides another intriguing model for
elucidating the molecular basis of ribosome function. The mitoribosome
has a smaller sedimentation coefficient of 55 S (14) consisting of
large 39 S subunit and small 28 S subunits; the former contains 16 S,
and the latter contains 12 S rRNAs as RNA components without bacterial
5 S rRNA counterpart. Total length of the mitochondrial rRNA is about
half that of the bacterial ribosome. Recently, the physiochemical
properties of rat mitoribosome were precisely determined (15). Rat
mitoribosome has a large molecular mass (3.57 MDa) compared with that
of E. coli ribosome (2.49 MDa), which is explained by the
fact that the protein to RNA ratio is completely reversed (16). This
suggests that large parts of the RNA domains are replaced by protein
components during the process of mitochondrial evolution from an
eubacteria-like endosymbiont in a progenitor of eukaryotic cells.
Functional equivalency of the mammalian mitoribosome to E. coli ribosome has been suggested by the fact that mitochondrial translation factors are able to function efficiently on E. coli ribosomes. Mammalian mitochondrial elongation factors (EF-Tu
mt1 and EF-G mt) can be
replaced by bacterial factors and maintain their efficient activities
on the E. coli ribosome (17, 18), and even bacterial and
chloroplast EF-Tus can cooperate with the bovine mitoribosome and
E. coli tRNAs to some extent (17). Furthermore, initiation
factor 2 from bovine mitochondria also functions on the bacterial
ribosome (19, 20). The observations indicate that the mitochondrial
factors are exchangeable with bacterial factors on bacterial ribosome.
Systematic analysis of protein components in the mitoribosome is
necessary to elucidate the molecular basis of exchangeability of these
factors. O'Brien and co-workers (21) were the first to isolate
proteins from bovine mitoribosome by two-dimensional PAGE. They
identified 52 protein spots in the large subunit and 33 protein spots
in the small subunit (21). The increased number of proteins in the
mitoribosome as compared with the E. coli ribosome suggested
a structural and functional compensation of the shortened rRNA in the
mitoribosome by the protein moiety. Peptide analysis and cDNA
cloning of each protein component is required to characterize the
mitoribosome in more detail. Until 1998, only a few protein sequences
had been reported (22, 23). Based on the human genome data base and EST
sequences, more than 20 mitoribosomal proteins have been identified
from rat and bovine mitoribosomes using N-terminal sequencing and data
base screening (24-29).
In this study, we systematically analyzed the large subunit proteins of
bovine mitoribosome by peptide analysis using mass spectrometry and
N-terminal sequencing. We identified 31 mitoribosomal proteins
including 15 new members, with their complete human and mouse cDNA
sequences. A striking result was that the mitoribosomal proteins whose
binding sites on rRNA are shortened or lost carry an N- or C-terminal
extension, whereas the proteins with conserved rRNA binding sites have
similar molecular weights when compared with those of the E. coli counterparts. Three-dimensional orientations of the enlarged
proteins and the concomitantly lost or shortened rRNA domains were
mapped on the crystal structure of the 50 S subunit to investigate how
proteins cover the shortened RNA domains to construct the functional
mitoribosome. The enlarged proteins together with several extra
proteins that are specifically found in the mitoribosome obviously
provide both structural and functional compensation for the deficit of
rRNA in mitoribosome, so that the whole molecular architecture of the
ribosome is conserved in bacteria and mitochondria. The mitoribosome
thus serves as a good model system for defining the functional domains
of rRNA in more detail and also for an experimental verification of the concept of transition from the "RNA world" to the "RNP world" in the early process of evolution of life.
Purification of Mitoribosome from Bovine Liver--
Mitochondria
were prepared from a fresh bovine liver according to the literature
(46). Crude mitoribosomes were prepared as reported (20) and stored in
the 55 S buffer: 20 mM Tris-HCl (pH 7.6), 20 mM MgCl2, 80 mM KCl, and 6 mM 2-mercaptoethanol. The 55 S mitoribosome was purified by
the sucrose density gradient centrifugation in the 55 S buffer
containing sucrose. The concentrations of sucrose were 6% for the top
layer and 38% for the bottom layer. Before centrifugation, a linear
sucrose gradient was formed in the centrifuge tubes (Seton) by Gradient
Mate model 117 (BioComp) according to the user's manual. Eighty
A260 units of mitoribosome, dissolved in
0.6 ml of the 55 S buffer without sucrose, were layered onto the
gradient. Centrifugation was run at 20,000 rpm for 18 h using the
SW28 rotor (Beckman). The gradient was fractionated from the top to the
bottom using the Piston Gradient Fractionater (BioComp). Fractions
containing 55 S mitoribosomes were pooled and collected by the
ultracentrifugation at 40,000 rpm for 24 h using the 70 Ti rotor
(Beckman). The purified 55 S mitoribosome was dissociated into 39 S and
28 S subunits in a buffer containing 20 mM Tris-HCl (pH
7.6), 2 mM MgCl2, 200 mM KCl, and 6 mM 2-mercaptoethanol. Each subunit was purified by sucrose
density gradient centrifugation at 20,000 rpm for 16 h using the
SW28 rotor. The purified 55 S, 39 S, and 28 S ribosomal subunits were
dissolved in the 55 S buffer and stored at Separation and Isolation of Mitoribosomal Proteins by Radical
Free High Reducing (RFHR) Two-dimensional PAGE--
Total ribosomal
proteins were extracted from the purified 55 S mitoribosome by stirring
for 1 h at 4 °C in 600 µl of the solution containing 50 mM magnesium acetate, 50%(v/v) acetic acid; 13.3 A260 units (425 pmol) of mitoribosome
were used. After removing the insoluble fraction by centrifugation,
total soluble proteins were precipitated in 5 times the volume of
acetone at In-gel Digestion of Mitoribosomal Proteins with Trypsin--
The
protein spots on RFHR two-dimensional gel were visualized by Coomassie
Brilliant Blue staining, excised, and completely dried in
vacuo. The dried gel pieces were then rehydrated with 5-10 µl
of the trypsin digest solution (0.2 M
NH4HCO3, 15 ng/ml L-1-tosylamido-2-phenylethyl chloromethyl ketone-trypsin
(Pierce)). After the gel pieces absorbed the liquid, 10-20 µl of 0.2 M NH4HCO3 was added to the gel.
Complete digestion was carried out overnight at 30 °C. The digested
peptides were extracted from the gel by shaking in 200 µl of 60%
acetonitrile and 0.1% TFA solution for 20 min. This step was repeated
two more times. The collected fractions containing peptides were
batched together and dried in a speedvac, and the obtained sample was
dissolved in 80 µl of 0.1% formic acid.
For the proteins separated by SDS-PAGE, the gel piece was soaked in a
buffer containing 0.2 M NH4HCO3
with 50% acetonitrile and incubated at 30 °C for 30 min to remove
SDS from the proteins. To assure the complete removal of SDS, the step
was repeated twice. Subsequent treatment of the sample was same as
described above.
Mass Spectrometry and Protein Identification--
A Finnigan
LCQ ion trap mass spectrometer (ThermoQuest) equipped with an
electrospray ionization source was used for the peptide analysis of
mitoribosomal proteins. The LC/MS analysis was performed using an
ODS reverse-phase column (Monitor C18, 0.1 × 15 cm; Michrom BioResource) connected on-line to the electrospray interface. A solvent
system consisting of 0.1% formic acid in H2O (A) and in
acetonitrile (B) was developed from 0% to 70% B in 35 min at a flow
rate of 50 µl/min by using the Magic 2002 high performance liquid
chromatography system (Michrom BioResource). The flow rate of sheath
gas and the capillary temperature were kept at 55 arbitrary units and 235 °C, respectively. The zoom scan analysis and MS/MS experiment by collision-induced dissociation using the data dependent scan (triple play) were performed in the range of 300-2000
m/z throughout the separation. An uninterpreted
data set of the peptide product ion for each protein generated from the
triple play analysis was searched against the nonredundant protein data
base and human and mouse EST data bases using the SEQUEST search
program (32, 48).
Determination of N-terminal Sequence--
The excised gel pieces
were dried in vacuo and soaked in a buffer consisting of 10 mM CAPS-NaOH (pH 11) and 10% methanol for 1 h. The
each protein in a gel was blotted onto polyvinylidene difluoride
membrane (Fluorotrans, Paul) by electroelution using the Electroelutor
(Nihon Eido, Japan). Amino acid sequencing was performed using a
gas-phase protein sequencer (PPSQ-21, Shimadzu).
Separation of Bovine Mitoribosomal Proteins from 55 S Mitoribosome
and 39 S Large Subunit--
To understand the compensation mechanism
of the RNA deficit by proteins to maintain ribosomal function, we first
analyzed the protein content in the large subunit of the mitoribosome. Since the crude ribosome fraction obtained by ultracentrifugation of
the mitoplast extract contained various proteins for metabolisms and
amino acid biosynthesis (data not shown), 55 S mitoribosomes were
purified by sucrose density gradient centrifugation. Furthermore, the
large 39 S subunit was isolated from the purified 55 S mitoribosome by
sucrose density gradient centrifugation under a low Mg2+
concentration. The proteins of the purified 55 S mitoribosome were
isolated by RFHR two-dimensional PAGE (30, 31), which has a wide
separation range in the basic region and, thus, applies well for
ribosomal proteins. RFHR two-dimensional PAGE requires no SDS, thereby
simplifying the peptide analysis of gel-purified protein by LC/MS
spectrometry. More than 70 protein spots were identified in the gel
(Fig. 1A). Each protein
component of the 39 S subunit was analyzed by one-dimensional SDS-PAGE,
as shown in Fig. 1B.
Peptide Analysis of the Large Subunit Proteins and Identification
of Their cDNA Sequences--
Each protein spot from the 55 S
mitoribosome and 39 S large subunit was treated with trypsin in the
gel. The tryptic peptides were subjected to mass spectrometric analysis
by LC/MS/MS as described under "Experimental Procedures." A total
of 27 proteins were identified as ribosomal proteins from the large 39 S subunit (Fig. 1B).
An example (L11mt) for the investigation of the LC/MS/MS analysis is
shown in Fig. 2A. The triple
play analysis by a data-dependent scan was performed for
each bovine protein to obtain the information on the molecular mass
(upper panel), charge state (middle panel), and
fragment patterns for each peptide (lower panel) within one analysis (see "Experimental Procedures"). The data set of the LC/MS/MS analysis was matched against the nonredundant protein data
base of human (and mouse) EST data base by the algorithm SEQUEST (32),
and the partial cDNA sequences encoding the analyzed proteins were
obtained. High homology in the amino acid sequences of mammalian
mitoribosomal proteins led to many primary hits in the human (and
mouse) EST data base. A series of related EST sequences were retrieved
by BLASTN search (33), with the hit sequence as a query and assembled
in silico to make the longest possible cDNA sequences.
Sequencing errors were identified and corrected by comparing
overlapping EST clones. Since ribosomal proteins are relatively small
in general, highly reliable cDNA sequences containing complete open
reading frames were obtained. Taking the analysis of L11 homologue
(bMRP-32) as an example, two EST sequences from human (AI188527) and
mouse (AA272356) were obtained from an initial SEQUEST search. The
possible longest cDNA sequence was obtained by assembling numerous
related EST sequences. A complete open reading frames of L11 homologue
was encoded in the sequence as shown in Fig. 2B. Many
peptide ions derived from this protein sequence were identified in the
mass spectra and assigned in the mass chromatogram (Fig. 2A,
left upper panel). Furthermore, a partial or complete
sequence of each peptide was assigned in the collision-induced
dissociation spectrum, as shown in Fig. 2A (right
lower panel). For four small proteins, namely bMRP-59b, -66, -68, and -69, N-terminal sequences were directly determined by the peptide
sequencing and the EST data base analysis using the TBLASTN program
(33). The human EST clones with significant homology were
successfully retrieved for each protein, as shown in Table
I.
The protein sequences for L3, L23, and L33 homologues were previously
reported not as mitochondrial ribosomal proteins, although their
sequence similarity with prokaryotic counterparts had been implied
(34-36). Three protein spots were identified as the above three
proteins. The specific EST clones (listed in Table I) from human or
mouse were initially retrieved for each protein by SEQUEST or
TBLASTN search. With 16 proteins having identical sequences to the
previously reported proteins, 12 new sequences were assembled in
silico comparing many related EST sequences. The protein sequences derived from each cDNA sequence of human or mouse were used as queries to search against other data bases to retrieve homologous proteins in Drosophila melanogaster, Caenorhabditis
elegans, and yeast mitochondria (Table I). Sequence alignment of
each subunit with respective homologues from other species is shown
in Fig. 3.
In addition to 27 species that we identified from the large 39 S
subunit, four protein homologues of L3, L33, L34, and L36 were found in
the 55 S mitoribosome in our two-dimensional PAGE analysis. The small
components, such as L33mt, L34mt and L36mt, could not be detected in
the SDS-PAGE analysis of the 39 S subunit for technical reasons. But
even the relatively large L3mt could not be detected on either the 39 S
nor the 28 S subunits for unknown reasons. We suggest that these
additional four protein homologues, which have significant sequence
similarity with the bacterial counterparts, belong to the large subunit.
A total of 31 proteins was identified in the large 39 S subunit,
including 15 new members (Table I, Fig. 3). Fourteen of 15 newly
identified protein sequences were recognized as prokaryotic homologues
of ribosomal proteins given their sequence similarity. The remaining
one sequence, bMRP-36a, had no homology to any of ribosomal proteins
but had significant similarity to B8 subunit of NADH-ubiquinone
oxidoreductase (complex I) (Fig. 3).
To determine the cleavage site of the signal sequence for the
mitochondrial import, N-terminal sequencing was performed. The N-terminal sequences for nine proteins were determined, whereas six
proteins may have blocked termini, since we were unable to determine
the sequence (Fig. 3). The molecular weight of each protein was
calculated by its amino acid sequence of the matured form (listed in
Table I). For the proteins with the blocked N termini, apparent
molecular weights were estimated by SDS-PAGE.
Protein Components in the 39 S Large Subunit of the
Mitoribosome--
Among the 31 protein components in the large 39 S
subunit, 24 proteins were identified as prokaryotic homologues of
ribosomal proteins (Table I). The amino acid sequence homology between the human mitoribosomal protein and its counterpart from other species
was shown in Fig. 3. The significant homology of mitoribosomal proteins
throughout the sequence indicates similar topological orientations of
the proteins in the mitoribosome to those of the prokaryotic ribosome.
The partial conservation of the specific interactions between the rRNA
and protein components in the mitoribosome is suggested. High homology
was seen among animal and insect mitochondrial proteins, whereas yeast
mitoribosomal proteins have lower similarity to human mitochondrial
counterparts, which is explained by the fact that no reduction in
length of rRNA was observed in the yeast mitoribosome. As shown in the
sequence alignment in Fig. 3, the mitoribosomal proteins show the
characteristic N- or C-terminal extension that results in the increased
molecular mass when compared with E. coli counterparts.
Although all proteins in the large subunit are yet to be identified,
the average molecular mass of ribosomal proteins, both from E. coli and human mitochondria, was calculated using the identified
24 proteins that belong to the family of prokaryotic homologs. The
calculated average molecular mass for these ribosomal proteins were
21.1 kDa for mitochondrial proteins and 13.8 kDa for E. coli
proteins. Mitochondrial ribosomal proteins thus show a 1.5-fold
increase in the average of their molecular masses when compared with
those of the E. coli counterparts. In addition to these
enlarged protein homologues, new proteins specific for the mitoribosome
participate in the compensation of the decrease of the rRNA moiety in
the mitoribosome.
Ribosomal proteins L1 and L9 form the L1 ridge together with helices
76-79 in domain V of 23 S rRNA (3). The extended C terminus of human
mitochondrial L1mt is possibly replacing helices 77 and 78 that are
lacking in the mitoribosome. The molecular mass of L1mt extends for
about 1.5-fold the E. coli counterpart (Table I).
Interestingly, in the C. elegans mitoribosome, which lacks
helix 76 in addition to helices 77 and 78, the C terminus of the
C. elegans L1 homologue is even longer than the C terminus of human mitochondrial L1mt (Fig. 3). In addition, both extended termini of L9mt seem to compensate the collar region of L1 ridge. Several ribosomal protein homologues that are indispensable for peptidyltransferase activity (37-39) have been identified in this study. L3mt shows a significant homology to its E. coli
counterpart over its entire sequence but has elongated N and C termini
that seem to compensate for the missing binding sites in domain VI (see
below). In the case of the C. elegans mitoribosome
having short domain VI rRNA, the L3 homologue contained two
characteristic insertions and the longest C terminus (Fig. 3). L4mt
shows one of the most prominent enlargement. L16mt also has an extended C terminus with a relatively low homology to the E. coli
counterpart. L11 is possibly involved in GTP hydrolysis by translation
factors in collaboration with L8 complex (L10· (L7/12)4)
and the GTP-associated region (helices 42-44). The high homology of
L11mt to the counterparts from other E. coli organisms can
be explained by the fact that the binding site for L11 on helices 43 and 44 is well conserved in all mitochondrial rRNA.
Since the C. elegans mitoribosome contains shorter rRNA (952 bases) in the large subunit than the corresponding rRNA (1558 bases) of
human mitoribosome, the progressive compensation of lost rRNA with
enlarged protein in C. elegans is a tempting speculation. We
detected four protein homologues in C. elegans, L1, L3, L13 and L20, having the longest C terminus among the aligned species (Fig.
3). The findings indicate a correlation between the shortening of rRNA
and the enlargement of proteins in the animal mitoribosome.
Structural Compensation for Deficit of Mitoribosomal RNA with
Enlarged Protein Components--
To visualize the global image of the
mammalian mitoribosome composed of shortened rRNAs and enlarged
proteins, mitoribosomal proteins were mapped on the secondary structure
of rRNA (Fig. 4). The secondary structure
of human mitochondrial rRNA (red line) of the large subunit
was superimposed on the corresponding E. coli 23 S rRNA
(black line) (40). Mitoribosomal proteins with well
conserved binding sites on mt rRNA have similar molecular masses as
those of the prokaryotic counterparts. In contrast, the proteins having
small (reduced) or no distinct binding sites on mt rRNA showed
increased molecular masses due to N- or C-terminal extensions.
According to the crystal structure of the large subunit, the helices
66, 61, 53, 43/44, and 95 in rRNA are the main binding sites for L2,
L22, L23, L11, and L14, respectively. These helices are well conserved
in mt rRNA, and the mitochondrial protein homologues have similar
molecular masses with the E. coli counterparts (Fig. 4,
Table I). L24, L4, and L15 have many contacts with domains I and II in
Haloarcula marismortui 50 S subunits; their binding sites
are almost lost in the mt rRNA. The molecular masses of L24mt, L4mt,
and L15mt, however, are increased by more than 15 kDa when compared
with the bacterial counterparts. Similar compensation was also observed
in the L19mt homologue, where most of the binding sites were lost in mt
rRNA. The lack of the helices 77 and 78 in mt rRNA may be substituted
for by the extended termini of L1mt and L9mt. The L3 binding sites in
domain VI (helices 94 and 100) are also missing in mt rRNA; L3mt shows
long N- and C-terminal extensions. Taken together, these observations
strongly suggest that enlarged mitoribosomal proteins may have
compensated for the sequence losses of the rRNAs in the mitoribosome.
As already mentioned, we also identified a new protein component,
bMRP-36a, which has a high homology with B8 subunit of NADH-ubiquinone
oxidoreductase (complex I). The rRNA binding site as well as the
structure and function of this new ribosomal protein is unknown.
In 1970, Margulis proposed the symbiosis theory stating that
mitochondria have evolved from eubacteria-like endosymbionts (41);
Rickettsia-like alpha proteobacteria (42) are
considered to be the closest relative of the endosymbionts today. The
most primitive mitochondrial DNA so far was found in a protozoan
Reclinomonas americana (43), in which the largest number of
97 genes coding for proteins and RNAs were identified in the total 64 kilobase pair of mtDNA. Three ribosomal RNA genes including 5 S rRNA
have similar lengths with those of eubacterial genes. In addition to the genes related to the respiration, as many as 27 ribosomal protein
genes were encoded in the mtDNA. Furthermore, the gene organization and
the way of gene expression are also eubacterial-like. Thus that
R. americana mtDNA seems to closely resemble the ancestral mitochondrial genome. In the mammalian mitochondrial translation system, the mitoribosome has been categorized as a bacteria-type ribosome with regard to antibiotic susceptibility and sequence similarity of ribosomal proteins, translation factors, and
aminoacyl-tRNA synthetases. The bacterial ribosome, therefore, might
well be considered as a direct ancestor for mammalian mitoribosome.
Mammalian mitochondria contain small mtDNA (16 kilobase pairs) only
capable of encoding 13 proteins, 22 tRNAs, and 2 shortened rRNAs,
suggesting that mitochondria would have managed to transfer a large
portion of mtDNA to the nuclear genome during the process of
mitochondrial evolution (42). Although the molecular mechanism for this
gene transfer is unclear, the repairing process of double-strand breaks
in yeast chromosomes by mtDNA may be crucial evidence for the gene
transfer (44). The mammalian mitoribosome is composed of 2 rRNAs
derived from mtDNA and more than 70 different mitochondrial proteins
that are all encoded in the nuclear genome. Furthermore, the 5 S rRNA
gene and about half-lengths of both large and small rRNA genes were
removed from the mtDNA. The result of our analysis shows that the
bacteria-type ribosome was build by covering the gaunt backbone of the
shortened rRNAs in the mitoribosome by enlarged ribosomal proteins of
prokaryotic homologues and/or new protein families encoded in the
nuclear genes. Thus, the structural and functional compensation of the
lost rRNA with protein would have resulted by adaptiogenesis.
Because we have identified 14 new proteins in the large subunit of
mitoribosome that belong to the family of prokaryotic ribosomal protein
in this study, at least 24 homologues of prokaryotic ribosomal proteins
of the large subunit are so far reported at this moment. Therefore,
ribosomal proteins might be important to maintain ribosomal functions.
A molecular mechanism for rRNA-catalyzed peptidyl transfer has been
derived (9, 12), and it is also known that several protein components
are indispensable for the catalytic activity (37, 45). Mitoribosomal
proteins may serve as a good model to investigate the function of
ribosomal proteins in peptide synthesis.
We constructed three-dimensional models for mitochondrial rRNAs
from human, C. elegans, and C. fasciculata (Fig. 5) based on
the 50 S crystal structure of H. marismortui (8). The
shortened mt rRNA forms a spherical cluster at the center of the
peptidyltransferase, whereas several lost rRNA portions, which are
localized discontinuously in the secondary structure (Fig. 4), form
large missing domains at the bottom, the back, the central
protuberance, and the left side of the crown view. Recently, Brimacombe
and co-workers (3) report a similar model of human mitochondrial rRNA
based on their own 23 S rRNA structure. The topological orientation of
rRNA helices in their model is in good agreement with the present model
based on the 50 S crystal structure. In the case of mammalian
mitoribosome (Fig. 4), the missing bottom domain was composed of H47
(domain II), H54-58 (domain III), H62/63 (domain IV), and H99-101
(domain VI). The helices 83-85 (domain V), H38 (domain II), and 5S
rRNA necessary for forming the central protuberance are all lost. The missing back and the left domains are composed mostly of domain I,
H25/28-31/41/42/45/46 (domain II), H52 (domain III), H66/68 (domain
IV), H77-79/88 (domain V), and H97 (domain VI). A number of ribosomal
proteins are localized in rRNA portions that are not present in the
mitoribosome. rRNA portions of ribosomal protein binding sites
that seemed to be shortened are replaced by certain lengthened
ribosomal proteins; ribosomal proteins fill up the empty space by
extending their N or C terminus as observed in this study. The binding
sites for L2, L22, L23, L11, and L14, whose mitochondrial homologues
have similar molecular masses (Fig. 4, Table I), are also conserved in
the three-dimensional model (Fig. 5). Typical enlarged protein
homologues for L4, L15, L24, and L19 are localized in the region where
rRNA is missing (Fig. 5). The elongated peptide sequences of these
proteins may contribute in substituting the functional deficit of the
lost rRNA. Further study is necessary to clarify in detail how
proteins replace the missing rRNA both structurally and
functionally.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
70 °C.
80 °C for 1 h. The total proteins were
centrifuged, and the protein pellet was dried. The proteins were then
denatured and reduced at 40 °C for 30 min in 60 µl of the reaction
mixture consisting of 8 M urea, 0.2 M
2-mercaptoethanol, 0.074% acetic acid, 0.012 N KOH, 0.015% acridine orange, and 0.015% pylonine G. The resultant sample was subjected to RFHR two-dimensional PAGE analysis according to the
literature (30, 47).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Electrophoretograms of bovine mitoribosomal
proteins. A, RFHR two-dimensional PAGE analysis of
total proteins from 55 S mitoribosome. 31 spots corresponding
to the protein moiety of the large subunit were identified. To overcome
difficulty in isolating the spots in a boxed area, prolonged separation
was carried out. The result of the magnified area is shown in the
window. B, SDS-PAGE analysis of 39 S large mitoribosomal
components in a 40-cm-long gel. A total of 27 protein bands was
identified as large ribosomal components by mass spectrometric
analysis.
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Fig. 2.
Peptide analysis by LC/MS/MS using
electrospray ionization/ion trap mass spectrometry. A,
triple play analysis for bMRP-32 (L11mt). Mass chromatograms of total
ion current (TIC) are shown in the left panels.
Triple play analysis for full scan, zoom scan, and MS/MS are shown in
the top, middle, and bottom panels on
left-hand side, respectively. Peptide fragments derived from tryptic
digestion as shown in B were assigned in the chromatogram of
full scan (peptide numbers are in red; left top
panel). Mass spectra of scan number 311-313 (peptide
10, shown as the blue sequence in B
are shown in the right panels. Top,
middle, and bottom panels in the right hand side
exhibit an m/z value, charge state, and fragment
pattern for the peptide 10, respectively. Many fragment ions
of peptide 10 were identified as a-,
b-, or y-type ions and their deaminated ions (indicated by
the asterisk) according to the literature (49) in the
collision-induced dissociation spectrum (right bottom
panel). B, amino acid sequence of human L11mt deduced
from the complete cDNA assembled from related EST sequences. The
boxed region indicates the corresponding EST sequence of
AI188527, which was initially retrieved by SEQUEST as described under
"Experimental Procedures." Colored sequences were
identified in the peptide analysis as shown in A (left
top panel).
Protein components in the large ribosomal subunit from human
mitochondria
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Fig. 3.
Comparison of amino acid sequences for 15 large mitoribosomal proteins of human and mouse with those of various
organisms. Each sequence was aligned with a set of protein
homologues from C. elegans (Celeg), D. melanogaster (Dmela), Saccharomyces
cerevisiae (Scere), E. coli (Ecoli),
Bacillus subtilis (Bsubt), Bacillus
stearothermophilus (Bstea), Thermus
thermophilus (Tther), Kluyveromyces
lactis (Klact), Mycoplasma
pneumoniae (Mpneu), Thermotoga
maritima (Tmari), R. americana
(Ramer), Helicobacter pylori (Hpylo)
or Haemophilus influenzae (Hinfl). Multiple
alignment of each sequence has been carried out by CLUSTAL W (50) and
displayed by Genedoc multiple sequence alignment editor (51). The
homology values between human mitoribosomal protein sequence and others
shown at the side of the alignment were calculated by using Genedoc.
Protein sequence of bovine B8 subunit of complex I (CI-B8) was also
aligned with bMRP-36a homologues. The colored boxes
indicated a degree of the sequence similarity. The N-terminal sequence
of each bovine mitoribosomal protein was obtained by peptide
sequencing. Red letters represent N-terminal sequences with
the indicated cleavage site for the importation signal peptide.
View larger version (76K):
[in a new window]
Fig. 4.
Secondary structures for large ribosomal RNAs
of bacterial and mammalian mitochondria shown with interacting
ribosomal proteins. The secondary structure of human mitochondrial
16 S rRNA (red line) was superimposed on the secondary
structure of E. coli 23 S rRNA (black line) (40),
which is described according to the format of Ban et al.
(8). The 5' region of mitochondrial 16 S rRNA (about 160 bases) could
not be aligned with domain I of bacterial 23 S rRNA. Ovals
that represent large ribosomal proteins are mapped on the secondary
structure of rRNA, with interactions indicated by gray
arrows. Solid arrows show interaction maps that were
identified by the crystal structure of the 50 S subunit (8).
Broken arrows indicate the interaction maps obtained from
biochemical studies (52, 53). The oval size for each protein represents
the relative molecular weight. The degree of protein size enlargement
as compared with the E. coli counterpart (Table I) was
indicated with colors: red, more than 15 kDa;
orange, 10-15 kDa; yellow, 5-10 kDa;
green, less than 5 kDa.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (55K):
[in a new window]
Fig. 5.
The three-dimensional models for large
mitoribosomal RNA based on the crystal structure of the 50 S
subunit. All atom coordinates of the H. marismortui 50 S subunit were obtained from the Protein Data Bank (accession number is
1FFK (8)). The model structures for three mitoribosomes were
constructed by removal of nonconserved rRNA portions from the atom
coordinates of 1FFK, based on the secondary structure of large
mitoribosomal RNA from mammalian (B), C. elegans
(C), and Crithidia fasciculata (D)
(40). The three-dimensional structures were displayed by Rasmol Version
2.6 (54). The outline shows an edge line of the crystal
structure of the 50 S subunit from the crown view. Some functional rRNA
domains were colored: red, P loop; blue, A loop;
green, S/R loop; light blue, and L2 binding helix
(H66). Topological orientation of the ribosomal protein is shown on the
model for the mammalian mitoribosome (B).
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ACKNOWLEDGEMENTS |
---|
We are grateful to Dr. Linda L. Spremulli (University of North Carolina) for helpful advice on the preparation of the mitoribosome and kind help in setting up our meat homogenizer. We thank Hiroaki Nishiuchi, Ng Ching Ging, Fumiko Negayama, Takashi Nagaike, Akimasa Kajiura (University of Tokyo), Masumi Yamamoto, Yoko Saito, Hirotoshi Doi, and Yoshimi Arai (Gakushuin University) for kind help in the mitoribosome preparation. We are grateful to Prof. Kin-ichiro Miura (Gakushuin University) for encouragement in this project. Special thanks are due to Dr. Knud H. Nierhaus (Max-Planck-Institut, Germany) for critical reading of the manuscript and valuable comments.
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FOOTNOTES |
---|
* This work was supported by a grant-in-aid for Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture, Japan.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB049474 and AB049632-AB049657.
¶ To whom correspondence should be addressed. Tel.: 81-3-5800-6950; Fax: 81-3-5800-6950; E-mail: suzuki@kwl.t.u-tokyo.ac.jp; kw@ kwl.t.u-tokyo.ac.jp.
Published, JBC Papers in Press, February 21, 2001, DOI 10.1074/jbc.M100432200
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ABBREVIATIONS |
---|
The abbreviations used are: mt, mitochondrial; PAGE, polyacrylamide gel electrophoresis; RFHR, radical free high reducing; LC/MS, liquid chromatography/mass spectroscopy; CAPS, 3-(cyclohexylamino)propanesulfonic acid.
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