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J Biol Chem, Vol. 273, Issue 52, 34828-34836, December 25, 1998
From the Max-Planck-Institute for Molecular Genetics, Ihnestrasse
63, D-14195 Berlin, Germany and the ** Institute for Genetics, Free
University of Berlin, Arnimallee 7, D-14195 Berlin, Germany
The integrity of healthy mitochondria is supposed
to depend largely on proper mitochondrial protein biosynthesis.
Mitochondrial ribosomal proteins (MRPs) are directly involved in this
process. To identify mammalian mitochondrial ribosomal proteins and
their corresponding genes, we purified mature rat MRPs and determined 12 different N-terminal amino acid sequences. Using this peptide information, data banks were screened for corresponding DNA sequences to identify the genes or to establish consensus cDNAs and to
characterize the deduced MRP open reading frames. Eight different
groups of corresponding mammalian MRPs constituted from human, mouse,
and rat origin were identified. Five of them show significant sequence similarities to bacterial and/or yeast mitochondrial ribosomal proteins. However, MRPs are much less conserved in respect to the amino
acid sequence among species than cytoplasmic ribosomal proteins of
eukaryotes and bacteria.
Intact mitochondrial protein biosynthesis has been shown to be
indispensable for the maintenance of mitochondrial DNA in yeast (1).
Nearly all of the mitochondrial ribosomal proteins
(MRPs)1 investigated so far
are essential for proper mt protein synthesis (2). Knock-out mutants of
yeast MRP genes lose their respiratory capacity and change to
Determinaton of Rat N-terminal MRP Peptide
Sequences--
Preparation of mitochondrial ribosomes from rat liver
was accomplished according to Ref. 3. Proteins were extracted with acetic acid and lyophilized (22), and were separated by two-dimensional PAGE as described (23). Proteins from the second dimension gels were
transferred to polyvinylidene difluoride membranes by Western blotting
(24). N-terminal sequencing of blotted proteins was done as described
(24).
Computing--
Sequence searches were performed with the BCM
Search Launcher program2
(25). First, the rat MRP N-terminal peptide sequences were compared
with EST sequences with the "general protein sequence/pattern searches" and the "TBLASTN/dbest query versus
6-frame translation of dbest with Entrez & SRS links
(NCBI/BCM)" subprogram. Nucleic acid sequences obtained were selected
for overlapping and extension, and a consensus sequence was established
using the "pileup" program (26). The putative ORF (open reading
frame) was localized using the "translate" program (26). The
resulting peptide sequence was aligned to the rat N-terminal peptide
sequence by the "bestfit" program (26). In the case of ORFs open at
their 5' and/or 3' end, the first and the last 50 nucleic acids,
respectively, of the established consensus sequence were subjected to a
second search using the "nucleic acid sequence search" (25). The
search was performed with the "BLASTN/dbest with
Repeat Masker and Entrez & SRS links (NCBI/UW/BCM)"
subprogram. Newly identified nucleic acid sequences were aligned with
the consensus sequence. The extended sequence was translated for
detection of a complete ORF. Complete ORFs were analyzed using the
"peptidesort," "peptidestructure," and "map" programs (26).
Comparison of similar proteins from different species was performed
using the "pileup" and "bestfit" programs (26).
Deduced protein sequences were compared with sequences from the
SWISSPROT data base using the "wordsearch" program (26). Further,
the sequences were analyzed by comparison to any possible ORF deducible
from the genomic sequence of Saccharomyces cerevisiae (25).
The ORFs were also compared with known yeast MRP sequences as listed in
Ref. 2.
N-terminal Sequencing of Purified Rat MPRs and Computational
Analysis--
Several rat MRPs were purified from mt r-protein
mixtures. The proteins were identified according to the two-dimensional
map of rat MRPs (27), and their isoelectric points were determined (Table I). The proteins were blotted from
two-dimensional gels onto polyvinylidene difluoride membranes and
N-terminally sequenced. Twelve different N-terminal sequences were
obtained (Table I). The sequences of MRP-L22rat and
MRP-L24rat are almost identical, and thus they are expected
to be two differently modified forms of the same protein, separated by
the two-dimensional PAGE technique applied. The N-terminal sequences
obtained were compared with EST data base sequences. For eight
proteins, groups of corresponding EST sequences from human, mouse, and
rat were identified. Consensus cDNA sequences were established by
multiple sequence comparisons, and the ORFs deduced were characterized
and compared with each other, and to the determined N-terminal
sequences of the mature rat MRP (Fig. 1).
N-terminal extensions of the deduced ORFs compared with the mature rat
N-terminal peptide sequences were postulated to be putative signal
sequences for mt protein import. Putative signal peptides were further
analyzed for general properties of mt import sequences according to
Refs. 28 and 29.
MRP-L8--
In the primary search using the obtained
MRP-L8rat peptide sequence as screening probe, 12 and 5 "primary hits" of mouse and human ESTs were found, respectively.
The consensus cDNA sequence of MRP-L8mouse was
assembled by multiple searches from many different EST sequences (Table
II). A complete ORF of 261 amino acid
residues was identified. Positions 28-46 of this ORF correspond to the MRP-L8rat N-terminal peptide (Table I, Fig. 1a,
present report). A cleavable mitochondrial import signal peptide (MISP)
of 27 amino acid residues is postulated for MRP-L8mouse.
Correspondingly, a cDNA for MRP-L8human was assembled
encoding an ORF of 261 amino acid residues (Table II, Fig.
1a, present report). The deduced MRP-L8mouse and
MRP-L8human show a high degree of sequence identity to each
other over their entire length. An MISP of 28 amino acid residues is
postulated in comparison to the rat N-terminal L8 peptide and in
accordance with (28, 29). Both the mouse and the human putative MISPs
are highly hydrophobic; they contain only positively charged and no
negatively charged amino acid residues, and very few hydroxylated amino
acid residues. The arginine (R) residues in position
Significant sequence similarities of the mammalian L8 MRPs were
detected by computer search with the yeast MRP YmL11 and bacterial r-proteins of the L10 family (Fig. 1a, Table
III). However, the percentages of
sequence similarities and sequence identities are low (Table III) and
might be below the threshold. The family of the bacterial L10
r-proteins itself is very heterogeneous, and a comparison of the citrus
greening disease-associated bacterial L10 with the E. coli
L10 r-protein shows only 45% similarity and 32.4% identity over a
stretch of 153 amino acid residues, respectively. These values are very
low, but nonetheless the membership of similar proteins from different
bacterial species to the L10 r-protein family is supported,
e.g. by the similar location of the corresponding genes
within the same operons, respectively. However, the similarities of the
mammalian MRP-L8s to the L10 r-proteins class are the only ones to be
picked up by the computer. Accordingly, we assign the mammalian MRP-L8
proteins as members of the L10 family of r-proteins.
MRP-L22/24--
Fourteen and 14 primary hits for
MRP-L22human and MRP-L22mouse were found,
respectively. Additionally, a single EST sequence of rat origin was
identified (Table II). The incomplete ORF deduced for
MRP-L22rat matches the N-terminal sequence of the mature
MRP-L22rat from positions 42 to 70 (Fig. 1b,
present report). An N-terminal MISP of 41 amino acids is postulated for
MRP-L22rat. For MRP-L22mouse, a complete
cDNA consensus sequence of 826 base pairs was determined (Table
II). An ORF of 201 amino acid residues was deduced from this cDNA,
for which the N-terminal 80 amino acid residues are almost identical to
the MRP-L22rat protein. Further, MRP-L22mouse shows approximately 80% identity to the deduced
MRP-L22human sequence over its entire length (Fig.
1b; see below). An MISP of 41 amino acid residues is
postulated for MRP-L22mouse. The ORF of
MRP-L22human was deduced from a consensus cDNA of 775 base pairs (Table II). MRP-L22human consists of 206 amino
acid residues, which are highly conserved as compared with the rat and
mouse MRP-L22 sequences (Fig. 1b, present report). By
comparison with the rat mature peptide sequence, an MISP of 46 amino
acid residues is postulated for MRP-L22human. According to
Ref. 29, the putative MISPs of rat, mouse, and human MRP-L22 belong to
the R-none class of MISPs. All three show the typical properties of
MISPs such as many hydrophobic, positively charged, and hydroxylated
amino acid residues.
In addition, genomic sequences were identified for
MRP-L22human in the data banks. The gene for
MRP-L22human was located on chromosome 22q11 cosmid clone
102 g9 (accession no. AC000068) from nucleotides 28098 to 31642 in
reverse complement orientation. The exon/intron structure of the
MRP-L22human gene, spanning approximately 4.5 kilobase
pairs, is presented in Fig. 2. The last
20 nucleotides of the consensus cDNA are mainly adenosines, which
do not fit to the corresponding positions of the genomic DNA. A perfect
polyadenylation signal AATAAA was identified at positions 736-741 of
the cDNA, and accordingly the deduced 3' end of the consensus
cDNA was assumed to represent the true 3' end of the
MRP-L22human mRNA.
MRP-L25--
Three, 14, and 17 primary hits are found in the EST
data bases for rat, human, and mouse MRP-L25, respectively. A truncated ORF of 91 amino acid residues was deduced for MRP-L25rat
from a single EST. Amino acids 34-59 of this ORF match the N-terminal peptide of MRP-L25rat obtained by amino acid sequencing
(Fig. 1c, present report). The consensus cDNA sequences
for MRP-L25mouse and MRP-L25human,
respectively, were assembled from many different EST sequences (Table
II). The identity of some nucleotides could only be clarified by
comparison of the deduced amino acid sequences, in order to avoid
frameshifts that otherwise would cause total disagreement between the
supposedly closely related mouse and human sequences. The incomplete
ORF of MRP-L25mouse spans 212 amino acid residues, which
are in very good agreement with the truncated MRP-L25rat
(Fig. 1c, present report). The N-terminal 180 amino residues
of MRP-L25mouse also correspond to the respective N-terminal amino acid residues of the human MRP-L25human
(Fig. 1c, present report). The consensus cDNA sequence
of 824 nucleotides for MRP-L25human was assembled from
three different ESTs (Table II). To avoid a frameshift as compared with
the deduced amino acid sequence of MRP-L25mouse, base pair
225 of EST H87659, which is an "n," was omitted. The complete ORF
deduced spans 223 amino acid residues. A polyadenylation signal AATAAA
is found 3' to the stop codon. MRP-L25human and
MRP-L25mouse are closely related proteins except for their
respective C termini (Fig. 1c). MISPs 33 amino acids in
length are postulated for all three rat, mouse, and human MRP-L22s. The
signal peptides belong to the R-none class according to Ref. 29.
Similarity of the human and mouse proteins to the E. coli
L22 r-protein has been postulated (EST accession no. AA101598). More
than 30 different L22 r-proteins were picked out from the data bases
using human or mouse MRP-L25 amino acid sequence as a screening probe.
When MRP-L25human is compared with EcoL22 at the amino acid
residue level, a sequence similarity of only 42%, and a sequence
identity of 31.8% was detected, covering a region of 84 amino acid
residues (Table III). However, when the complete MRP-L25human sequence was compared with different members
of the L22 protein family, several amino acids were identified that are identical among all of them (Fig. 1c). Thus, the affiliation
of the mammalian MRP-L25 proteins to the EcoL22 r-protein family is confirmed.
MRP-L27--
Four and 27 primary hits were found for human and
mouse MRP-L27, respectively. Complete ORFs of 136 and 134 amino acid
residues for MRP-L27human and MRP-L27mouse were
deduced from consensus cDNAs (Table II, Fig. 1d, present
report). Both deduced ORFs are quite similar to each other. The ORF of
MRP-L27mouse starts with an ATG, which is surrounded by the
appropriate nucleotides that are common for eukaryotic translational
start codons (data not shown). An in-frame stop codon precedes this
translational start, thus making the N terminus of the
MRP-L27mouse ORF highly probable. The start codon of
MRP-L27human lacks one nucleotide of the appropriate consensus sequence for eukaryotic start codons. No in-frame stop codon
is found 5' of the translational start, since the 5' end of the
assembled consensus cDNA does not extend far enough into 5'
direction. Nevertheless, we assume this to be the true start codon of
MRP-L27human due to the amino acid sequence similarity to
MRP-L27mouse downstream, and the complete disagreement of
the deduced amino acid sequences upstream of the start codon. Thus, in
comparison to the mature N terminus of rat MRP-L27 for
MRP-L27human and MRP-L27mouse, MISPs of 13 amino acid residues are postulated, respectively (see Fig.
1d). Both MISPs are highly hydrophobic with few (two in
human, one in mouse) positively charged and two hydroxylated (mouse)
amino acid residues. Both signal peptides belong to the R-none class of
signal peptides according to Ref. 29. The consensus cDNAs of human
and mouse show polyadenylation signals ATTAAA closely located
downstream of the respective stop codons. The nucleotide environments
of these signals are highly conserved between the
MRP-L27mouse and MRP-L27human consensus cDNAs.
Sequence comparison revealed weak but significant sequence similarities
of the mammalian MRP-L27 to the N-terminal portion of the yeast YmL27
MRP (Fig. 1d, Table III). Although the sequence similarities
are not high, they are comparable to the values obtained for other
mammalian MRPs similar to bacterial and yeast mitochondrial r-proteins
(Table III). The yeast YmL27 shows no sequence similarity to any known
r-protein (31), and thus the discovery of the mammalian counterparts of
YmL27 defines the first MRP family that is not similar to known
r-proteins from other sources.
MRP-L28--
One, 6, and 48 primary hits were found for rat,
human, and mouse MRP-L28, respectively. For MRP-L28rat, an
incomplete ORF was identified. The last 11 amino acid residues of the
ORF deduced are in complete agreement with the first 11 amino acid
residues of the mature MRP-L28rat protein, as determined by
amino acid sequencing (Table I, Fig. 1e, present report).
Furthermore, most of this ORF shows strong sequence similarities to the
ORFs deduced for MRP-L28human and MRP-L28mouse,
respectively (Fig. 1e). Interestingly, among the human
MRP-L28 ESTs, two different groups of consensus cDNA sequences were
identified and assembled. MRP-L28human1 (Table II, Fig.
1e) encodes an ORF of 162 amino acid residues. The consensus cDNA of MRP-L28human2, which is supported by four
independent ESTs, is not complete (Fig. 1e). Strikingly, it
contains a stop codon (*) followed by a frameshift (-) caused by a
missing nucleotide in the cDNA coding for the putative signal
peptide (see Fig. 1e). The stop codon as well as the
frameshift are found in all of the four detected ESTs. Further, the
four ESTs of MRP-L28human2 are all of fetal liver spleen
origin, whereas the ESTs identified for MRP-L28human1 are
products of mRNA isolated from different tissues such as retina,
melanocyte, fetal liver spleen and fetal heart, and parathyroid tumor.
Thus, irrespective of whether the MRP-L28human2 mRNA is
a product of a pseudogene not being translated in vivo, it
seems that for MRP-L28human at least two different genes do
exist that are transcribed in a tissue-specific manner.
The MRP-L28mouse consensus cDNA codes for an ORF, which
is quite similar in sequence to the MRP-L28rat,
MRP-L28human1, and MRP-L28human2, respectively
(Fig. 1e). However, within the 5' part of the consensus
cDNA (Table II), a single frameshift is found. This frameshift is
supported by six out of six ESTs identified for this region. The
frameshift causes an alternative N terminus of MRP-L28mouse
without a translational start codon, which is not similar to the
corresponding N termini of rat and human MRP-L28s (Fig. 1e).
If the frameshift is not taken into account, the N terminus of
MRP-L28mouse corresponds well to the respective rat and
human sequences (Fig. 1e). It might be speculated that the apparent frameshift is caused by sequencing errors, or that the MRP-L28mouse cDNA is the MRP-L28human2
"homologue" rather than the MRP-L28human1 homologue.
For all MRP-L28s (neglecting the frameshifts), MISPs of 34 amino acid
residues are postulated. For MRP-L28human1 and
MRP-L28human2, stop codons 5' to the translational start
support this assignment. In the case of rat and mouse MRP-L28s, there
are no stop codons preceding the starting methionine, due to the short
5' sequences of the respective consensus cDNAs (Table II). The 5'
encoded amino acid sequence is shown in the case of MRP-L28rat (Fig. 1e), but is very unlikely to be
a true part of the MISP. All four MISPs are characterized by a high
proportion of hydrophobic and positively charged amino acid residues.
With the exception of MRP-L28human2, they belong to the
R
The MRP-L28mouse and MRP-L28human1 sequences
showed a weak but significant sequence similarity to the yeast YmL33
MRP and the EcoL30 r-protein, respectively. The regions of sequence
similarity span the total length of the EcoL30 protein, and 49 amino
acid residues of the N terminus of YmL33, and the middle part of
MRP-L28mouse, respectively (Fig. 1e, Table III).
Interestingly, the overall size of the yeast MRP is considerably less
than that of the mammalian MRPs. The EcoL30 protein itself is only
two-third the size of the yeast YmL33. Thus, the putative "core" of
these different r-proteins is much smaller than the mitochondrial
representatives of unicellular and multicellular eukaryotes.
MRP-L31--
Three, 11, and 28 primary hits were found for rat,
human, and mouse MRP-L31, respectively. For MRP-L31rat, an
incomplete ORF of 127 amino acid residues was deduced from the cDNA
lacking some amino acid residues of the putative MISP (Fig.
1f, Table III). Amino acid residues 18-40 correspond to the
N-terminal amino acid sequence of the mature MRP-L31rat
obtained by amino acid sequencing. The MRP-L31rat sequence
is very similar to the deduced amino acid sequences of
MRP-L31mouse and MRP-L31human (Table II, Fig.
1f, present report). MISPs of 31 and 32 amino acid residues,
respectively, are postulated (Fig. 1f). The MISPs are highly
hydrophobic and positively charged, and contain hydroxylated but no
negatively charged amino acid residues. The (truncated)
MRP-L31rat MISP belongs to the R MRP-L32--
For the MRP-L32 proteins 2, 10, and 38, primary hits
were identified for rat, human, and mouse in the EST data bases,
respectively. An incomplete ORF of 101 amino acid residues was deduced
from a single rat EST sequence (Table III). From amino acid residues 2 to 59, this ORF corresponds to the amino acid sequence of the mature
MRP-L32rat determined by amino acid sequencing (Fig.
1g). The complete ORFs deduced for both the
MRP-L32mouse and MRP-L32human proteins are
quite similar except for their extreme N termini (Fig. 1g,
present report). Both proteins show a very good sequence correspondence
to the MRP-L32rat sequence (Fig. 1g). MISPs of 30 and 64 amino acid residues are postulated for
MRP-L32mouse and MRP-L32human, respectively.
However, the elongated form of MRP-L32human seems to be
unlikely, since the surrounding of the second ATG codon perfectly
matches the consensus sequence for the start of eukaryotic translation
(as the mouse ATG does), whereas the first ATG does not. The
MRP-L32mouse start codon is preceded by an in-frame stop
codon. Both the mouse and human N termini show general properties of
MISPs, such as a high content of hydrophobic, positively charged, and
hydroxylated amino acid residues, but no negatively charged residues.
The mouse MISP belongs to the R
The mammalian L32 MRPs show significant sequence similarities as
compared with E. coli r-protein L14 and the corresponding YmL38 MRP of yeast (Fig. 1g, Table III). The N termini of
the latter two correspond to the postulated mature N termini of the
mammalian L32 MRPs. Interestingly, the YmL38 lacks a cleavable MISP
(2). Thus, the N terminus of the mature YmL38 corresponds to the
N termini of the mature (i.e. after mt import and
processing) mammalian MRP-L32s (Fig. 1g).
MRP-S13--
Astonishingly, only three and one ESTs were found in
the primary search for rat and mouse MRP-S13, respectively. Human ESTs corresponding to this MRP were identified using the mouse cDNA sequences as "screening probes." For MRP-S13rat, an ORF
was deduced that lacks an N-terminal start codon and an N-terminal
extension as compared with the mouse and human MRP-S13s (Table II, Fig. 1h, present report). Although 50 different ESTs of rat
origin were identified by using the extreme 5' end of EST AI0455711r,c (see Table II) as screening probe, none of them extends more than 15 base pairs in the 5' direction. Because the MRP-S13rat ORF presented in Fig. 1h is preceded by three in-frame stop
codons, and because the deducible amino acid residues at the very N
terminus do not correspond to the respective MRP-S13mouse
amino acid sequence in the same position, we assume that the 5' end
nucleotide sequence of EST AI0455711r,c represents an intron that was
not reverse transcribed during EST creation and/or sequenced. The
mature MRP-S13rat as compared with the N-terminal sequence
obtained by direct amino acid sequencing is highly conserved, as
compared with the respective mouse and human MRP-S13 sequences (Fig.
1h). For MRP-S13mouse, an ORF of 200 amino acid
residues was deduced from a consensus cDNA of 977 nucleotides
(Table II). The pre-mRNA of MRP-S13mouse contains at
least three introns, two at positions 107/108 and 154/155, and a third
intron of 80 nucleotides at position 363/364 of the consensus cDNA.
This was deduced by comparison of EST sequences derived from
incompletely spliced mRNA molecules with the mature consensus
cDNA. At position 925-930, a polyadenylation signal AATAAA was
found, and 28 consecutive adenine residues mark the location of the
poly(A) tail from position 943 onward. The deduced ORF is highly
conserved as compared with the mature MRP-S13rat sequence
(Fig. 1h, present report). For MRP-S13human, a
consensus cDNA was assembled, and the corresponding genomic DNA was
localized 3' of the GnRH-II gene (Ref. 32; accession no. AF036329). The
EST consensus cDNA sequence corresponds to that of the genomic DNA
from nucleotide 3763 to nucleotide 4424 of the latter. Two short
introns are covered by this region (data not shown). However, the
genomic DNA sequence does not cover the entire MRP-S13human sequence. Accordingly, the C terminus was completed by adding EST
derived consensus cDNA sequences (Table II).
For both the MRP-S13mouse and the MRP-S13human,
an MISP of 27 amino acid residues is postulated, respectively. Although
not identical in sequence, both peptides are quite similar in specific properties such as a high content of hydrophobic amino acid residues and a net positive charge. The MRP-S13mouse MISP belongs to
the R
Altogether, sequences of 23 different mammalian MRPs have been
identified by comparison with the N-terminal peptide sequences of
purified rat MRPs determined by biochemical methods (Table I). The
significance of the deduced ORF sequences is influenced by the
inaccurate EST sequencing results. However, although all the ORF
sequences deduced need further confirmation by classical cDNA
isolation and sequencing, the deduced amino acid sequences are reliable
in terms of consensus cDNA assembling and sequence comparison with
similar proteins of the same r-protein family. Thus, eight classes of
mammalian MRPs were characterized in this work.
Our understanding of mt genetics linked to mutations in nuclear
genes suffers from a lack of knowledge of the influence of nuclear
encoded proteins on mt maintenance and function. This is also a crucial
point in the elucidation of molecular mechanisms for nuclear-inherited
mitochondrial diseases. Nuclear-encoded MRPs are good candidates for
proteins involved in mt genetics, as has been shown in yeast (1, 2). In
the present report, we describe the identification of eight groups of
mammalian MRPs from rat, mouse, and human.
It might seem surprising that the numbers of ESTs picked from the data
bases at least as primary hits vary so much among the different MRPs
investigated. One could assume that ESTs respective mRNAs of
different gene products, which are present in stoichiometric amounts
within the cell (the mt ribosome), would be represented in the data
bases in similar ratios. In yeast the expression of MRP genes is
differentially regulated at the mRNA, translational, and protein
stability levels. Thus, equal amounts of MRP mRNAs are not present
(for a review, see Ref. 2). Additionally, it should be noted that ESTs
derive from different healthy and tumor tissues. The degree of
respiration and the mitochondrial content of different tissues differs
remarkably, causing a different level of expression of nuclear genes
for mt proteins. The automatic processing of mRNAs yielding ESTs
may also influence the occurrence of individual ESTs by selective
preferences for the oligonucleotides and reverse transcriptases used.
Therefore, it seems rather unlikely that the relative amounts of MRP
ESTs detected would mirror any (assumed) molecular ratios within an
"ideal" cell.
By comparison of the deduced MRP ORFs with the rat N-terminal amino
acid sequences of the mature MRPs isolated, we postulated several
MISPs. These postulated peptides were further analyzed according to the
criteria of common features for such import sequences (28, 29).
However, no consensus sequence in the sense of a canonical amino acid
sequence has been found for MISPs, and it is unlikely that such a
consensus will emerge. Thus, in general, the mammalian MRP MISPs do
indeed fit the postulated properties such as a highly hydrophobic
character, a net positive charge, and hydroxylated amino acid residues
(28). The assignment of the mammalian MRP MISPs to various classes of
substrates for the mt processing proteases is common to MRPs. This has
been shown for more than 30 different yeast MRPs (2). Although the
MISPs presented in this study are on average 30-40 amino acid residues in size, shorter ones such as the MISPs of the mammalian MRP-L27s (13 amino acid residues) are not unusual. In yeast short MISPs (between 14 and 7 amino acid residues) have been identified for MRPs (2). The lack
of the mammalian MRP MISPs for hydroxylated amino acid residues may be
a specific property of this class of mammalian proteins imported into mitochondria.
Five of the different mammalian MRP classes show significant sequence
similarities to r-proteins from other mt and/or bacterial sources.
However, the calculated values for sequence similarities and identities
are low as compared with most of the homogeneous classes of r-proteins
from other sources (33). In this context it should be noted that a
comparison of yeast MRPs to r-proteins of defined classes reveals that
MRPs in general seem to be much more divergent (for a review, see Ref.
2). MRPs show N- and C-terminal and/or internal sequence elongations as
compared with eubacterial/chloroplast/eukaryotic cytoplasmic
r-proteins. MRPs are less similar to other members of the same
r-protein family and among species (2). Mammalian and yeast MRPs are as
much divergent as mammalian MRPs and E. coli r-proteins.
This is in sharp contrast to rat and yeast cytoplasmic r-proteins,
which on average share 60% identical amino acid residues (34). On the
other hand, the specific large subunit r-proteins picked out from the
data bases by comparison to mammalian large subunit MRPs point to a
reduced but still reliable sequence conservation of mammalian MRPs and
bacterial r-proteins. It should be mentioned that, in certain families
of bacterial r-proteins, some of the members are so divergent that they
are not assigned as similar by a direct sequence comparison, but only
by "intermediates" from other sources (see "MRP-L8" under
"Results"). Thus, mammalian MRPs also fit to these divergent
classes of r-proteins.
Since mt ribosomes contain many more proteins than cytoplasmic
ribosomes, it is not surprising that several MRPs were identified for
which no similar proteins, e.g. in E. coli
ribosomes, exist. These "new" MRPs define new classes of
r-proteins. They may represent the "molecular excess" of proteins
as compared with cytoplasmic ribosomes. In yeast the majority of MRPs
are not similar to any other r-protein (2). However, the fact that only
one of the mammalian MRPs which are not similar to E. coli r-proteins shares sequence similarity with yeast MRPs
(YmL27/MRP-L27mammalian) (i) may result from the still
incomplete characterization of all yeast MRPs, and/or (ii) may be the
consequence of further reaching divergences of yeast and mammalian mt
ribosomes in their respective protein compositions. It should be noted
at this point that yeast and mammalian mt ribosomes differ strongly in
their protein/RNA ratio, although they seem to possess the same total
molecular mass (35). The mammalian MRP-L32s, MRP-L31s, and MRP-S13s
seem to represent such additional proteins, as compared with E. coli and yeast mt r-proteins. Due to the stringent washing and
purification methods applied, it can be excluded that these additional
proteins represent mt translational factors loosely attached to the mt
ribosome rather than true MRPs. The purification methods for yeast and
rat MRPs are comparable, and no mt translational factor has so far been found as a contaminant of mt ribosome preparations (36). Only two-dimensional PAGE spots, which appear reproducibly in stoichiometric amounts in repetitive experiments, are counted as true MRPs (27).
In general, MRPs are much less conserved from one species to another
than cytoplasmic r-proteins. This finding raises questions concerning
the molecular mechanisms that have allowed MRPs such a divergent
evolution while keeping the mitochondrial protein biosynthesis
machinery intact. Furthermore, is it possible to generalize results
about MRPs to the same extent as has been done for bacterial and
eukaryotic cytoplasmic r-proteins? Obviously, the use of yeast MRPs as
a model system for mammalian MRPs suffers from the low conservation of
sequences and proposed functions. A practical consequence is that it
will not be possible to identify most of the mammalian MRP genes simply
by comparison of yeast MRP sequences with the increasing number of
unknown mammalian EST and genomic DNA sequences. Peptide sequences
obtained from mammalian MRPs are much more helpful for this purpose.
Short peptide sequence data correspond to DNA sequence data from the
same species or close relatives strongly enough to identify the
corresponding genes unambiguously. This method has been successfully
applied to yeast MRPs (24, 36). Further, the correspondence of human cytoplasmic r-proteins separated by two-dimensional PAGE to sequenced r-protein genes has been proven (30). In this work, we have applied
this approach for the first time to mammalian MRPs and their
corresponding genes.
We thank R. Brimacombe for carefully reading
the manuscript.
*
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 N-terminal peptide sequences reported in this paper have been
deposited in the MIPS Data Base with accession nos. S78411 (MRP-L8rat), S78412 (MRP-L22rat), S78413
(MRP-L24rat), S78414 (MRP-L25rat), S78415
(MRP-L27rat), S78416 (MRP-L28rat), S78417
(MRP-L31rat), S78418 (MRP-L32rat), S78419
(MRP-L33rat), S78420 (MRP-L41rat), S78421
(MRP-S13rat), and S78422 (MRP-S20rat),
respectively.
§
Present address: Faculty of Science, Dept. of Biology, University
of Kobe, Nada-Ku, 657 Kobe, Japan.
¶
Present address: Max-Delbrück-Center for Molecular
Medicine, Robert-Rössle-Strasse 10, D-13125 Berlin-Buch, Germany.
The abbreviations used are:
MRP, mitochondrial
ribosomal protein; EST, expressed sequence tag; MISP, mitochondrial
import signal peptide; mt, mitochondrial; ORF, open reading frame; PAGE, polyacrylamide gel electrophoresis; r-protein, ribosomal protein.
2
This program is available via the World Wide Web
(http://kiwi.imgen.bcm.tmc.edu:8088/search-launcher/launcher.html).
Mammalian Mitochondrial Ribosomal Proteins
N-TERMINAL AMINO ACID SEQUENCING, CHARACTERIZATION, AND
IDENTIFICATION OF CORRESPONDING GENE SEQUENCES*
,
, and
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
or o mt genetic status by successive
losses of mt DNA (1). In higher eukaryotes, the knowledge about
comparable functions of MRPs is only rudimentary, since only a few MRPs
have been characterized on the molecular level. The protein composition
of mammalian mt ribosomes has been studied extensively (3-5). Some
properties of mt ribosomes such as structure (6), binding of
nucleotides and RNA (7-11), and interaction with different factors
have been studied (12-14). However, only 3 of the approximately
80-100 different human MRPs have been described at the molecular level
so far. MRL3, which is the EcoL3 counterpart in human mt ribosomes, was identified as an overexpressed r-protein in Mahlavu hepatomic cells
(15). Later, it was postulated to be a true MRP by virtue of its
sequence similarity to the corresponding yeast MRP YmL9 (16). MRPL12
was identified as a delayed-early response gene similar in sequence to
the Escherichia coli L7/L12 r-protein (17). The metazoan
mitochondrial counterpart of EcoS12 has been characterized in
Drosophila, human, and mouse (18, 19). In
Drosophila a mutation of mt S12 causes abnormal behavior.
This is the first case reported so far of affection of the status of an
animal by an MRP mutation (18). Diseases affecting mitochondria are
known in humans, and are caused by nuclear mutations responsible for the loss of mt DNA as a secondary effect by a so far unknown mechanism (20, 21). Mutations of MRPs are good candidates affecting mt genetic
and/or physiological status. To characterize mammalian MRPs and to
compare their biochemical properties with that of their (essential)
counterparts, e.g. of yeast, we identified several mammalian
MRPs and their corresponding gene sequences. We used N-terminal
sequence information obtained from purified rat MRPs to screen DNA data
banks and to characterize identified MRPs of rat, human, and mouse.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
N-terminal amino acid sequences of mature MRPs of R. norvegicus
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Fig. 1.
Alignment of deduced mammalian MRP amino acid
sequences with rat MRP N-terminal peptide sequences.
Numbers give the respective amino acid position.
Lines (|) mark identical amino acid positions,
colons (:) mark strongly conserved amino acid residues, and
points (·) mark weakly conserved amino acid residues.
Dashes (-) show N- or C-terminal ends of incomplete amino
acid sequences, and asterisks (*) mark stop codons. Amino
acid residues written in lowercase letters are
uncertain in their determination by amino acid sequencing (see Table
I). x marks unidentified amino acids although the positional
number is valid. a, alignment of mammalian MRP-L8 protein
sequences with the citrus greening disease-associated bacterial L10
(accession no. M94319, rpIJ gene). b, alignment
of mammalian MRP-L22 protein sequences. c, alignment of
MRP-L25 protein sequences with the E. coli EcoL22 sequence
(accession no. sw:rl22_ecoli). Section marks (§)
in the alignment of MRP-L25human and EcoL22 mark amino acid
residues that are absolutely conserved among nearly all members of the
EcoL22 r-protein family. d, alignment of mammalian
MRP-L27 MRP sequences with the yeast YmL27 sequence (accession no.
S77888). e, alignment of the mammalian MRP-L28 sequences
with the yeast YmL33 sequence (accession no. D90217), and the E. coli EcoL30 sequence (accession no. sw:rl30_ecoli). f,
alignment of MRP-L31 sequences. g, alignment of the
mammalian MRP-L32 sequences with the E. coli EcoL14 sequence
(accession no. sw:rl14_ecoli), and the yeast YmL38 sequence (accession
no. S38000). h, alignment of the mammalian MRP-S13
sequences.
2
(MRP-L8mouse) and 10 and 2 (MRP-L8human) classify the peptides as R2 (MRP-L8mouse) and R10/R2
(MRP-L8human), respectively, according to Ref. 29.
Assembly of identified EST sequences: consensus cDNA in 5' to 3'
direction
Sequence comparison of similar ribosomal proteins from mitochondria,
and bacteria
View larger version (8K):
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Fig. 2.
Genomic map of the exon/intron structure of
the MRP-L22human gene. The genomic DNA is shown as a
horizontal line, and restriction sites are
represented by vertical lines. Boxes
mark location of cDNA sequences; black boxes
represent translated cDNA sequence, and open
boxes show untranslated cDNA sequences.
2 class of cleavable signal sequences according to Ref. 29. In
MRP-L28human2, the arginine in position 2 is replaced by
a histidine (Fig. 1e).
2 class, whereas the
mouse and human MRP-L31 MISPs belong to the R-none class of mt import
peptides according to Ref. 29. No significant sequence similarity to
any known protein was found. Thus, the mammalian L31 MRPs define a new
class of MRPs.
10 class according to Ref. 29. In the
human sequence, the arginine (R) at position 10 is replaced by a
histidine (H). However, it has not been shown so far that histidine can
functionally replace arginine in MISP processing.
2 class, and the MRP-S13human MISP belongs to the
R3 class of signal peptides according to Ref. 29. In general, the
MISPs of the mammalian MRPs presented in this study are quite similar to each other. This conclusion is not based on the primary amino acid
sequences, which are heterogeneous; instead, an analysis of the
properties shows common features. The MISPs of the MRPs are between 27 and 46 amino acids in length. The only exceptions are the MRP-L27 MISPs
of 13 amino acid residues. Nearly all of them show a structure
prediction of an N-terminal 
-helix joined to a C-terminal 
-sheet.
The putative MISP of MRP-L32human of 64 amino acid residues
shows the -helix--sheet motif twice in a row. All MRP MISPs are
highly hydrophobic, they have a net positive charge, and they contain
less hydroxylated amino acid residues than is common for other MISPs
(28).
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
ACKNOWLEDGEMENT
FOOTNOTES
Present address: Dept. of Biochemistry, Tel Aviv University, 69978 Ramat-Aviv, Tel Aviv, Israel.
Present address: BioInside GmbH, Warthestrasse 21, D-14513
Teltow, Germany.
To whom correspondence should be addressed. Tel.:
49-30-838-2629; Fax: 49-30-838-3649; E-mail:
graack{at}zedat.fu-berlin.de.
REFERENCES
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Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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