From the Génétique des Interactions
Macromoléculaires, CNRS (URA2171), ¶ Génétique
Moléculaire des Levures, CNRS (URA2171), and ** Chimie Structurale
des Macromolécules, CNRS (URA2185), Institut Pasteur, 25-28 Rue
du Dr. Roux, 75724 Paris Cedex 15, France
Received for publication, December 1, 2000, and in revised form, January 18, 2001
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ABSTRACT |
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Mitochondrial ribosomal proteins were studied
best in yeast, where the small subunit was shown to contain about 35 proteins. Yet, genetic and biochemical studies identified only 14 proteins, half of which were predictable by sequence homology with
prokaryotic ribosomal components of the small subunit. Using a recently
described affinity purification technique and tagged versions of yeast
Ykl155c and Mrp1, we isolated this mitochondrial ribosomal subunit and identified a total of 20 proteins, of which 12 are new. For a subset of
the newly described ribosomal proteins, we showed that they are
localized in mitochondria and are required for the respiratory competency of the yeast cells. This brings to 26 the total number of
proteins described as components of the mitochondrial small ribosomal
subunit. Remarkably, almost half of the previously and newly identified
mitochondrial ribosomal components showed no similarity to any known
ribosomal protein. Homologues could be found, however, in predicted
protein sequences from Schizosaccharomyces pombe. In more
distant species, putative homologues were detected for Ykl155c, which
shares conserved motifs with uncharacterized proteins of higher
eukaryotes including humans. Another newly identified ribosomal
protein, Ygl129c, was previously shown to be a member of the DAP-3
family of mitochondrial apoptosis mediators.
In yeast mitochondria, the majority of the characterized ribosomal
proteins are essential for protein synthesis (for review see Ref. 1).
Homologues of about 18 of the 21 prokaryotic small ribosomal subunit
proteins can be identified by similarity searches in the yeast complete
genomic sequence. However, the number of ribosomal proteins is larger
in the small subunit of yeast mitochondria, estimated to be 33 or 36 (2, 3).
Thus far, yeast mitochondrial ribosomal proteins have been
identified either by the study of mutant strains with mitochondrial dysfunction (pet mutants; for review see Ref. 4) or by direct biochemical approaches involving isolation of mitochondria,
purification of mitochondrial ribosomes, and protein separation
followed by microsequencing (5). Mitochondrial ribosome purification
has thus far been technically difficult, because the ribosomal proteins represent only 2-3% of the mitochondrial proteins (2). To date, these
approaches have identified only a subset of the total number of
mitochondrial ribosomal proteins. In yeast, the eukaryote in which most
of the studies were performed, only 14 proteins of the small
mitochondrial ribosomal subunit have been characterized experimentally
(1, 6).
We were originally interested in the study of the yeast
YKL155C gene because it was found in a two-hybrid exhaustive
screen to interact with the Prp11 splicing factor (7). To test whether this protein was associated with splicing factors under physiological conditions, we used the recently described tandem affinity purification technique (8) that allows the isolation of protein complexes by two
successive affinity purification steps under mild conditions. Instead
of containing splicing factors, the purified complex associated with
Ykl155c was found to consist of the mitochondrial small ribosomal subunit. We took advantage of this rapid and efficient affinity purification strategy to identify and characterize twelve novel proteins of the small ribosomal mitochondrial subunit.
Plasmids and Strains--
Strains used in this study are listed
in Table I. Gene deletions were made by
replacing the entire open reading frame by a TRP1 or KanMX6 cassette
(9). The strains containing green fluorescent protein
(GFP)1 fusion proteins or
TAP-tagged fusion proteins were constructed by genomic
insertion of the tag together with the TRP1 marker downstream of the
affected genes to obtain C-terminally tagged fusion proteins. The
Tag/TRP1 markers were generated by polymerase chain reaction
either from pFA6a-GFP(S65T)-TRP1 for the GFP fusion (9) or pBS1479 for
the TAP-tagged fusion proteins (8) using oligonucleotides
designed according to the authors. The YGL129C-containing vector pAH028.1R was constructed by cloning a
BamHI/NotI polymerase chain reaction fragment of
YGL129C into a pCM190-derived plasmid (10).
Complex Purification--
Complex purification was done
essentially by the method described in detail by Rigaut et
al. (8) starting with 2 liters of yeast culture. Polyacrylamide
gradient gel electrophoresis was done in the Tris-Tricine system (11).
A gradient of 5-20% acrylamide was used in all cases. Protein bands
were visualized by Coomassie Blue G-250 staining (12).
Mass Spectrometry--
In-gel digestion of the proteins was
performed by the protocol of Shevchenko et al. (13), using
bovine trypsin (Roche Molecular Biochemicals). The generated peptides
were cleaned on a reversed-phase support using Millipore Zip-Tip C18 or
Poros R2 (Perseptive Biosystems). The mixture of peptides was analyzed
by matrix-assisted laser desorption ionization time of flight
(MALDI-TOF) mass spectrometry on a Voyager DE-STR system
(Perseptive Biosystems) using
Tandem mass spectrometry was performed on a triple quadrupole API 365 mass spectrometer (Sciex) equipped with a nanoelectrospray source
(Protana). Protein identification was done using the PeptideScan 3.02 software part of the BioToolBox 2.3 package (PE-Sciex) for Macintosh.
Protein data bases were searched for matching entries using the peptide
sequence tag methodology (14). Identification of a given protein was
considered to be accurate if two or more fragmented peptides gave the
same polypeptide match or one fragmented peptide and a peptide mass map
identified the same protein.
Fluorescence Microscopy--
Fluorescence microscopy was done
using a Leica DMRB fluorescence microscope equipped with a
Hamamatsu C4880 cooled digital camera and a 100-watt UV
excitation lamp. MitoTracker Red CMXRos from Molecular Probes was used
as a specific mitochondrial marker following the manufacturer's instructions.
Informatics--
Image manipulation was done with Photoshop
version 5 (Adobe). For sequence homology search, we used the PSI-BLAST
2.1 program (15) from the NCBI web site with an expected E-value
of 10 for the first BLAST search. The PSI-BLAST iterations were
initiated after manual selection of similar sequences based on the
presence of potential sequence motifs. The same homologues were also
detected using the precalculated Blastp alignments accessible as the
Blink feature of the GenPept entries. A similarity between sequences coming from two different organisms was considered significant if
searches starting with either sequence found the other as one of the
best matches. The search for yeast homologues of prokaryotic ribosomal
proteins was facilitated by the available clusters of orthologous
groups (16). Multiple sequence alignments were made using the CLUSTALX
program (17) and prepared for publication using GeneDoc version
2.6.
Ykl155c Is Associated with Components of the Small Ribosomal
Subunit of Mitochondria--
A yeast strain deleted for
YKL155C was viable but unable to grow on glycerol as the
sole carbon source (respiration-deficient; data not shown). We
constructed the TAP-tagged chromosomal fusion of YKL155C
according to Rigaut et al. (8). This construction ensured a
natural level of tagged protein expression, and the strain was
respiration-competent, indicating that the modified protein was
functional. The TAP affinity purification of the Ykl155c-associated complex was performed under standard conditions as described (8).
The proteins that remained associated with the Ykl155c-TAP-tagged
protein after the two successive affinity purification steps were
separated on a denaturing polyacrylamide gradient gel (see "Experimental Procedures"). After Coomassie Blue staining, about 30 different bands were visible, with various intensities (Fig. 1A, lane 1). This
variability of band intensities could result from different factors
such as uneven protein recovery after trichloroacetic acid
precipitation, dye binding dependent on protein sequence and length,
bands that contain more than one protein, components of the complex
that are not equimolar, or partial degradation.
The bands were excised from the gel, and the proteins were identified
by MALDI peptide fingerprinting and nanoelectrospray tandem mass
spectrometry (see "Experimental Procedures" and Fig. 2 for an example). A number of the
identified proteins were known components of the small subunit of the
mitochondrial ribosome, namely Nam9, Mrp4, Mrps28, Mrp2, Var1,
Pet123, Mrp51, and Mrp1 (Fig. 1 and Table
II). These findings, together with the
respiratory phenotype that follows the deletion of the
YKL155C gene, strongly suggested that Ykl155c was associated
with the small subunit of the mitochondrial ribosome.
Ykl155c and Mrp1 Are Associated with the Same Complex--
We
wanted to confirm that the complex isolated by affinity purification
with the Ykl155c-tagged protein indeed corresponded to the small
subunit of the yeast mitochondrial ribosome. Therefore, we examined the
protein profile of the affinity-purified complex obtained using the
TAP-tagged version of the small subunit mitochondrial ribosomal protein
Mrp1 (Mrp1-TAP). When compared, the band patterns of the two purified
complexes were identical, with the exception of the tagged proteins,
which migrated about 5 kDa higher than their unmodified version as a
result of the remaining calmodulin binding domain tag fragment (Fig.
1). The presence of Ykl155c in the complex purified with Mrp1 was
confirmed by mass spectrometry. The intensity of the Ykl155c band
appears to be significantly lower in the Mrp1-TAP purification. It is
possible that part of the tagged form of Ykl155c is not associated with
the ribonucleoprotein complex, and therefore additional free
protein was purified when using Ykl155c-TAP.
To search for RNA components in the purified complex, the RNAs
extracted from the purified fractions were heat-denatured, spotted on a
filter, and hybridized with either a 21 S (Fig. 1B) or a 15 S (Fig. 1C) yeast rRNA-specific probe. As expected, the 15 S
probe for the small subunit mitochondrial rRNA hybridized specifically
to the RNA associated with the Ykl155c and Mrp1 complexes, whereas the
21 S-specific probe did not.
Finally, we analyzed the cosedimentation of Ykl155c with Mrp1 and the
15 S rRNA by ultracentrifugation on a 15-30% sucrose gradient under
dissociating conditions (0.5 M salt). Whole yeast soluble
crude extracts from the Ykl155c-TAP- and Mrp1-TAP-tagged strains were
mixed and loaded on top of the gradient. After ultracentrifugation, the
fractions were analyzed for their protein content by denaturing gel
electrophoresis and immunoblot specific for the protein A component of
the tag. In addition, the RNAs of an aliquot from each fraction were
spotted on a filter and probed with the 21 S- or 15 S-specific probe.
Cosedimentation of Ykl155c with Mrp1 and the 15 S mitochondrial rRNA is
shown in Fig. 3.
In addition to the proteins previously described as components of the
mitochondrial small ribosomal subunit, we found 12 proteins that had
not been previously biochemically characterized. Only 6 of these 12 proteins had clear known ribosomal prokaryotic homologues (Table
II).
The nomenclature of the mitochondrial ribosomal proteins is as variable
as the methods employed for their characterization, but MRP
(for mitochondrial ribosomal protein) is the most frequently used.
However, this name does not allow one to discriminate between small and
large subunit proteins and thus may be misleading. For example, Mrp2 is
not the homologue of the prokaryotic S2 ribosomal proteins but belongs
to the S14p family. Accordingly, we preferred to designate the newly
identified genes RSMxx genes, where RSM stands
for ribosomal small subunit of
mitochondria, and xx is the number of the
corresponding prokaryotic protein family (see Table II). The genes that
encode proteins that are not significantly similar to prokaryotic
proteins were named with the same prefix RSM followed by a
number that begins with 22, because there are 21 prokaryotic small
ribosomal subunit protein families. Thus, the proposed name of
the YKL155c gene is RSM22 (Table II).
A large amount of functional data was generated in the course of
genetic screenings and functional genomic studies in yeast. Hence, for
a number of the identified proteins, some cellular or functional data
were already available in public data bases. In addition, for some of
the less well characterized proteins, we analyzed the cellular
localization of the GFP C-terminally tagged proteins and the
respiratory competency of strains disrupted for the corresponding
genes. These data are summarized in Table II and described below.
Functional Data on the New Proteins That Belong to Known Ribosomal
Protein Families--
Ribosomal protein families S7p and S10p are
represented in yeast mitochondria by the corresponding homologues
Yjr113c and Ydr041w. Mammalian homologues of S7 (19) and S10 (20) were also recently described as components of the bovine small mitochondrial ribosomal subunit. As expected, the fluorescently labeled yeast proteins localized to mitochondria (Fig.
4).
The C-terminal quarter of the Yer050c sequence is similar to
prokaryotic S18 ribosomal proteins (16). In the case of the haploid
strain LMA139 (YER050C-GFP; Table I), no strong
mitochondrial signal is visible either in the case of the specific
mitochondrial marker or in the case of the GFP (Fig. 4), and the strain
is respiration-deficient. Mitochondria are visible in a heterozygous
diploid strain (obtained by crossing LMA139 and wild-type BMA64
Mat
A similar case was observed after the GFP tagging of Ynr037c, a
homologue of the S19 prokaryotic ribosomal proteins. However, in a
heterozygous diploid strain, we were able to detect a weak GFP signal
that matches the mitochondrial marker (Fig. 4, bottom).
Novel Proteins Not Similar to Prokaryotic Ribosomal Components Are
Essential for Mitochondrial Function--
Given that six newly
identified ribosomal proteins have no obvious similarity with
previously known ribosomal proteins, it was important to determine the
cellular localization and the involvement of these proteins in
mitochondrial function. The result of the cellular localization
of the GFP C-terminally tagged proteins is shown in Fig. 4 and
summarized in Table II. With the exception of Yjr101w, which was not
analyzed, all these proteins were unambiguously localized to the
mitochondria. Ykl155c, Ygl129c, Ydr175c, and Yjr101w were previously
reported in the literature or public data bases to be essential for
mitochondrial function (see Table II for references). We confirmed the
direct involvement of Ykl155c in mitochondrial function by showing that
the defective growth on glycerol resulting from the deletion of its
gene could be complemented by a plasmid expressing a wild-type version
of the protein (data not shown). For Yil093c, no previously reported
functional data were available. We deleted the gene, which
proved to be essential for growth on a medium containing glycerol as
the sole carbon source (data not shown). All these data confirmed that
the newly identified proteins were localized to mitochondria and/or
were essential for mitochondrial function.
Affinity Purification of the Ribosomal Subunit Is Complementary to
Previously Described Methods--
The number of protein components of
the yeast small mitochondrial ribosomal subunit was estimated by
mitochondrial purification, preparative ultracentrifugation, and
two-dimensional gel electrophoresis to lie between 33 (2) and 36 (3).
We used an affinity purification method with tagged versions of yeast
Ykl155c and Mrp1 that allowed us to identify, by mass spectrometry, 20 different proteins of this ribosomal particle. Some of the previously
described components of the small subunit were not found (Table II).
Our affinity-purified complex is unlikely to correspond to a specific
particle, distinct in vivo from the bona fide
small mitochondrial ribosomal subunit, because of the identical
patterns of proteins obtained with either Ykl155c or Mrp1 (a previously
described mitochondrial ribosomal protein). Thus, the discrepancies
observed between the presented method and previously described
techniques might result from the loss of specific proteins during the
purification procedure or from our inability to identify some of the
components. Conversely, at least Ykl155c, with an apparent molecular
mass of 68 kDa, is conspicuously absent from the two-dimensional
gels previously published for the mitochondrial small ribosomal subunit
in which no proteins with an apparent mass above 60 kDa were revealed. The two approaches may thus be regarded as complementary.
Yeast and Mammalian Mitochondrial Ribosomal Proteins--
An
international consortium for the study of mammalian mitochondrial
ribosomal proteins has been created recently (Mammalian Mitochondrial
Ribosomal Consortium). A number of mammalian homologues of the
prokaryotic ribosomal proteins have been identified by using the bovine
ribosome as a source for the isolation and purification of the
mitochondrial ribosomes (19-22). Mammalian proteins belonging to
prokaryotic families S7 (19), S12 (21), and S10 and S15 (20) were
described this way. We were able to identify in yeast, along with other
mitochondrial homologues of prokaryotic ribosomal proteins, the
corresponding proteins for the S7 (Yjr113c/Rsm7) and S10 family
(Ydr041w/Rsm10) and also the previously identified S15 homologue,
Mrps28 (23).
The analysis of the bovine and yeast mitochondrial ribosome shows that
an important number of proteins have no similarity with known ribosomal
proteins from prokaryotes. For this group of yeast proteins,
significant similarities can be detected with sequences of the
relatively distant fungi Schizosaccharomyces pombe. In some
cases, similar proteins from other fungi may also be found, and one
example is a hypothetical protein from Neurospora crassa
(NCBI GenPept data base gi 7899415) that is similar to the yeast
Ydr175c/Rsm24 protein. Yjr101w/Rsm26 is similar to the yeast
mitochondrial small ribosomal subunit protein Mrp1. Interestingly, both
proteins also share similarities with proteins of the superoxide dismutase family.
No prokaryotic homologues of Ykl155c/Rsm22 may be detected, but we
found significant similarities of its sequence with hypothetical proteins of yet unknown function in several higher eukaryotes, including humans (Fig. 5). It is tempting
to speculate that these proteins are true homologues of the yeast
Ykl155c/Rsm22 ribosomal protein and are components of the corresponding
mitochondrial ribosomes.
Ygl129c/Rsm23 was found by similarity search to be a member of the
DAP-3 family of apoptosis mediator proteins (24). Just like
Ygl129c/Rsm23, the mouse mDAP-3 protein is localized into the
mitochondrial matrix. Ygl129c/Rsm23 is the yeast sequence most similar
to the DAP-3 proteins, but this similarity is weak (17% identity with
the mouse mDAP-3). Nevertheless, a BLAST search using the S. pombe Ygl129c homologue sequence (SPBC29A3.15c) also finds the
human hDAP-3 sequence. Moreover, mDAP-3 is able to partially complement
a mitochondrial DNA loss phenotype observed in yeast strains deleted
for YGL129C (24). If Ygl129c/Rsm23 is the true homologue of
hDAP-3, these observations would suggest an interesting link between
modulation of apoptosis and the mitochondrial protein synthesis machinery.
In conclusion, an efficient affinity purification technique allowed us
to characterize novel ribosomal components and to bring to 26 the total
number of proteins identified as components of the yeast mitochondrial
small ribosomal subunit. These findings and the discovery of the yeast
Ykl155c/Rsm22 and Ygl129c/Rsm23 as essential components of the
mitochondrial ribosome and members of conserved eukaryotic protein
families contribute to extending the classification of mitochondrial
ribosomal proteins to three different classes. A first group, which
probably exists in every eukaryotic organism, consists of proteins
similar to prokaryotic ribosomal components. Another group contain
proteins with no prokaryotic homologues but only conserved in related
species. Finally, a third group that includes the Ykl155c and Ygl129c
protein families comprises mitochondrial ribosomal proteins that are
conserved across diverse eukaryotic species.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Yeast strains used in this study
-cyano-4-hydroxycinnamic acid as
matrix. The software used for data base searches was a local copy of
the MS-Fit 3.2 part of the Protein Prospector package (University of
California, San Francisco, Mass Spectrometry Facility). A
minimum of four peptides was considered necessary for an accurate protein identification.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Proteins that copurify with Ykl155c are
components of the small mitochondrial ribosomal subunit.
A, the proteins that were isolated by tandem affinity
purification using either a tagged version of Ykl155c (lane
1) or Mrp1 (lane 2) were separated by a Tris-Tricine
5-20% gradient gel electrophoresis. MW, molecular weight
marker. Protein band identifications were done by mass spectrometry
(see "Experimental Procedures"). B and C, RNA
dot-blot analysis of the RNA that copurifies with Ykl155c (lane
1) or Mrp1 (lane 2) using 2 µg of total RNA as a
control (T). Hybridization was done using radioactively
labeled probes specific for the large subunit 21 S mitochondrial rRNA
(B) and the small subunit 15 S mitochondrial rRNA
(C).
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Fig. 2.
Mass spectrometry analysis of the tryptic
digest for the band that contained Ygl129c. A, after
in-gel digestion the resulting peptides were analyzed by MALDI-TOF mass
spectrometry. Marked peaks correspond to tryptic fragments of Ygl129c.
The measured masses of the peptides are within 150 ppm of the
corresponding predicted peptide masses. B, schematic
representation of the peptides covering the Ygl129c. Top,
peptides identified by MALDI-TOF. Bottom, peptides
unambiguously identified by nanoelectrospray-MS/MS. Center,
the thin line represents the protein sequence derived from the coding
sequence. The thick sections summarize the sequences covered
by peptides identified by MALDI-TOF and nanoelectrospray-MS/MS. No
peptide was found between residues 1 and 88; this N-terminal fragment
is predicted to undergo proteolytic cleavage (Psort II, Ref. 25).
C, part of the MS/MS fragmentation spectrum for the most
N-terminal sequenced peptide, which begins at position 89 in the
predicted protein sequence. A search using these values as y" in a data
base containing all the predicted open reading frame sequences for the
yeast genome finds the Ygl129c protein. The matched peptide has the
sequence VTPGSLYK. The differences between the masses of the marked
peaks correspond to the masses of amino acid residues Gly, Pro, and
Thr. D, the fragmentation spectrum for the peptide that is
found within residues 400-408 in the predicted sequence of
Ygl129c shows a very good signal-to-noise ratio. Fragmentation spectra
that were generated for other peptides show a signal-to-noise ratio
between the two extremes shown in C and D.
Summary of small mitochondrial ribosomal subunit proteins
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Fig. 3.
Ykl155c is associated with the small subunit
of the mitochondrial ribosome. 0.5 ml of a mix of total yeast
extracts of strains that contained TAP-tagged versions of Ykl155c and
Mrp1 was separated by ultracentrifugation on a 38-ml high salt (50 mM Tris-HCl, pH 7.4, 0.5 M ammonium chloride,
10 mM magnesium acetate, and 1 mM
dithiothreitol) 15-30% sucrose gradient in the presence of protease
inhibitors (Roche Molecular Biochemicals). The arrow
indicates the direction of sedimentation. Western blot analysis (1/5000
dilution of peroxidase-antiperoxidase antibodies from Sigma in
phosphate-buffered saline with 0.2% Tween 20 and revealed with an ECL
kit from Amersham Pharmacia Biotech) of proteins from fractions 10-29
shows that Ykl155c (A) cosediments with the known small
subunit ribosomal protein Mrp1 (B). A substantial amount of
both Ykl155c and Mrp1 may be seen in the fractions of the upper part of
the gradient and may represent forms of the tagged proteins that are
not tightly bound to the ribonucleoprotein particle. C,
integrated PhosphorImager signal obtained by dot-blot analysis of the
RNAs from fractions 6-29 hybridized to probes specific for the large
( ) and small (
) subunit rRNA. The second peak seen at fraction 25 may represent a small ribosomal subunit depleted in proteins due to the
high ionic strength of the ultracentrifugation buffer, as previously
reported for bacterial ribosomes (26). The shoulder seen in the large
subunit peak most likely results from experimental variations in RNA
extraction and sampling.
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Fig. 4.
Mitochondrial localization for novel
components of the small ribosomal subunit. GFP(S65T)-tagged
genomic versions of the genes were obtained by integrative
recombination ("Experimental Procedures"). Each row
corresponds to a given strain as indicated on the left. The
columns were labeled as follows: Nomarski for
differential interference contrast images of cells,
Mitotracker for images of a specific mitochondrial
marker in living cells, and GFP for the image of the
fluorescently tagged proteins in the same cells. The respiratory
phenotype of the strains is shown at the right of the figure
as an indication of the functionality of the fusion proteins.
*, Ygl129c localization was visualized using a plasmid carrying the
GFP-tagged version of the gene under a doxycycline-regulated promoter
("Experimental Procedures"). Images were obtained in a diploid
strain after 12 h of doxycycline derepression. , image obtained
in a diploid strain obtained by crossing wild-type BMA64 (Mat
) and
the haploid strain LMA134 (Mata) carrying the chromosomal
YNR037C GFP-tagged gene. The LMA134 strain was
respiration-deficient and showed no active mitochondria when using the
Mitotracker marker. WT, strain not modified, wild
type.
), which can also grow on glycerol as the sole carbon source, but
no GFP signal could be detected (data not shown). Hence, the GFP tag
interferes with the function of the protein. Although this precludes
the determination of its cellular localization, it shows that the
protein is essential for mitochondrial function, because the haploid
strain LMA139 is respiration-deficient.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 5.
Ykl155c is similar to eukaryotic human,
nematode, or fruit fly proteins of unknown function. The presented
alignment depicts the conserved motifs of predicted proteins from
Saccharomyces cerevisiae Ykl155c (Sc) (gi
6322694), Drosophila melanogaster CG13126 (Dm)
(gi 7297582), Homo sapiens (Hs) (gi 10436813),
and Caenorhabditis elegans SwissProt P91862
(Ce) (gi 7500302). The sequence identification numbers
"gi" are from the NCBI GenPept data base. Similar residues
conserved in all sequences are shaded in black, residues
that are conserved in three of four sequences are shown in
white on gray, and those that are conserved in
only two sequences are shaded gray; if large insertions were
present, the number of residues is shown between
parentheses. The succession of conserved residues of the
type CXHX3CXnC is
reminiscent of a zinc finger-like structure that may be involved in
protein-RNA or protein-protein interactions.
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ACKNOWLEDGEMENTS |
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We thank B. Séraphin for providing plasmid pBS1479, V. Galy for help on fluorescence microscopy, L. Decourty for help on strain constructions, P. Legrain and O. Bârzu for critical reading of the manuscript, B. Dujon for discussions, and J.-C. Rousselle for help with mass spectrometry.
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FOOTNOTES |
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* This work was supported by grants from Groupe d'Interet Publique-Aventis, CNRS, and Ministere de l'Education Nationale (France).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.
§ Recipient of financial support from Groupe d'Interet Publique- Aventis.
Supported by a grant (EUROFAN2 Bio4-CT97-2294) from the
European Economic Community.
To whom correspondence should be addressed. Tel.: 33 140 613 205; Fax: 33 145 688 790; E-mail: jacquier@pasteur.fr.
Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M010864200
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ABBREVIATIONS |
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The abbreviations used are: GFP, green fluorescent protein; TAP, tandem affinity purification; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MALDI-TOF, matrix-assisted laser desorption ionization time of flight.
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