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Mol Cell Biol, April 1998, p. 1826-1834, Vol. 18, No. 4
0270-7306/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Functional Interactions between Yeast Mitochondrial
Ribosomes and mRNA 5' Untranslated Leaders
Noelle S.
Green-Willms,
Thomas D.
Fox, and
Maria C.
Costanzo*
Section of Genetics and Development, Cornell
University, Ithaca, New York 14853-2703
Received 27 May 1997/Returned for modification 21 July
1997/Accepted 22 December 1997
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ABSTRACT |
Translation of mitochondrial mRNAs in Saccharomyces
cerevisiae depends on mRNA-specific translational activators that
recognize the 5' untranslated leaders (5'-UTLs) of their target mRNAs.
We have identified mutations in two new nuclear genes that suppress translation defects due to certain alterations in the 5'-UTLs of both
the COX2 and COX3 mRNAs, indicating a general
function in translational activation. One gene, MRP21,
encodes a protein with a domain related to the bacterial ribosomal
protein S21 and to unidentified proteins of several animals. The other
gene, MRP51, encodes a novel protein whose only known
homolog is encoded by an unidentified gene in S. kluyveri.
Deletion of either MRP21 or MRP51 completely
blocked mitochondrial gene expression. Submitochondrial fractionation
showed that both Mrp21p and Mrp51p cosediment with the mitochondrial
ribosomal small subunit. The suppressor mutations are missense
substitutions, and those affecting Mrp21p alter the region homologous
to E. coli S21, which is known to interact with mRNAs.
Interactions of the suppressor mutations with leaky mitochondrial initiation codon mutations strongly suggest that the suppressors do not
generally increase translational efficiency, since some alleles that
strongly suppress 5'-UTL mutations fail to suppress initiation codon
mutations. We propose that mitochondrial ribosomes themselves recognize
a common feature of mRNA 5'-UTLs which, in conjunction with
mRNA-specific translational activation, is required for organellar
translation initiation.
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INTRODUCTION |
While mitochondrial ribosomes
exhibit distinct similarities to bacterial (eubacterial) ribosomes
(30, 69), the yeast mitochondrial translation system has
many intriguing differences from bacterial systems. Mitochondrial
ribosomes have more proteins than do bacterial ribosomes (23,
43). Of the mitochondrial ribosomal proteins whose sequences are
known, some are simple homologs of their bacterial counterparts, others
have domains homologous to bacterial ribosomal proteins attached to
domains with no recognizable homology to any known proteins, and still
others are completely unrelated to bacterial ribosomal proteins
(reviewed in reference 32). Saccharomyces
cerevisiae mitochondrial mRNAs generally have long, A+U-rich 5'
untranslated leaders (5'-UTLs) lacking a typical Shine-Dalgarno sequence (11, 27, 31). While the mechanism of start site selection remains obscure in this system, translation initiation on
most or all yeast mitochondrial mRNAs requires membrane-bound mRNA-specific activator proteins whose targets lie in the 5'-UTLs (reviewed in reference 27). These mRNA-specific
activators appear to play a dual role in mitochondrial gene expression:
tethering the synthesis of the very hydrophobic mitochondrial gene
products to the inner membrane (27) and modulating the
translation levels of individual mRNAs (63).
We have focused on translation of the COX2 and
COX3 mRNAs, which encode subunits II and III of cytochrome
c oxidase, respectively. Previous studies have identified
their mRNA-specific translational activators and established functional
interactions among activator proteins, their mRNA targets, and other
components of the mitochondrial translation system. The
COX2-specific translational activator protein is encoded by
the nuclear gene PET111 (46, 54), while the
COX3-specific activator is a complex containing three
proteins encoded by the nuclear genes PET54,
PET122, and PET494 (6, 9, 14, 38).
Using suppressor analysis, we have shown that one subunit of the
COX3-specific activator, Pet122p, interacts functionally
with the small subunit of mitochondrial ribosomes (33, 35,
42) and that each translational activator interacts functionally
with the 5'-UTL of its target mRNA (12, 45, 67). These
findings suggested that yeast mitochondrial ribosomes were unable to
recognize mRNAs unless the ribosome-mRNA interaction was mediated by
translational activators that recognized sites unique to each mRNA.
Here we report that certain mutations in the COX2 and
COX3 mRNA 5'-UTLs that are suppressible by alterations of
mRNA-specific activators can also be suppressed by mutations in nuclear
genes encoding two mitochondrial ribosomal small-subunit proteins. The suppression is allele specific, indicating that the ribosomes play an
active role in the recognition of translation start signals. Surprisingly, however, suppression is not gene specific, indicating that the ribosomes are recognizing features of the 5'-UTLs that are
common to at least several mRNAs. One of these yeast mitochondrial ribosomal proteins is homologous to bacterial S21 and to the products of unidentified genes from several animals.
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MATERIALS AND METHODS |
Yeast strains, media, and genetic methods.
The S. cerevisiae strains used in this study are listed in Table
1. All the strains were isogenic or
congenic to the wild-type strain D273-10B (ATCC 25657). The media and
genetic methods used were as described previously (60).
Respiratory growth was assessed on YPEG medium (3% ethanol, 3%
glycerol, 1% yeast extract, 2% Bacto Peptone, 2% agar). Second-site
suppressors of 5'-UTL mutations were selected in the strains JJM120,
JJM156, MCC199, and MCC200 (Table 1).
Plasmid manipulations, nucleotide sequencing, and computer
analysis.
Plasmids were constructed and transformed into
Escherichia coli DH5
F'IQ by standard techniques
(58). Nucleotide sequencing was performed either with the
Sequenase version 2.0 DNA-sequencing kit (U.S. Biochemicals) or by DNA
Services, Cornell University, with an ABI 371 DNA sequencer. Nucleotide
sequence data were analyzed with Lasergene biocomputing software
(DNAStar, Inc.). The Basic Local Alignment Search Tool (BLAST)
(2) program was accessed through the National Center for
Biotechnology Information or the Saccharomyces Genome
Database to search for nucleotide and protein sequence similarities.
Cloning of the MRP21 and MRP51
genes.
Nuclear DNA from a strain carrying both the
MRP21-1 and MRP51-3 suppressor genes (MCC267
[Table 1]) was prepared as described previously (28) and
partially digested with Sau3AI; the partial digestion
products were separated by size in a 10 to 40% sucrose gradient as
described by Rose and Broach (56), and 6- to 10-kb fragments
were pooled. The genomic DNA fragments were ligated to
BamHI-cleaved YEp24 (5) to create a library of
approximately 20,000 independent E. coli transformants,
which was amplified by standard methods (56).
To clone
MRP21-1, the
cox3-15 mutant strain TF210
(Table
1) was transformed with the MCC267 library. Transformants were
selected
on minimal medium and then printed to YPEG medium and
incubated
at 13.5°C. The transformants that grew on YPEG medium at
13.5°C
were analyzed to verify that cold resistance segregated with
the
plasmid. Six plasmids carrying a region of the genome near the
ROX3 gene and 13 plasmids carrying
PET494 were
isolated (see Results).
To determine whether
MRP21 was on
the plasmids carrying the
ROX3 region, a 4.4-kb
BamHI-
EcoRI fragment from this region was
subcloned
from plasmid pBSROX3BR (
57) into the integrating
vector YIp5
(
64), which carries the
URA3 gene,
creating the plasmid pMC327.
pMC327 was cut at a single
XbaI
site in the insert and integrated
into the genome of strain TF210 by
transformation and homologous
recombination; the integrant strain was
crossed to the
MRP21-1 cox3-15 strain MCC211, and
respiratory growth of the meiotic progeny
was analyzed.
To determine the nucleotide sequences of the
MRP21
suppressor alleles, the
MRP21 coding sequence was PCR
amplified from genomic
DNA of strains carrying each of the suppressor
alleles. The nucleotide
sequence of the entire PCR product from each
strain was determined,
and in each case a single nucleotide difference
from the wild-type
sequence (GenBank accession no.
Z35851) was
observed.
MRP21-1 and
MRP21-2 alleles had the
same mutation, a G-to-A change at
nucleotide 343 of the
MRP21 coding region. In the
MRP21-3 allele
position 363 of the
MRP21 coding sequence was changed from C
to
G.
To clone
MRP51-3, the
cox2-12 mutant strain
JJM158 was transformed with the MCC267 library. The transformants were
selected
on minimal medium and then printed to YPEG medium. Plasmids
were
isolated from respiratory-competent transformants and transformed
back into
cox2-12 and
cox2-11 strains to verify
that suppression
was plasmid linked. Fourteen overlapping plasmids were
isolated.
To determine whether
MRP51 was on the suppressing
plasmids, a
1,975-bp
SalI fragment, including 276 bp of the
vector, YEp24,
was subcloned from the suppressing library plasmid pB-14
into
the
BamHI site of the integrating vector pRS306
(
62), creating
plasmid pNSG17. pNSG17 was cut at either a
unique
AflII site or
a unique
MunI-
MfeI site in the insert and used for
integrative
transformation of an
MRP51-5/MRP51 cox2-11
diploid strain (JJM173
× PTY22rho0). The integrant strain was
sporulated, and the meiotic
progeny were analyzed.
The sequence of
MRP51-3 was determined by direct sequencing
of the suppressing library plasmid. It corresponded to the wild-type
sequence (YPL118W; coordinates 16771 to 17805 of GenBank no.
U43503)
except for the single nucleotide change from C to G at position
782 of
the coding sequence. The other
MRP51 suppressor alleles
(except
MRP51-8) were cloned by gap repair (
52),
and the ability
of gap-repaired plasmids to suppress
cox2-11
was confirmed. For
MRP51-2 and
MRP51-4, the
sequence of the entire open reading frame
was determined, partly from
the gap-repaired plasmids and partly
from PCR products amplified from
the genomic DNAs of the suppressor
strains; for
MRP51-1 and
MRP51-5, the sequence of the entire gene
except the 3' 114 bp was determined in the same manner. For
MRP51-8,
the
sequence was determined from PCR products amplified from genomic
DNA.
In
MRP51-1, position 704 was changed from T to C; in
MRP51-2,
position 721 was changed from A to C;
MRP51-4 had the same change
as the independently isolated
MRP51-3 allele (see above); in
MRP51-5,
position
779 was changed from C to T; and in
MRP51-8, positions
835 and 836 were changed from GA to AG.
In vivo labeling of mitochondrial translation products.
In
vivo labeling was performed as described previously (28).
Cells were grown in galactose-containing minimal medium containing the
35S-labeled E. coli hydrolysate labeling reagent
Tran 35S-label (ICN Radiochemicals) in the presence of
cycloheximide. Crude mitochondria were subjected to electrophoresis on
16% polyacrylamide gels (prepared from a stock solution containing
29.2% acrylamide and 0.8% bisacrylamide) containing 10% glycerol in
the presence of 0.1% sodium dodecyl sulfate. The gels were dried and
autoradiographed.
Generation of null alleles, and epitope tagging of Mrp21p.
An mrp21 null allele was generated by removing a 459-bp
ClaI-BglII fragment internal to the structural
gene and inserting a
hisG::URA3::hisG
cassette (1). An mrp51 null allele was generated by removing an internal 550-bp MunI-BglII
fragment and inserting the same cassette. To tag the MRP21
gene with three copies of the sequence encoding the influenza virus
hemagglutinin (HA) epitope (25, 65) at its 3' end, we used
the plasmid pCS124 (59), an integrative plasmid carrying
three copies of the HA sequence and the TRP1 gene. The 3'
294 bp of MRP21 was amplified by PCR and inserted into
pCS124 in frame with the HA sequence. The resulting plasmid, pMC343,
was cut at a unique EcoRI site within the MRP21 coding sequence, and the linearized DNA was used to transform strain
PTY11 to Trp+. In the resulting integrative transformant,
MCC291, the only complete copy of MRP21 was the tagged
allele, MRP21-HA.
Mitochondrial isolation and fractionation.
Mitochondria were
prepared from cells grown to late exponential phase in complete medium
(yeast-peptone [YP] medium) containing 2% galactose as described
previously (29), except that spheroplasts were disrupted
with a Parr-Bomb (Parr Instrument Co., Moline, Ill.) as described
previously (17). Mitochondria were purified by equilibrium
density gradient centrifugation in Nycodenz
[5-(N-2,3-dihydroxypropylacetamido)-2,4,6-triiodo-N,N'-bis(2,3-dihydroxypropyl)-isophthalimide; Sigma, St. Louis, Mo.] step gradients as described previously (29). Mitochondrial ribosomes were analyzed by sucrose
gradient centrifugation (15 to 30% sucrose in 0.5 M NH4Cl,
10 mM Tris [pH 7.4], 10 mM magnesium acetate, 7 mM
-mercaptoethanol, 0.5 mM phenylmethylsulfonyl fluoride, and 2 µg
each of the protease inhibitors antipain, aprotinin, bestatin,
chymostatin, E-64, leupeptin, pepstatin A, and phosphorhamidon per ml)
directly from disrupted mitochondria as described previously
(53). The clarified lysate obtained from 2 mg of whole
mitochondria was applied to a 36-ml gradient; 1-ml fractions were
collected, and 0.2 ml of each was precipitated with trichloroacetic
acid and subjected to sodium dodecyl sulfate-polyacrylamide gel
electrophoresis on 12% polyacrylamide gels.
Antisera and immunological methods.
Mouse monoclonal
antibodies against Mrp7p (24) and Mrp13p (53)
were obtained from T. L. Mason. Mouse monoclonal antibody 12CA5
against the HA epitope was purchased from BAbCo (Berkeley, Calif.).
Anti-Mrp51p polyclonal rabbit antiserum was prepared as described
previously (
36) with histidine-tagged Mrp51p as an antigen.
An
MRP51 gene with six His codons at the 3' end of the
coding
sequence was generated by PCR and inserted into pQE-30 (Qiagen),
with an additional six His codons added to the 5' end of the coding
sequence. The resulting plasmid, pNSG29, was transformed into
E. coli and induced with 2 mM
isopropyl-
-
D-thiogalactopyranoside
(IPTG), and the
fusion protein was affinity purified with Ni-nitrilotriacetic
acid
resin as directed by the manufacturer (Qiagen) (
37).
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western
blotting were performed by standard techniques (
36).
Antigen-antibody complexes were visualized by using horseradish
peroxidase-conjugated goat anti-mouse immunoglobulin G or goat
anti-rabbit immunoglobulin G (Gibco BRL, Bethesda, Md.) secondary
antibody and the enhanced chemiluminescence system (Amersham Life
Science Inc., Arlington Heights, Ill.).
| |
RESULTS |
Selection of second-site suppressors of cox2 and
cox3 5'-UTL mutations.
The cox2-11 mutation
is a single-base deletion (deletion of the G residue at 
24) in the
COX2 5'-UTL that does not affect the stability of the
COX2 mRNA but greatly decreases its translation, causing
weak respiratory growth (45). In a previous study, six independent dominant nuclear suppressors of the cox2-11
mutation were isolated (45). One of these affected
PET111, the known specific translational activator for the
COX2 mRNA, while the other five did not (45). To
characterize further the unknown mutations, a strain carrying one of
the suppressors was crossed to each of the other four suppressor
strains. Analysis of the respiratory growth of the meiotic progeny
showed that the first suppressor was tightly linked to each of the
other four. Thus, all five cox2-11 suppressors were in a
single gene, now called MRP51 (MRP51-1,
MRP51-2, MRP51-3, MRP51-4, and
MRP51-5). Respiratory growth of the cox2-11
strains containing these suppressors remained dependent upon the
function of the PET111 gene.
The
cox3-15 mutation, which consists of two deletions in the
COX3 5'-UTL, causes cold-sensitive respiratory growth
(
12).
The respiratory defect is due to cold-sensitive
translation, since
the
cox3-15 mRNA is present at wild-type
levels in cells grown
in the cold but Cox3p is not synthesized
(
12). Translation of
the
cox3-15 mRNA at the
permissive temperature is still dependent
on the
COX3-specific translational activator complex
(
12).
Six spontaneous cold-resistant revertants of a
cox3-15
mutant strain were isolated previously (
12), and for this
study an
additional 20 revertants were isolated. The revertant strains
were characterized genetically as previously described (
12)
to determine whether the dominant suppressor mutations were nuclear
or
mitochondrial and whether they were linked to any known genes
whose
products are involved in translational activation. Of the
26 revertants
analyzed, 17 had nuclear suppressor mutations. Nine
of these
suppressors mapped to the
PET122 gene, which encodes
a
subunit of the
COX3-specific translational activator
(
10,
12,
38); three mapped to a new gene we have called
MRP21 (
MRP21-1,
MRP21-2, and
MRP21-3); and one mapped to the
MRP51 gene
(
MRP51-8),
also identified above as a suppressor of the
cox2-11 mutation
(the four remaining nuclear suppressors
were relatively weak and
were not studied further). The wild-type
function of the
COX3-specific
translational activators
PET54,
PET122, and
PET494 was required
for respiratory growth of the
cox3-15 strains carrying
MRP21 or
MRP51 suppressor alleles. Thus, the
selection of suppressors of
5'-UTL mutations that specifically blocked
the translation of
particular mitochondrial mRNAs yielded not only
mutations in the
corresponding specific translational activators
(
12,
45) but
also mutations in two previously unidentified
genes whose products
interact functionally with the same regions of
these 5'-UTLs.
Specificity of the MRP21 and MRP51
suppressors.
To characterize the specificity of the two novel
suppressors, they were combined with a variety of nuclear and
mitochondrial mutations affecting translation and the double-mutant
phenotypes were analyzed. Respiratory growth, a phenotype we have
repeatedly found to be a sensitive indicator of Cox2p and Cox3p
synthesis in mutant strains (6, 12, 13, 22, 26, 45), was
assessed for all combinations of alleles (Tables
2 and 3;
Fig. 1). To confirm that the observed
respiratory growth phenotypes reflected the synthesis rates of
mitochondrial proteins, in vivo labeling of mitochondrial translation
products in the presence of cycloheximide was performed for selected
strains (Fig. 2).
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FIG. 1.
Suppression of selected cox2 and
cox3 5'-UTL mutations by MRP21 and
MRP51 mutations. Cells were grown on glucose-containing
medium (YPD), printed to nonfermentable medium (YPEG medium
supplemented with 0.02 mg of adenine per ml), and incubated at 30°C
for 2 days (A) or at 13.5°C for 12 days (B). Relevant genotypes are
shown. Where it is not indicated otherwise, MRP21 and
MRP51 are wild type.
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FIG. 2.
Effects of the MRP51-3 suppressor on Cox2p
and Cox3p synthesis. Mitochondrial translation products were
radioactively labeled in the presence of cycloheximide and subjected to
electrophoresis as described in Materials and Methods. The positions of
Cox2p and Cox3p are indicated. Strain names and relevant genotypes are
as follows: lane 1, DL1 (wild-type); lane 2, NSG78
(MRP51-3); lane 3, JJM120 (5'-UTL mutation
cox2-11); lane 4, NSG50 (MRP51-3 cox2-11); lane
5, JJM113 (initiation codon mutation cox2-10); lane 6, NSG59
(MRP51-3 cox2-10); lane 7, LSF75 (initiation codon mutation
cox3-1); lane 8, NSG83 (MRP51-3 cox3-1).
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None of the suppressor alleles had a detectable phenotype when combined
with the wild-type mitochondrial genome. However,
some suppressor
alleles at both
MRP21 and
MRP51 suppressed some
5'-UTL mutations in both
COX2 and
COX3 (Tables
2
and
3; Fig.
1 and
2), suggesting that Mrp21p and Mrp51p are involved in
the
translation of at least these two mitochondrial mRNAs.
One possible mechanism for suppression of 5'-UTL mutations in both the
COX2 and
COX3 mRNAs is that the alterations in
Mrp21p
and Mrp51p cause a general increase in the level of
mitochondrial
translation. To test this possibility, we asked whether
the
MRP21 and
MRP51 suppressor alleles would
improve the respiratory growth
of strains with leaky initiation codon
mutations (AUG to AUA)
in these mitochondrial mRNAs (
cox2-10
and
cox3-1). Since the respiratory
growth of these mutants
is responsive to levels of translational
activity (
26,
47),
we would expect that all of the
MRP21 and
MRP51
suppressor alleles would suppress the initiation codon mutations
if the
mechanism of suppression were an overall increase in translation.
We
might also expect that the strength of suppression of 5'-UTL
mutations
would correlate with the strength of suppression of
initiation codon
mutations for each particular suppressor allele:
a strong suppressor of
5'-UTL mutations would strongly suppress
initiation codon mutations,
and the converse. However, this was
not the case. Some alleles that
suppressed 5'-UTL mutations failed
to affect the initiation codon
mutants, others suppressed them,
and still others reduced their
respiratory growth, causing a synthetic
defect (Tables
2 and
3).
In the experiment in Fig.
2, this phenomenon is illustrated at the
level of mitochondrial protein synthesis for the
MRP51-3 allele.
MRP51-3 had a dramatic effect on the
cox2-11 5'-UTL mutation:
in the unsuppressed strain, the
level of Cox2p was greatly reduced
(Fig.
2, lane 3), while in an
MRP51-3 cox2-11 strain, Cox2p was
synthesized at wild-type
levels (lane 4). In a leaky
cox2 initiation
codon mutant
strain, Cox2p levels were reduced from wild-type
levels, whether or not
the strain carried
MRP51-3 (lanes 5 and
6). Similarly,
MRP51-3 had no effect on Cox3p synthesis from the
leaky
cox3 initiation codon mutant allele (lanes 7 and 8). Levels
of Cox2p and Cox3p synthesis in these strains correlated with
their
respiratory growth phenotypes, confirming that
MRP51-3
strongly
suppressed the 5'-UTL mutation while having no effect on the
initiation
codon mutations.
In vivo labeling of mitochondrial translation products in
MRP21-1 strains with
cox2 and
cox3
initiation codon mutations (not
shown) yielded similar results. The
MRP21-1 allele, a strong suppressor
of the 5'-UTL mutation
cox3-15, had no effect on Cox2p synthesis
in the
cox2 initiation codon mutant strain. In combination with
the
cox3 initiation codon mutation,
MRP21-1 caused
reduced Cox3p
synthesis, consistent with the synthetic respiratory
defect observed
in the double-mutant strain (Table
2).
Molecular cloning and nucleotide sequence analysis of the
MRP21 and MRP51 genes.
The dominant
suppression phenotypes of the MRP21 and MRP51
suppressors were used to clone both genes. A genomic library was constructed in a multicopy plasmid from a strain (MCC267; Table 1)
carrying both the MRP21-1 and MRP51-3 alleles, to
isolate both genes from a single library (Materials and Methods). To
clone MRP21, the cox3-15 mutant strain TF210
(Table 1) was transformed with the library and transformants with
cold-resistant respiratory growth were selected. To clone
MRP51, the cox2-12 mutant strain JJM158 (Table 1)
was transformed with the library and respiring transformants were
selected.
Plasmids that conferred cold-resistant respiratory growth on the
cox3-15 mutant strain TF210 fell into two distinct sets
based
on restriction mapping and hybridization analysis. The nucleotide
sequences of small fragments from each class were determined to
localize the plasmid inserts in the genome. One class of plasmids
was
found to carry
PET494, which encodes a subunit of the
COX3-specific
translational activator (
9,
10) and
is known to suppress
cox3-15 when overexpressed
(
12). The other class of plasmids
conferring cold-resistant
respiratory growth carried a region
of DNA near the
ROX3
gene (
57) from chromosome II. To test whether
the
MRP21 gene was located in this region of the genome, the
URA3 gene was integrated into the
ROX3 region of
a strain carrying
the
cox3-15 allele and the integrant
strain was crossed to an
MRP21-1 strain also carrying
cox3-15 (see Materials and Methods).
Among the meiotic
progeny of this cross, all the spores that were
able to respire at
13.5°C were Ura
whereas all Ura
+ spores had
cold-sensitive respiratory growth: thus, the
MRP21 gene is
tightly linked to the
ROX3 region.
A 2.9-kb
BamHI fragment which included 1.8 kb from the
region common to all six plasmids obtained from the genomic library
carried the complete
MRP21-1 gene, as judged by its ability
when
subcloned to confer cold-resistant respiration on the
cox3-15 strain TF210 as well as did the original library
plasmids. This
fragment carried two open reading frames, but only one
was located
entirely within the region of overlap of the library
plasmids,
identifying it as
MRP21. The
MRP21 open
reading frame (YBL090W;
GenBank accession no.
Z35851) encodes a
strongly basic (net
charge of +18) 177-amino-acid protein with a
predicted molecular
mass of 20.4 kDa. Homology searches with the Basic
Local Alignment
Search Tool (BLAST) program (
2) revealed
weak similarity between
the C-terminal region of Mrp21p and predicted
proteins of unknown
function from humans, mice, and
Caenorhabditis elegans, all of
which are highly homologous.
The metazoan proteins exhibit a clear
similarity to the small-subunit
ribosomal S21 protein from bacteria.
Alignment of the Mrp21p, higher
eukaryotic, and bacterial sequences
(Fig.
3) reveals clear similarities between
Mrp21p and S21. Interestingly,
the sequences of the suppressor alleles
(see Materials and Methods)
revealed that they caused missense
substitutions in a small region
of the S21-homologous C-terminal domain
of Mrp21p. The independently
isolated
MRP21-1 and
MRP21-2 alleles were identical (see Materials
and Methods),
both causing a Glu-to-Lys change at amino acid 118.
The
MRP21-3 allele changed Asn to Lys at amino acid 124, increasing
the similarity to bacterial S21 proteins.
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FIG. 3.
Alignment of the C-terminal region of Mrp21p with
bacterial ribosomal S21 proteins and with sequences from higher
eukaryotes. Black boxes, identities between Mrp21p and either or both
of the bacterial S21 proteins; gray boxes, identities between Mrp21p
and the metazoan proteins not shared by the bacterial S21 proteins;
white boxes, identities between the metazoan proteins and the bacterial
S21 proteins not found in Mrp21p. The percentages of identical plus
similar amino acids for selected pairwise comparisons are as follows:
Mrp21p-E. coli S21, 23.9%; Mrp21p-Myxococcus
xanthus S21, 28.1%; Mrp21p-human, 36.1%; Mrp21p-mouse, 32.5%;
Mrp21p-C. elegans, 25.3%; human-E. coli S21,
34.3%; human-M. xanthus S21, 39.1%. The accession numbers
of the sequences are as follows: Mrp21p, Z35851; E. coli
rpsU gene, V00346 (40); M. xanthus rpsU
gene, U20669; human, coordinates 447 to 710, U79258; mouse, coordinates
73 to 336, AA050698; C. elegans gene F29B9.10, U70849.
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To clone
MRP51, the
cox2-12 mutant strain JJM158
was transformed with the MCC267 library and respiring transformants
were
selected. Fourteen overlapping plasmids which conferred
respiratory
growth were isolated. To test whether
MRP51 was
linked to this
region, a fragment from this plasmid was cloned into an
integrating
vector carrying the
URA3 gene and transformed
into a
MRP51-5/MRP51 diploid (see Materials and Methods).
When independent diploid
transformants were sporulated and tetrads were
dissected, the
ability to respire segregated either with the
Ura
+ marker (integration into the suppressor chromosome) or
opposite
to the Ura
+ marker (integration into the wild-type
chromosome). Thus, the
plasmid-borne sequences were tightly linked to
MRP51.
Sequence analysis of the smallest library plasmid revealed two complete
open reading frames: the
IDI1 gene encoding isopentenyl
diphosphate-dimethylallyl diphosphate isomerase, an enzyme of
the
isoprenoid biosynthetic pathway (
3), and an unidentified
gene. To determine whether the unidentified open reading frame
was
MRP51, it was subcloned from the suppressing plasmid after
PCR amplification (Materials and Methods). The resulting plasmid,
pNSG19, had suppressor activity in
cox2-11 and
cox2-12 mutant
strains, identifying this gene as
MRP51. The
MRP51 open reading
frame (YPL118W;
coordinates 16771 to 17805 of GenBank sequence
no.
U43503) encodes a
344-amino-acid protein with a predicted
molecular mass of 39.5 kDa.
Mrp51p is predicted to be a strongly
basic protein with a net charge of
+22. The DNA sequences of the
suppressor alleles (see Materials and
Methods) revealed that they
were associated with amino acid
substitutions in a limited region
of Mrp51p:
MRP51-1, Val to
Ala at position 235;
MRP51-2, Asp to
His at position 241;
MRP51-3 and
MRP51-4, Pro to Arg at position
261;
MRP51-5, Pro to Leu at position 260;
MRP51-8, Glu
to Arg
at position 279. No proteins of known function are homologous
to
Mrp51p. However, Mrp51p is 46% identical to an unidentified
open
reading frame of
S. kluyveri (
66) (coordinates
290 to 378
of GenBank sequence no.
U83662 and coordinates 2569 to 1543
of EMBL sequence no.
Z14125).
Construction and characterization of mrp21 and
mrp51 null mutations.
To inactivate MRP21
and MRP51, internal fragments of both genes were removed and
replaced with a
hisG::URA3::hisG
cassette (1) (see Materials and Methods). DNA fragments
carrying each deleted and disrupted gene were used, separately, to
transform a diploid strain homozygous for a ura3 mutation.
In each case, the Ura+ diploid transformants respired well,
but when the diploids were sporulated and tetrads were dissected, each
tetrad had two respiratory-competent Ura spores and two
respiratory-deficient Ura+ spores. The Ura+
spores were unable to produce respiring diploids when mated to a
nuclearly wild-type, rho0 tester strain (lacking
mitochondrial DNA), indicating that deletion of either MRP21
or MRP51 caused the cells to lose their mitochondrial DNA.
The destabilization of mitochondrial DNA is a hallmark of mutations
that block all mitochondrial translation (24, 34, 49, 50).
This suggested that both Mrp21p and Mrp51p might be required generally
for mitochondrial translation.
Subcellular and submitochondrial localization of Mrp21p and
Mrp51p.
To detect Mrp21p, it was tagged at the carboxy terminus
with three copies of the influenza virus HA epitope (25, 65)
(see Materials and Methods), which is recognized by the 12CA5 mouse monoclonal antibody. HA-tagged Mrp21p was only partially functional: strains in which the only copy of MRP21 carried the HA tag
showed a mild respiratory defect. To detect Mrp51p, we raised a rabbit polyclonal antiserum to a version of Mrp51p carrying amino- and carboxy-terminal six-histidine tags (37), purified after
expression in E. coli (see Materials and Methods).
Wild-type yeast (PTY11) and a strain carrying a chromosomally
integrated gene encoding HA-tagged Mrp21p (MCC291) were grown
and
fractionated into mitochondrial pellets and postmitochondrial
supernatant fractions, after which the mitochondria were purified
by
buoyant density gradient centrifugation (see Materials and
Methods).
The fractions were analyzed by gel electrophoresis and
Western
blotting, probing either with the anti-HA monoclonal antibody
or with
the polyclonal anti-Mrp51p antiserum (Fig.
4). As expected,
both Mrp21p and Mrp51p
were associated specifically with mitochondria.
View larger version (67K):
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|
FIG. 4.
Mrp21p and Mrp51p are located in mitochondria. (A and B)
Subcellular location of Mrp21p-HA (arrow). A 50-µg portion of protein
was applied to each lane of the gel. Western blots in both panels were
probed with monoclonal anti-HA antibody. (A) Subcellular fractions of
an MRP21-HA strain (MCC291). Mrp21p-HA is present in
whole-cell extract (lane T) and gradient-purified mitochondria (lane M)
but not in the cytosol (lane S). (B) Corresponding fractions from a
wild-type strain (PTY11). (C) The polyclonal anti-Mrp51p antibody
detects an approximately 39-kDa protein in whole-cell extract from the
wild type (MRP51; strain DAU1) that is absent in a null
mutant (mrp51 ; NSG63) and overproduced in a strain
carrying MRP51 on a high-copy-number plasmid
(MRP51 2µm; plasmid pNSG22 in strain DAU1), identifying
this band as Mrp51p. Approximately 10 µg of total-cell protein was
applied per lane. (D) Subcellular location of Mrp51p (arrow). The
anti-Mrp51p antibody detects Mrp51p in whole-cell extract (lane T) and
gradient-purified mitochondria (lane M) but not in the cytosol (lane S)
of a wild-type strain (PTY11). The amounts of protein applied to the
gel were 50 µg (lane T), 20 µg (lane S), and 20 µg (lane M).
|
|
The phenotypes of the
mrp21 and
mrp51 null
mutations suggested that they were required for all mitochondrial
translation.
This, as well as the similarity between Mrp21p and
bacterial S21
proteins, raised the possibility that they were
components of
the mitochondrial ribosome. To test this possibility,
gradient-purified
mitochondria were solubilized with detergent and the
contents
were sedimented into a sucrose gradient in the presence of
high
salt concentrations (0.5 M NH
4Cl; see Materials and
Methods) to
separate the subunits of mitochondrial ribosomes. The
gradient
fractions were analyzed for absorbance at 260 nm, to locate
the
rRNAs, and by gel electrophoresis and Western blotting (Fig.
5 and
6).
In the experiment in Fig.
5, mitochondrial ribosomes from
wild-type
(PTY11) cells were subjected to this analysis. The position
of Mrp51p
coincided with that of the ribosomal small subunit,
as identified by
the smaller of two peaks of absorbance at 260
nm and by the presence of
Mrp13p, a known small-subunit constituent
(
53). In the
experiment in Fig.
6, a similar analysis was performed
on mitochondrial
ribosomes isolated from a strain (MCC291) in
which the only functional
MRP21 gene carried the HA tag at its
3' end. As noted above,
the HA-tagged Mrp21p did not function
as well as the wild type, and the
experiment in Fig.
6 reveals
the probable reason for this. The small
subunit of mitochondrial
ribosomes was partially destabilized in this
strain, as shown
by the dramatic decrease in the peak of absorbance for
the small
rRNA relative to that for the large rRNA. Nevertheless, the
peak
of Mrp21p-HA coincided with the small ribosomal subunit. Indeed,
the fact that an alteration which decreased the function of Mrp21p
specifically affected the small subunit of mitochondrial ribosomes
strongly supports the idea that Mrp21p is a small-subunit component.
View larger version (16K):
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|
FIG. 5.
Mrp51p cosediments with the small subunit of
mitochondrial ribosomes. Nycodenz gradient-purified mitochondria of
strain PTY11 were disrupted with deoxycholate, and the soluble contents
were centrifuged into a sucrose gradient in the presence of 0.5 M salt
(see Materials and Methods). (Top) Absorbance at 260 nm
(A260) of alternate fractions. (Bottom) Western blots of
alternate fractions probed with antisera against Mrp51p, the known
small-subunit protein Mrp13p (53), and the known
large-subunit protein Mrp7p (24).
|
|
View larger version (24K):
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[in a new window]
|
FIG. 6.
Partially functional Mrp21p-HA cosediments with the
small subunit of mitochondrial ribosomes but destabilizes it. Nycodenz
gradient-purified mitochondria of strain MCC291 were analyzed as
described in the legend to Fig. 5, except that the fractions were also
probed with the anti-HA monoclonal antibody.
|
|
| |
DISCUSSION |
We have identified two nuclear yeast genes encoding previously
unidentified mitochondrial ribosomal small-subunit proteins, Mrp21p and
Mrp51p. These genes can mutate to suppress defects in the 5'-UTLs of
two different mitochondrial mRNAs, COX2 and COX3,
but do not bypass the mRNA-specific translational activation system.
The 5'-UTL mutations are known from previous genetic analysis to alter
the targets of the COX2 and COX3 mRNA-specific
translational activators (12, 46). However, the functions of
the MRP21 and MRP51 products are not mRNA
specific. Suppressor alleles at each of these two nuclear genes were
able to improve the respiratory growth of certain 5'-UTL mutations, but
not others, affecting both the COX2 and COX3
mRNAs. Furthermore, deletion of either MRP21 or
MRP51 prevented mitochondrial translation globally.
Suppression of 5'-UTL mutations might occur by any alteration that
caused a general increase in mitochondrial translational activity.
However, this does not appear to be the mechanism by which the
MRP21 and MRP51 suppressors work, since many of
the suppressors failed to increase the respiratory growth of strains bearing leaky COX2 and COX3 initiation codon
mutations (cox2-10 and cox3-1) and also failed to
increase Cox2p or Cox3p synthesis in these strains. The initiation
codon mutations reduce the translation of mRNAs bearing otherwise
wild-type 5'-UTLs roughly five- to sevenfold without altering the sites
of initiation (26, 47). Furthermore, the growth phenotypes
they cause are influenced by the levels of their respective
mRNA-specific translational activators, indicating that they are
sensitive to translational activity (26, 47). While some of
the other suppressor alleles did improve the growth of initiation codon
mutants, MRP21-1 and MRP51-8 actually reduced the
respiratory growth of cox3-1 and cox2-10 mutants, respectively. Thus, we conclude that the MRP21 and
MRP51 mutations do not generally increase the activity of
mitochondrial ribosomes. Instead, the patterns of suppression by the
MRP21 and MRP51 mutations, which are allele
specific but, surprisingly, gene nonspecific, suggest that yeast
mitochondrial ribosomes may recognize a common feature in mRNA 5'-UTLs.
According to this hypothesis, the structure of the common element was
altered by mutations in the COX2 and COX3 5'-UTLs
and the suppressors altered the ribosomal small subunit to compensate
for the defects.
Mrp21p resembles several other yeast mitochondrial small-subunit
ribosomal proteins (4, 18, 19, 39) in that it has a domain
lacking homology to any known protein and a domain identifiably homologous to a bacterial ribosomal protein. The amino-terminal 99-amino-acid sequence of Mrp21p is not similar to currently known sequences, but the carboxy-terminal 78-residue sequence exhibits clear
similarity to a metazoan sequence, which in turn is clearly similar to
those of bacterial ribosomal S21 proteins. This family of proteins is
absent in eukaryotic cytoplasmic ribosomes (68) and those of
known members of the Archaea (7). The limited homology is convincing when taken together with the facts that both
Mrp21p and S21 are small-subunit ribosomal proteins and that our
suppressors are missense substitutions in a small region of the
S21-homologous C-terminal domain of Mrp21p. The functions of the
metazoan Mrp21p homologs are unknown, but it is likely that they are
also mitochondrial ribosomal proteins involved in translation
initiation.
The available evidence is consistent with the idea that Mrp21p and
bacterial S21 may have similar functions in promoting mRNA-ribosome interactions. Our genetic data suggest that Mrp21p may interact directly with the 5'-UTLs. Ribosomal protein-mapping studies indicate that E. coli S21 protein is in the platform region of the
small subunit, the site of Shine-Dalgarno and codon-anticodon
interactions (8). S21 is in very close proximity to both the
16S rRNA and the initiation region of mRNAs, as shown by cross-linking
and resonance energy transfer experiments (15, 21, 48).
However, the in vivo function of S21 has not been studied genetically. The only reported alleles of the E. coli gene encoding S21,
rpsU, have no effect on translation (16).
Like several other mitochondrial ribosomal small-subunit proteins
(33, 42, 49, 53), Mrp51p exhibits no clear homology to any
known ribosomal proteins. The only known homolog is the product of an
unidentified open reading frame in the yeast S. kluyveri,
which probably also encodes a mitochondrial ribosomal protein.
Interestingly, the missense substitutions caused by our five different
MRP51 suppressor alleles are clustered within a 45-amino-acid region of the protein, which could be involved in mRNA
interactions.
The mechanism by which yeast (and other) mitochondrial ribosomes
identify translation initiation sites is not clear, largely owing to
the lack of suitable in vitro systems (20). However, it does
not involve either a classical Shine-Dalgarno interaction or a simple
scanning mechanism (27). AUG codons clearly play a role in
start site selection, but additional information is also used (26,
47). Genetic studies have strongly supported a model in which
mRNA-specific translational activators mediate the mRNA-ribosome
interaction leading to initiation and possibly influence start site
selection (reviewed in reference 27).
Our present data demonstrate that, in conjunction with mRNA-specific
activators, the yeast mitochondrial ribosome itself plays an active
role in recognizing translatable mRNAs. The pattern of suppression
observed suggests that the ribosomes may recognize a feature common to
all yeast mitochondrial mRNA 5'-UTLs. A candidate for such a common
feature, the octanucleotide sequence UAUAAAUA, has recently
been identified based on a functional analysis of the COX2
mRNA 5'-UTL and comparisons with other 5'-UTLs (22). While
this sequence is not directly altered in the suppressible alleles
studied here, cox2-11 and cox3-15, it is within
10 bases upstream of both mutations. This octanucleotide is
complementary to several sites in the mitochondrial small-subunit rRNA
and could thus be involved in mRNA-rRNA base pairing. Clearly, Mrp21p
and Mrp51p could play a role in establishing such an mRNA-rRNA
interaction. However, this putative interaction would not closely
resemble the Shine-Dalgarno mechanism (55, 61), since the
octanucleotide does not occur at fixed distances from translation
initiation codons of mRNAs and its complement is not located at the 3'
end of the rRNA. Alternatively, yeast mitochondrial ribosomes could interact with mRNAs purely through protein-mRNA contacts, possibly involving Mrp21p and Mrp51p directly.
| |
ACKNOWLEDGMENTS |
We thank C. Shamu and J. Nunnari for providing us with the
plasmid pCS124, R. Zitomer for providing plasmids carrying the ROX3 region, T. L. Mason for gifts of antisera, and P. Nagley for the gift of strain h45.
This work was supported by National Institutes of Health research grant
GM29362. N.S.G.-W. was supported by National Institutes of Health
predoctoral training grant GM07617.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Section of
Genetics and Development, Biotechnology Building, Cornell University,
Ithaca, NY 14853-2703. Phone: (607) 254-4834. Fax: (607) 255-6249. E-mail: mcc9{at}cornell.edu.
| |
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Abstract
Introduction
