|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 275, Issue 42, 32585-32591, October 20, 2000
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Received for publication, April 27, 2000, and in revised form, August 2, 2000
Mammalian mitochondrial small subunit ribosomal
proteins were separated by two-dimensional polyacrylamide gel
electrophoresis. The proteins in six individual spots were subjected to
in-gel tryptic digestion. Peptides were separated by capillary liquid chromatography, and the sequences of selected peptides were obtained by
electrospray tandem mass spectrometry. The peptide sequences obtained
were used to screen human expressed sequence tag data bases, and
complete consensus cDNAs were assembled. Mammalian mitochondrial
small subunit ribosomal proteins from six different classes of
ribosomal proteins were identified. Only two of these proteins have
significant sequence similarities to ribosomal proteins from
prokaryotes. These proteins correspond to Escherichia coli S10 and S14. Homologs of two human mitochondrial proteins not found in
prokaryotes were observed in the genomes of Drosophila melanogaster and Caenorhabditis elegans. A homolog of
one of these proteins was observed in D. melanogaster but
not in C. elegans, while a homolog of the other was present
in C. elegans but not in D. melanogaster. A
homolog of one of the ribosomal proteins not found in prokaryotes was
tentatively identified in the yeast genome. This latter protein is the
first reported example of a ribosomal protein that is shared by
mitochondrial ribosomes from lower and higher eukaryotes that does not
have a homolog in prokaryotes.
Mammalian mitochondria carry out the synthesis of 13 polypeptides
that are essential for oxidative phosphorylation and, hence, for the
synthesis of the majority of the ATP used by eukaryotic organisms. The
protein synthesizing system of mammalian mitochondria has a number of
interesting features that are not observed in the corresponding systems
in prokaryotes or the cell cytoplasm (1). The ribosomes present in
mammalian mitochondria are 55-60 S particles and are composed of small
(28 S) and a large (39 S) subunits (2). They are characterized by a low
percentage of rRNA and a compensating increase in the number of
ribosomal proteins (3).
Considerable progress has been made on the identification of the
mitochondrial ribosomal proteins in yeast. About 50 different mitochondrial ribosomal proteins
(MRPs)1 have been identified
in this organism (4). Additional protein components in yeast
mitochondrial ribosomes remain to be determined. Surprisingly, less
than half of the mitochondrial ribosomal proteins in yeast show
significant sequence identities to the ribosomal proteins of other
systems (4). Analysis of the protein composition of mammalian
mitochondrial ribosomes indicates that they have more proteins than
observed in bacterial ribosomes (5). Limited information is available
on the identities of these proteins and on their relationships to
bacterial ribosomal proteins. Recently, 18 proteins of the large
subunit and 5 proteins of the small subunit of the mammalian
mitochondrial ribosome have been characterized primarily by peptide
sequencing coupled to the extensive use of the EST data bases to deduce
the full-length cDNAs and the corresponding amino acid sequences
(6-11). Of these proteins, 12 from the large subunit and 2 from the
small subunit are homologs of bacterial ribosomal proteins. The
remainder fall into new classes of ribosomal proteins. In the current
work, peptide sequence information has been obtained for six new
mitochondrial small subunit ribosomal proteins. The cDNAs and amino
acid sequences have been assembled using EST data bases, and the
genomes of Saccharomyces cerevisiae, Drosophila
melanogaster, and Caenorhabditis elegans have been searched for homologs. Only two out of the six new small subunit proteins found in mammalian mitochondrial ribosomes are similar to
prokaryotic ribosomal proteins. The remaining four small subunit proteins fall into new classes of ribosomal proteins.
Preparation of Bovine Mitochondrial Ribosomal Proteins for
Two-dimensional Gel Electrophoresis--
Bovine mitochondria and 28 S
subunits were prepared as described previously by Matthews et
al. (5), and the 28 S subunits were collected by centrifugation at
48,000 rpm for 6 h in a Beckman Type-50 rotor. The pellet
containing about 5 A260 (approximately 420 pmol)
(12) was resuspended in isoelectric focusing gel buffer containing 9.5 M urea, 2% Triton X-100, 2% ampholytes (consisting of
1.6% (v/v) pH 5-7 and 0.4% (v/v) pH 3-10), and 0.24 M
2-mercaptoethanol. The sample was prepared as described previously
prior to loading on non-equilibrium pH gradient tube gels as described
(9, 13). Following electrophoresis in the first dimension, gels were
equilibrated in buffer (10% glycerol, 2% sodium dodecyl sulfate, 1%
dithiothreitol, 62.5 mM Tris-HCl, pH 6.8) and subjected to
electrophoresis in the second dimension on 12% sodium dodecyl
sulfate-polyacrylamide gels (SDS-PAGE) (14). Gels were stained with
Coomassie Brilliant Blue G-250.
Peptide Sequencing by Mass Spectrometry--
Seven randomly
picked spots from the two-dimensional PAGE of the mitochondrial 28 S
subunit were excised and digested in-gel with trypsin (Roche Molecular
Biochemicals) in 10 mM Tris-HCl, pH 8.0, according to the
procedure of Shevchenko et al. (15) except that alkylation
of sulfhydryls was accomplished with 4-vinylpyridine.
Liquid chromatography-tandem mass spectrometric (LC/MS/MS) analyses of
in-gel digests were done using an Ultimate capillary liquid
chromatography system (LC Packings, San Francisco, CA) coupled to a
quadrupole time-of-flight mass spectrometer (Micromass, Manchester,
United Kingdom) fitted with a Z-spray ion source as described
previously (9). Uninterpreted peptide product ion spectra generated by
LC/MS/MS were searched against the nonredundant protein data base and a
human EST data base for exact matches using the Mascot search program
(16). High quality spectra that had no exact matches in either the
protein or EST data bases were sequenced de novo either
manually or with the aid of the PepSeq program (Micromass).
Computational Analysis--
Peptide sequences obtained from
Mascot searches of the protein and EST data bases and those obtained by
de novo sequencing from peptide product ion spectra were
searched against the nonredundant protein data base using the FASTA
algorithm (17). For peptides with no exact matches in the data bases,
sequences obtained by de novo sequencing were used for FASTA
searches. Because mass spectrometry cannot distinguish between the
isobaric (same nominal residue molecular weight) amino acids Leu and
Ile, initial data base searches were carried out using Leu in the
peptide sequences. Hits with an Ile at these positions were considered
exact matches. If no hits were obtained when Leu was present in the
search sequence, the search was redone with Ile. The isobaric amino
acids Phe and oxidized Met (a common artifact of PAGE) were
distinguished by diagnostic loss of methanesulfenic acid (64 Da) from
oxidized Met (18). Because the protease trypsin that cleaves on the
C-terminal side of Arg and Lys residues was used for in-gel digestion,
Lys residues could be distinguished with a fairly high certainty from isobaric Gln residues. EST data base and genomic DNA searches of the
peptide sequences were performed using the BLAST search program (19).
Sequence analysis and homology comparisons were done using the GCG DNA
analysis software package (Wisconsin Package version 10 (1999);
Genetics Computer Group, Madison, WI) and the results were displayed
using BOXSHADE (version 3.21, written by K. Hofmann and M. Baron).
Prediction of the cleavage sites for the mitochondrial signal sequence
was carried out using PSort and MitoProt II (20).
Characterization of Bovine Mitochondrial Ribosomal Proteins by
Tandem Mass Spectrometry--
As a first step toward understanding the
protein components of mammalian mitochondrial ribosomes, small subunit
proteins were separated by two-dimensional PAGE. The protein spots from
the two-dimensional PAGE were excised and subjected to in-gel digestion using trypsin. After digestion, the resulting peptide mixtures were
analyzed by nanoscale capillary LC/MS/MS using a quadrupole time-of-flight mass spectrometer. The instrument was operated in a
data-dependent MS to MS/MS switching mode where peptide
ions detected in an MS survey scan trigger a switch to MS/MS for
obtaining peptide product ion (fragmentation) spectra. Product ion
spectra of peptides contain primarily ions originating from either the peptide C terminus (y" ion series) or N terminus (b ion series), which
are formed by cleavage of amide bonds along the peptide backbone (21).
Adjacent y" or b ions differ by the corresponding amino acid residue
mass, enabling assignment of peptide amino acid sequence. A
representative spectrum for one of the peptides analyzed is shown in
Fig. 1. Initially, uninterpreted peptide product ion spectra were searched against the nonredundant protein and
human EST data bases using the Mascot program. This program searches
all entries in the data base for peptide sequences that would yield
product ion spectra with a fragmentation pattern identical to that
observed for peptide product ion spectra obtained by LC/MS/MS. In cases
where no exact matches were retrieved, spectra were subjected to manual
interpretation/sequence assignment (de novo sequencing). Peptide sequence matches obtained from Mascot data base searches and
sequences derived from de novo sequencing are shown in Table I.
Nomenclature--
Two-dimensional patterns and molecular weights
of bovine mitochondrial ribosomal small subunit proteins were reported
previously and the proteins thought to be present in these ribosomes
were designated S1 to S33, in order of decreasing molecular weight (5).
However, it is now clear that this system for designating mammalian
mitochondrial proteins does not provide a consistent way to define
them. For example, using this system, bovine MRP-S18 is the same
protein as rat MRP-S13. To simplify the nomenclature and to facilitate
a comparison between proteins in mammalian mitochondrial ribosomes and
in bacteria, we have adopted a nomenclature in which proteins are
designated by their prokaryotic homolog (for example S12 in bacteria is
designated MRP-S12 in mammalian mitochondria). Proteins without
bacterial homologs are given the next available number. Since there are
21 proteins in the bacterial ribosome, we have begun designating the
new mammalian mitochondrial ribosomal proteins beginning at MRP-S22.
This approach to the nomenclature of organellar ribosomal proteins has
recently been adopted for chloroplast ribosomal proteins (22, 23).
Overall Approach to the Assembly of Mammalian Mitochondrial
Ribosomal Protein Sequences from cDNA Clones--
Sequences of the
peptides obtained from bovine mitochondrial ribosomes (Table I) were
used to search the human EST data base using the tBLASTN program
(National Center for Biotechnology Information). A number of hits were
obtained for some but not all of the peptides used as virtual screening
probes. Overlapping clones for these hits were obtained using the
initial hit as a virtual probe to rescreen the human EST data base. For
all the peptide sequences obtained, consensus cDNAs were then
assembled by repetitive searching and comparison of EST sequences. The
sequence of the longest possible cDNA was assembled in
silico. Sequencing errors were corrected by comparison of
overlapping clones. The fully assembled human sequence was then used as
a query against entries in other data bases.
MRP-S22--
The sequences of five tryptic peptides were obtained
from this protein using mass spectrometry (Table I) and its full
sequence (360 amino acids) was deduced (Fig.
2). MitoProt II assigns a 71%
probability that MRP-S22 is localized in mitochondria and predicts
cleavage following residue 30 (20). Examination of the sequence of the
MRP-S22 using MotifFinder (software available via the World Wide
Web) indicates that this protein does not contain any of the
motifs found in the PROSITE data base including any known RNA binding
motifs. The Swiss-Prot and MitoProt II protein data bases were searched
using the full-length sequence of human MRP-S22 to find homologs of
this mitochondrial ribosomal protein. No significant similarities were
found to any known prokaryotic or eukaryotic protein in the data bases.
Therefore, MRP-S22 is categorized as a member of a "new" class of
ribosomal proteins.
A complete mouse homolog for MRP-S22 is present in the mouse EST data
base. Human and mouse MRP-S22 align well except in the region
corresponding to the mitochondrial import signal peptide. Overall,
these two proteins are 78.8% identical (Table
II). Homologs were also observed in
C. elegans and D. melanogaster (Fig. 2 and Table
II). Alignment of the C. elegans and D. melanogaster proteins with the human MRP-S22 shows that the
conserved regions are all located in the N-terminal and middle sections
of the protein (Fig. 2). Both the C. elegans and D. melanogaster proteins have C-terminal extensions not observed in
the mammalian proteins. These extensions do not share significant
homology, suggesting that they may not play an essential role in the
biological function of this protein. No sequence corresponding to the
MRP-S22 mitochondrial ribosomal protein could be detected in the yeast
genome when human, C. elegans, or D. melanogaster
sequences were used as virtual probes. This observation suggests that
prokaryotic and fungal mitochondrial ribosomes do not have this
particular ribosomal protein in common.
MRP-S26--
The sequence of only one peptide was obtained from
the tryptic digest of this protein (Table I). This peptide was derived from a previously identified protein designated MRP-S13 in rat and
MRP-S18 in bovine mitochondrial ribosomes (6). Therefore, no further
analysis was done for this protein.
MRP-S23--
The sequences of two peptides were obtained from
MRP-S23 by mass spectrometry (Table I). The protein encoded by this
cDNA is 190 amino acids in length (Fig.
3). Neither PSort nor MitoProt II
predicts a mitochondrial localization for the MRP-S23 protein. To help
ensure that this protein was actually a ribosomal protein present in
the small subunit, the intensity of this spot was examined in
two-dimensional gels of subunits prepared directly from crude ribosomes
on a single sucrose gradient and in 28 S subunit preparations obtained
in two sequential sucrose gradients. The first gradient was used to
prepare 55 S ribosomes, which were dissociated into subunits that were
purified in the second sucrose gradient. The intensity of the MRP-S23
was comparable in both types of preparations. The observation that
MRP-S23 protein is present in 55 S monosomes and in 28 S subunits
prepared from them is strongly suggestive that it is a bona
fide ribosomal protein.
A complete mouse cDNA was deduced from ESTs (Table I and Fig. 3).
Alignment of the human and mouse sequences gives 76% identity. A
homolog to human MRP-S23 is also present in the C. elegans
genome (Fig. 3). Searches of the D. melanogaster genome
detects a number of hits covering small stretches with similarity to
MRP-S23. However, most of these hits are not in predicted reading
frames. No sequence corresponding to this human mitochondrial ribosomal
protein could be detected in searches of the yeast genome using either
the human or C. elegans MRP-S23 protein as a probe.
Analysis of the amino acid sequence of the MRP-S23 using PROSITE does
not indicate the presence of any common motifs including RNA binding or
ribosomal protein motifs. A BLOCKS analysis provides several possible
poorly aligning blocks. One of these is to a region of the S8 family of
ribosomal proteins. However, alignment of the S8 sequences from several
sources with the sequence of the human MRP-S23 polypeptide gives
identities of less than 18%.
MRP-S10--
The sequences of two tryptic peptides obtained from
one spot allowed it to be identified as the bovine mitochondrial
homolog of bacterial S10. Mouse, C. elegans, and D. melanogaster MRP-S10 are also readily identified in the data bases
(Table II). Alignment of the human MRP-S10 with the corresponding
proteins from several prokaryotes indicates that the alignment begins
around residue 70 in the mammalian mitochondrial protein (Fig.
4). MRP-S10 is 24-36% identical to
various bacterial S10 proteins examined. Interestingly, the regions
most highly conserved between the prokaryotic S10 proteins do not
correspond to the regions that are most conserved between the
corresponding mitochondrial proteins. The plant mitochondrial homologs
known are 22-25% identical to the human sequence. No homolog for S10
can be detected in searches of the yeast genome when human MRP-S10 is
used as a query in a BLAST search. However, when the Escherichia
coli S10 sequence is used in a BLAST search, a mitochondrial
homolog (CAA90780) is detected with 29% identity to the E. coli sequence. Alignment of this yeast mitochondrial S10 homolog
and the human mitochondrial S10 using the GCG program GAP did not give
a convincing alignment (21% identity scattered throughout the sequence
with no more than three contiguous amino acids). This observation
indicates that the sequences of human and yeast mitochondrial S10
homologs are more closely related to the prokaryotic S10 proteins than
they are to each other.
MRP-S25--
Two peptides were generated from this protein (Table
I) and allowed the deduction of the sequence of the full-length protein from both human and mouse (Fig. 5).
Homologs are also present in C. elegans and D. melanogaster. No homolog to this protein could be detected in the
yeast genome using the human sequence as the query in an advanced Blast
or Psi-Blast search. However, when the D. melanogaster
sequence was used as a virtual screening probe, a yeast homolog with
31% sequence identity to the D. melanogaster sequence was
found. This yeast homolog is 24% identical to the human MRP-S25 (Table
II). No known ribosomal proteins from other sources show significant
similarities to MRP-S25. Therefore, this protein represents a new class
of ribosomal protein. No common protein sequence motifs can be
detected.
MRP-S24--
Three peptides were obtained from MRP-S24 (Table I),
and the complete sequences of the corresponding proteins from both
human and mouse were assembled from different ESTs (Fig.
6). A full-length genomic clone
(AC004985-2) was obtained and indicated the presence of three introns
and four exons.
The sequences of the MRP-S24 proteins from human, bovine, mouse, and
rat are strongly conserved (Fig. 6 and Table II). The sequence of the
mammalian MRP-S24 protein does not appear to be homologous to any of
the known prokaryotic ribosomal proteins. Searches of the D. melanogaster genome using the human MRP-S24 protein as a
cyberprobe indicates the presence of a homolog to the human protein.
However, no homolog can be detected in the C. elegans
genome. Thus, it appears that this ribosomal protein is not found in a
recognizable form in all animals. Alternatively, it is possible that
sequencing errors or a complex genomic organization might make it
difficult to locate the worm homolog. Finally, probing the yeast genome
with either the human or D. melanogaster MRP-S24 sequences
fails to locate a homolog (Table II).
MRP-S14--
One peptide was obtained allowing identification of
the human and mouse mitochondrial homolog of bacterial S14 (Fig.
7 and Table I). The chromosomal gene for
mitochondrial MRP-S14 has been located. It covers about 9 kilobase
pairs of DNA and consists of three exons and two introns. Cyberprobing
the C. elegans genome with the human MRP-S14 results in the
identification of the worm homolog (Swiss-Prot P49391). This protein is
40.7% identical to the human protein when the full sequences are
aligned (Table II). The D. melanogaster genome also has a
putative MRP-S14 homolog (AE003512). However, due to a probable
sequencing error, the D. melanogaster homolog could not be
obtained from the given coding sequence. Instead, the partial putative
S14 sequence is embedded in a long open reading frame of unknown
function (CG12211). Alignment of the animal and yeast S14 homologs
(Fig. 7) clearly shows that the animal proteins are more closely
related to each other than they are to yeast mitochondrial S14 as
expected. The animal proteins have clusters of conserved sequences in
blocks throughout the length of the protein. These highly conserved
regions are only partially conserved in yeast S14. The human
mitochondrial MRP-S14 is also well conserved when compared the
prokaryotic S14 proteins (Fig. 7, Table II). Once again the human S14
is more closely related to the prokaryotic proteins than to the yeast
MRP-S14. The prokaryotic S14 proteins are most highly conserved between
each other, and with human mitochondrial S14, in the C-terminal half of
the protein.
The identification and characterization of the proteins present in
mitochondrial ribosomes has been difficult due to their low abundance.
During the past several years, significant progress has been made in
identifying some of the proteins present using a combination of protein
sequencing and data base searching. In the present report, high
sensitivity peptide sequencing by mass spectrometry has been used to
identify seven different classes of mammalian mitochondrial ribosomal
proteins from the small subunit, only one of which had been
characterized previously (6). This work increases the number of
proteins identified in the small subunit to 11.
The small subunit of the mammalian mitochondrial ribosome is thought to
have about 33 different proteins. This number is considerably higher
than the 21 proteins found in the 30 S subunit of prokaryotic ribosomes
(24). Therefore, a number of the mammalian mitochondrial ribosomal
proteins are not expected to be homologs of prokaryotic ribosomal
proteins. Two of the six ribosomal proteins described in this paper
(MRP-S10 and MRP-S14) and two of the previously characterized ribosomal
proteins (MRP-S12 and MRP-S7) show significant sequence similarities to
bacterial ribosomal proteins (9, 10).
S10 is located in the head of the prokaryotic 30 S subunit (25, 26). It
is classified as a tertiary rRNA-binding protein since its assembly
into the small subunit is strongly dependent on the presence of other
proteins rather than arising from a tight direct interaction with the
small subunit rRNA. Foot-printing and UV cross-linking experiments
indicate that S10 is close to, or interacts with, helix 39 in the
3'-domain of 16 S rRNA (27, 28). A comparison of the secondary
structures of the E. coli 16 S rRNA and bovine mitochondrial
12 S rRNA indicates that the region corresponding to helix 39 is not
conserved in mitochondrial 12 S rRNA. This observation strongly
suggests that S10 interacts primarily with other ribosomal proteins in
the mitochondrial 28 S subunit. E. coli S10 can be
cross-linked to residue A9 in tRNAs bound to the A-site of the ribosome
(29). A similar proximity to the A-site is likely in the mammalian
mitochondrial ribosome.
The present work has also identified a human mitochondrial protein in
the S14 family of ribosomal proteins. Like S10, S14 is a tertiary
rRNA-binding protein located in the head of the small subunit. S14
gives weak rRNA footprints located in the 3' domain of the 16 S rRNA
(27). A zinc-finger motif found in several members of the S14 family
has been postulated to contribute to an interaction with rRNA (30, 31).
However, no zinc finger motif is present in mammalian mitochondrial
S14. Photoaffinity labeling of ribosomal proteins with puromycin
derivatives labels S14 in E. coli ribosomes suggesting that
this protein, although in the small subunit, is located in the
proximity of the peptidyltransferase center in the 50 S subunit.
Three of the four currently identified mammalian mitochondrial small
subunit ribosomal proteins that have prokaryotic homologs (S7, S10, and
S14) are located in the head of the small subunit. Of these, S7 is a
primary rRNA-binding protein (9). The assembly of both S10 and S14 into
the small subunit is dependent on the presence of S7. The location of
small subunit ribosomal proteins that are not homologs of known
prokaryotic ribosomal proteins will require an extensive investigation
of the structure of the mammalian mitochondrial ribosome.
We thank Mary Moyer (Glaxo-Wellcome) for
outstanding technical contributions to this work.
*
This work was supported by National Institutes of Health
Grant GM32734.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) P82649, P82650, and P82663-P82670.
Published, JBC Papers in Press, August 9, 2000, DOI 10.1074/jbc.M003596200
The abbreviations used are:
MRP, mitochondrial
ribosomal protein;
LC/MS/MS, liquid chromatography-tandem mass
spectrometric analysis;
MS, mass spectrometry;
EST, expressed sequence
tag;
PAGE, polyacrylamide gel electrophoresis.
A Proteomics Approach to the Identification of Mammalian
Mitochondrial Small Subunit Ribosomal Proteins*
,
Department of Chemistry and ¶ School of
Public Health, Environmental Science and Engineering, University of
North Carolina, Chapel Hill, North Carolina 27599-3290 and the
§ Department of Structural Chemistry, Glaxo Wellcome
Research and Development,
Research Triangle Park, North Carolina 27709-3398
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (26K):
[in a new window]
Fig. 1.
Product ion spectrum of the tryptic peptide
at m/z 921.4 from MRP-S25. The y" and b series
ions are labeled according to the nomenclature of Roepstorff and
Fohlman (21).
Amino acid sequences of mature MRPs of Bos taurus derived from data
base searching or de novo sequencing of peptide product ion spectra of
two-dimensional gel spots
View larger version (93K):
[in a new window]
Fig. 2.
Sequence of MRP-S22 and alignment with
homologs in other organisms. The sequence of the human cDNA
was assembled using ESTs AA313665, AA463594, W74092, and AI004657. The
mouse sequence was assembled from EST clones. The sequences of the
D. melanogaster (AAF56757) and C. elegans
homologs (CE24801) were obtained from the data bases.
Percentage identity of human mitochondrial ribosomal proteins to
homologs found in other species
View larger version (39K):
[in a new window]
Fig. 3.
Sequence of human MRP-S23 and alignment with
homologous proteins. The sequence of human MRP-S23 was assembled
using ESTs AW407459 and AW157159. The mouse protein was obtained from
assembled ESTs. C. elegans MRP-S23 homolog is Swiss-Prot
accession number P34748. Examination of the Swiss-Prot protein data
base reveals a postulated human protein (Q9Y3D9) that matches the
N-terminal 137 residues of human MRP-S23 (Table I). The sequence of
this protein was predicted based on an open reading frame (CGI-138)
observed in C. elegans. The sequence of this predicted
protein appears to be correct for the first 137 residues and then to
shift into an alternative frame due to an error in the cDNA
sequence used.
View larger version (54K):
[in a new window]
Fig. 4.
Amino acid sequence of human MRP-S10 and
alignment with homologous proteins from other species. The human
sequence was from NM_018141. The mouse protein was obtained from
assembled EST sequences. The C. elegans sequence is from
accession number CE20222; the D. melanogaster homolog is
CG4247, and the E. coli homolog is P02364.
View larger version (48K):
[in a new window]
Fig. 5.
Sequence of human MRP-S25 and alignment with
its homologs. The human sequence was assembled using EST data base
entries AW249185 and AI243774. The mouse protein was also obtained from
assembled EST sequences. The C. elegans sequence is CE22547;
the D. melanogaster homolog is CG14413, and the S. cerevisiae homolog is data base entry YKL167c.
View larger version (36K):
[in a new window]
Fig. 6.
Alignment of human MRP-S24 protein with its
homologs. The human sequence was assembled using EST data base
entries AI870515, AI086409, W46469, and AW022634. The mouse protein was
also obtained from assembled EST sequences. The D. melanogaster homolog is CG13608.
View larger version (45K):
[in a new window]
Fig. 7.
Amino acid sequence of human MRP-S14 and
alignment with homologous proteins. The human sequence was from
CAB16601. The mouse protein was obtained from assembled EST sequences.
The C. elegans sequence is from accession number P49391
(CE02309), the D. melanogaster homolog is CGI12211, and the
S. cerevisiae homolog is data base entry P10663.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed: Dept. of
Chemistry, University of North Carolina, Campus Box 3290, Chapel Hill, NC 27599-3290. Tel.: 919-966-1567; Fax: 919-966-3675; E-mail: linda_spremulli@unc.edu.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
1.
Pel, H.,
and Grivell, L.
(1994)
Mol. Biol. Rep.
19,
183-194
2.
O'Brien, T. W.,
and Matthews, D. E.
(1976)
in
Handbook of Genetics
(King, R. C., ed)
, pp. 535-580, Plenum, NY
3.
De Vries, H.,
and van der Koogh-Schuuring, R.
(1973)
Biochem. Biophys. Res. Commun.
54,
308-314
4.
Graack, H.-R.,
and Wittmann-Liebold, B.
(1998)
Biochem. J.
329,
433-448
5.
Matthews, D. E.,
Hessler, R. A.,
Denslow, N. D.,
Edwards, J. S.,
and O'Brien, T. W.
(1982)
J. Biol. Chem.
257,
8788-8794
6.
Goldschmidt-Reisin, S.,
Kitakawa, M.,
Herfurth, E.,
Wittmann-Liebold, B.,
Grohmann, L.,
and Graack, H.-R.
(1998)
J. Biol. Chem.
273,
34828-34836
7.
Graack, H.-R.,
Bryant, M.,
and O'Brien, T. W.
(1999)
Biochemistry
38,
16569-16577
8.
O'Brien, T. W.,
Fiesler, S.,
Denslow, N. D.,
Thiede, B.,
Wittmann-Liebold, B.,
Mouge, E.,
Sylvester, J. E.,
and Graack, H.-R.
(1999)
J. Biol. Chem.
274,
36043-36051
9.
Koc, E. C.,
Blackburn, K.,
Burkhart, W.,
and Spremulli, L. L.
(1999)
Biochem. Biophys. Res. Commun.
266,
141-146
10.
Mariottini, P.,
Shah, Z. H.,
Toivonen, J.,
Bagni, C.,
Spelbrink, J.,
Amaldi, F.,
and Jacobs, H.
(1999)
J. Biol. Chem.
274,
31853-31862
11.
O'Brien, T. W.,
Liu, J.,
Sylvester, J.,
Mourgey, E. B.,
Fischel-Ghodsian, N.,
Thiede, B.,
Wittmann-Liebold, B.,
and Graack, H.-R.
(2000)
J. Biol. Chem.
275,
18153-18159
12.
Hamilton, M. G.,
and O'Brien, T. W.
(1974)
Biochemistry
13,
5400-5403
13.
Cahill, A.,
Baio, D.,
and Cunningham, C.
(1995)
Anal. Biochem.
232,
47-55
14.
Laemmli, U. K.
(1970)
Nature
227,
680-685
15.
Shevchenko, A.,
Wilm, M.,
Vorm, O.,
and Mann, M.
(1996)
Anal. Chem.
68,
850-858
16.
Perkins, D. N.,
Pappin, D. J.,
Creasy, D. M.,
and Cotrell, J. S.
(1999)
Electrophoresis
20,
3551-3567
17.
Pearson, W. R.,
and Lipman, D. J.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
2444-2448
18.
Jiang, X.,
Smith, J. B.,
and Abraham, E. C.
(1996)
J. Mass Spectrom.
31,
1309-1310
19.
Altschul, S. F.,
Madden, T. L.,
Schaffer, A. A.,
Zhang, J.,
Zhang, Z.,
Miller, W.,
and Lipman, D. J.
(1997)
Nucleic Acids Res.
25,
3389-3402
20.
Claros, M. G.,
and Vincens, P.
(1996)
Eur. J. Biochem.
241,
770-786
21.
Roepstorff, P.,
and Fohlman, J.
(1984)
Biomed. Mass Spectrom.
11,
601
22.
Yamaguchi, K.,
and Subramanian, A. R.
(2000)
J. Biol. Chem.
275,
28466-28482
23.
Yamaguchi, K.,
von Knoblauch, K.,
and Subramanian, A. R.
(2000)
J. Biol. Chem.
275,
28455-28465
24.
Wittmann, H.
(1986)
in
Structure, Function and Genetics of Ribosomes
(Hardesty, B.
, and Kramer, G., eds)
, pp. 1-27, Springer-Verlag, New York
25.
Noller, H.,
Moazed, D.,
Stern, S.,
Powers, T.,
Allen, P.,
Robertson, J.,
Weiser, B.,
and Triman, K.
(1990)
in
The Ribosome: Structure, Function and Evolution
(Hill, W.
, Dahlberg, A.
, Garrett, R.
, Moore, P.
, Schlessinger, D.
, and Warner, J., eds)
, pp. 73-92, ASM Press, Washington, D. C.
26.
Wittmann-Liebold, B.
(1985)
in
Structure, Function and Genetics of Ribosomes
(Hardesty, B.
, and Kramer, G., eds)
, pp. 326-361, Springer-Verlag, New York
27.
Mueller, F.,
and Brimacombe, M.
(1997)
J. Mol. Biol.
271,
545-565
28.
Samaha, R. R.,
O'Brien, B.,
O'Brien, T. W.,
and Noller, H. F.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
7884-7888
29.
Abdurashidova, G. G.,
Tsvetkova, E. A.,
and Budowsky, E. I.
(1991)
Nucleic Acids Res.
19,
1909-1915
30.
Chan, Y. L.,
Suzuki, K.,
Olvera, J.,
and Wool, I. G.
(1993)
Nucleic Acids Res.
21,
649-655
31.
Tsiboli, P.,
Triantafillidou, D.,
Franceschi, F.,
and Choli-Papadopoulou, T.
(1998)
Eur. J. Biochem.
256,
136-141
Copyright © 2000 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  J. Kondo and E. Westhof The bacterial and mitochondrial ribosomal A-site molecular switches possess different conformational substates Nucleic Acids Res., May 1, 2008; 36(8): 2654 - 2666. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. Smits, J. A. M. Smeitink, L. P. van den Heuvel, M. A. Huynen, and T. J. G. Ettema Reconstructing the evolution of the mitochondrial ribosomal proteome Nucleic Acids Res., July 9, 2007; 35(14): 4686 - 4703. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Antonicka, F. Sasarman, N. G. Kennaway, and E. A. Shoubridge The molecular basis for tissue specificity of the oxidative phosphorylation deficiencies in patients with mutations in the mitochondrial translation factor EFG1 Hum. Mol. Genet., June 1, 2006; 15(11): 1835 - 1846. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I.-F. Chang, K. Szick-Miranda, S. Pan, and J. Bailey-Serres Proteomic Characterization of Evolutionarily Conserved and Variable Proteins of Arabidopsis Cytosolic Ribosomes Plant Physiology, March 1, 2005; 137(3): 848 - 862. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. J. Zhang, W. K. O'Neal, S. H. Randell, K. Blackburn, M. B. Moyer, R. C. Boucher, and L. E. Ostrowski Identification of Dynein Heavy Chain 7 as an Inner Arm Component of Human Cilia That Is Synthesized but Not Assembled in a Case of Primary Ciliary Dyskinesia J. Biol. Chem., May 10, 2002; 277(20): 17906 - 17915. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. C. Koc, W. Burkhart, K. Blackburn, M. B. Moyer, D. M. Schlatzer, A. Moseley, and L. L. Spremulli The Large Subunit of the Mammalian Mitochondrial Ribosome. ANALYSIS OF THE COMPLEMENT OF RIBOSOMAL PROTEINS PRESENT J. Biol. Chem., November 16, 2001; 276(47): 43958 - 43969. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Barakat, K. Szick-Miranda, I.-F. Chang, R. Guyot, G. Blanc, R. Cooke, M. Delseny, and J. Bailey-Serres The Organization of Cytoplasmic Ribosomal Protein Genes in the Arabidopsis Genome Plant Physiology, October 1, 2001; 127(2): 398 - 415. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Saveanu, M. Fromont-Racine, A. Harington, F. Ricard, A. Namane, and A. Jacquier Identification of 12 New Yeast Mitochondrial Ribosomal Proteins Including 6 That Have No Prokaryotic Homologues J. Biol. Chem., May 4, 2001; 276(19): 15861 - 15867. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Suzuki, M. Terasaki, C. Takemoto-Hori, T. Hanada, T. Ueda, A. Wada, and K. Watanabe Structural Compensation for the Deficit of rRNA with Proteins in the Mammalian Mitochondrial Ribosome. SYSTEMATIC ANALYSIS OF PROTEIN COMPONENTS OF THE LARGE RIBOSOMAL SUBUNIT FROM MAMMALIAN MITOCHONDRIA J. Biol. Chem., June 8, 2001; 276(24): 21724 - 21736. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Cavdar Koc, W. Burkhart, K. Blackburn, A. Moseley, and L. L. Spremulli The Small Subunit of the Mammalian Mitochondrial Ribosome. IDENTIFICATION OF THE FULL COMPLEMENT OF RIBOSOMAL PROTEINS PRESENT J. Biol. Chem., May 25, 2001; 276(22): 19363 - 19374. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||