EUKARYOTIC CELL, Nov. 2009, p. 1792–1802
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 8, No. 11
Mapping of the Saccharomyces cerevisiae Oxa1-Mitochondrial
Ribosome Interface and Identification of MrpL40,
a Ribosomal Protein in Close Proximity to Oxa1
and Critical for Oxidative Phosphorylation
Lixia Jia,†‡ Jasvinder Kaur,† and Rosemary A. Stuart*
Department of Biological Sciences, Marquette University, Milwaukee, Wisconsin 53233
Received 29 July 2009/Accepted 18 September 2009
The Oxa1 protein plays a central role in facilitating the cotranslational insertion of the nascent polypeptide
chains into the mitochondrial inner membrane. Mitochondrially encoded proteins are synthesized on matrix-
localized ribosomes which are tethered to the inner membrane and in physical association with the Oxa1
protein. In the present study we used a chemical cross-linking approach to map the Saccharomyces cerevisiae
Oxa1-ribosome interface, and we demonstrate here a close association of Oxa1 and the large ribosomal subunit
protein, MrpL40. Evidence to indicate that a close physical and functional relationship exists between MrpL40
and another large ribosomal protein, the Mrp20/L23 protein, is also provided. MrpL40 shares sequence
features with the bacterial ribosomal protein L24, which like Mrp20/L23 is known to be located adjacent to the
ribosomal polypeptide exit site. We propose therefore that MrpL40 represents the Saccharomyces cerevisiae L24
homolog. MrpL40, like many mitochondrial ribosomal proteins, contains a C-terminal extension region that
bears no similarity to the bacterial counterpart. We show that this C-terminal mitochondria-specific region is
important for MrpL40’s ability to support the synthesis of the correct complement of mitochondrially encoded
proteins and their subsequent assembly into oxidative phosphorylation complexes.
The mitochondrial genome encodes a small, but important,
number of proteins (8). These proteins are predominantly es-
sential components of the mitochondrial oxidative phosphory-
lation (OXPHOS) machinery. In the yeast Saccharomyces cer-
evisiae the proteins encoded by the mitochondrial DNA
(mtDNA) include cytochrome c oxidase subunits Cox1, Cox2,
and Cox3, cytochrome b of the cytochrome bc1complex, F1Fo-
ATP synthase subunits Atp6, Atp8, and Atp9, and the small
ribosomal subunit component Var1. With the exception of Var1,
these mitochondrially encoded proteins are integral membrane
proteins which become inserted into the inner membrane dur-
ing their synthesis on mitochondrial ribosomes tethered to the
inner membrane (11, 19, 29, 32, 34). The cotranslational mem-
brane insertion of these proteins is achieved by maintaining a
close physical association of the ribosomes to the inner mem-
brane at sites where the insertion machinery exists (19, 31, 32).
Oxa1 is an inner membrane protein that forms a central
component of the insertion machinery, whose presence is re-
quired for the cotranslational membrane insertion of the mi-
tochondrially encoded proteins (4–6, 15–17). The Oxa1 protein
has been shown to physically associate with the ribosomes and
more specifically with the large ribosomal subunit. Matrix-
exposed elements of the Oxa1 protein, such as its hydrophilic
C-terminal tail, support this Oxa1-ribosome interaction (19,
32). Furthermore, in intact mitochondria we have previously
demonstrated that Oxa1 can be chemically cross-linked to Mrp20,
a component of the large ribosomal subunit (19). Mrp20 is ho-
mologous to the bacterial ribosomal protein L23, a component
known from the structural analysis of the ribosomes to be located
next to the polypeptide exit site of the large ribosomal subunit (3,
10, 23, 27, 30). Thus, it was concluded that Oxa1, the site of
membrane insertion into the inner membrane, exists in close
physical proximity to the large ribosomal subunit and specifi-
cally to that region of the ribosomes where the nascent chain
emerges. This close physical relationship between ribosomal
components and the Oxa1 insertion site has been proposed to
support a tight coordination between the protein translation
and membrane insertion events (19, 31, 32). Given the strong
hydrophobicity of the OXPHOS complex subunits which are
encoded by the mitochondrial DNA and synthesized by these
ribosomes, a close coupling of the translation and insertion
events is proposed to ensure that the hydrophobic nascent
chains are directly inserted into the membrane during their
synthesis. The exposure of hydrophobic nascent chains to the
hydrophilic matrix space may promote their aggregation and
thus incompetency for subsequence membrane insertion.
In bacteria, the L23 protein has been implicated to play a
direct role in the cotranslational insertion of proteins into the
membrane (7, 13, 24, 33). Thus, it is possible that proteins
adjacent to the polypeptide exit site of mitochondrial ribo-
somes may be directly involved in targeting ribosomes to spe-
cific regions of the inner membrane where the membrane
* Corresponding author. Mailing address: Department of Biological
Sciences, Marquette University, 530 N. 15th Street, Milwaukee, WI
53233. Phone: (414) 288-1472. Fax: (414) 288-7357. E-mail: rosemary
† L.J. and J.K. contributed equally to this study.
‡ Present address: Department of Cell Biology and Physiology,
Washington University School of Medicine, St. Louis, MO 63112.
?Published ahead of print on 25 September 2009.
insertion and subsequent assembly events occur. The mito-
chondrial ribosomes resemble their prokaryotic ancestors in
some respects, e.g., antibiotic sensitivity, but they differ in a
number of important ways (1, 12, 22, 30). In general, the
protein content of the mitochondrial ribosomes is greater than
their bacterial counterparts. This increase in protein content is
largely attributed to the fact that the mitochondrial ribosomal
proteins are larger in size than their bacterial homologs. Over
the course of evolution, many of the mitochondrial ribosomal
proteins have acquired novel extensions, new domains, in ad-
dition to their bacterial homology domains. These acquired
extensions not only include N-terminal (often cleavable) sig-
nals to target these proteins (nuclear encoded) to the mito-
chondria but also in many instances large C-terminal exten-
sions, which are unique to the mitochondrial ribosomal proteins
and have thus been termed “mitospecific domains” (12, 30).
Largely uncharacterized, the functional relevance of these var-
ious mitospecific domains of the ribosomal proteins remains
unknown. It is speculated that some (or all) of these mitospe-
cific domains serve to ensure that the ribosome becomes as-
sembled and is translationally active while bound to the inner
In the present study we sought to further characterize the
interaction of the mitochondrial ribosome with the Oxa1 pro-
tein. We show here that MrpL40, a large ribosomal subunit
component, is physically close to both the Mrp20 and Oxa1
proteins, demonstrating the proximity of MrpL40 to both the
ribosomal polypeptide exit site and the Oxa1 membrane inser-
tion site. MrpL40 contains a large C-terminal mitospecific do-
main, which includes a predicted ?-helical region at its extreme
C-terminal end. The results presented here highlight that the
integrity of this domain of MrpL40 is crucial to ensure ribo-
some translational fidelity and subsequent OXPHOS complex
MATERIALS AND METHODS
Yeast strains. Unless otherwise stated, all strains used in this study were rho?.
Yeast strains used in this study were wild-type W303-1A (Mata, leu2 trp1 ura3
his3 ade2), rho0W303-1A (Mata, leu2 trp1 ura3 his3 ade2) (16), and the oxa1 null
mutant, ?oxa1 overexpressing the Oxa1Hisor nontagged Oxa1 protein (W303-1A
leu2 trp1 ura3 ade2 OXA1::HIS3 Yip351-GAL10-OXA1[?/?]His12-LEU2) (19).
The galactose-induced overexpression of Oxa1Hisin this manner does not per-
turb a wild-type cell’s ability to grow aerobically (results not shown). Construc-
tion of the strain expressing the C-terminal hemagglutinin (HA)-tagged MrpL40
protein was performed by homologous recombination at the MRPL40 gene locus
of wild-type cells, resulting in the introduction of a DNA sequence encoding one
HA epitope prior to the translational stop codon of the MRPL40 open reading
frame (ORF), followed by a new stop codon and the HIS3 auxotrophic gene.
Correct tagging of the MRPL40 gene in this manner and the insertion of the
HIS3 gene at the 3?end of the MRPL40 ORF were verified by PCR analysis of
the MRPL40 genomic region. Expression of the HA-tagged MrpL40 protein in
the resulting strain (MrpL40HA) was verified by sodium dodecyl sulfate-poly-
acrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis of the
mitochondria isolated from the MrpL40HAstrain and using an HA-specific
antiserum. The Oxa1Hisprotein was expressed in this strain by transforming the
MrpL40HAcells with the Yip351GAL10-OXA1Hisplasmid (19) and selecting for
LEU?transformants. Galactose-dependent Oxa1Hisexpression was verified in
the resulting strain (MrpL40HA?Oxa1His) by using Oxa1- and His-specific anti-
sera. The mrpL40?C strain was generated through homologous recombination in
the W303-1A wild-type yeast strain. A premature translational stop codon fol-
lowed by the HIS3 auxotrophic marker gene, resulting in a partial deletion (the
final 28 codons) on the 3? end of the MRPL40 open reading frame, was per-
formed essentially as previously described (35). Correct homologous recombi-
nation of the HIS3 gene at the MRPL40 gene locus was verified by PCR analysis
(results not shown).
Cross-linking assays. Mitochondria (200 ?g total protein) were suspended in
600 ?l SH buffer (0.6 M sorbitol, 20 mM HEPES-KOH, pH 7.2), and cross-
linking was performed with disuccinimidyl glutarate (DSG; 0.3 mM) or maleim-
idobenzoyl-N-hydroxysuccinimide (MBS; 0.5 mM), as indicated for 30 min on
ice. MBS cross-linking was performed in the presence of 3 mM NADH. Both
cross-linking reagents were dissolved in dimethyl sulfoxide, so the mock-treated
samples received dimethyl sulfoxide alone. Excess cross-linker was quenched by
adding glycine (80 mM, pH 8.0). Mitochondria were reisolated by centrifugation
and washed with SH buffer prior to further analysis. For direct analysis of the
proteins and cross-linked adducts the mitochondria were solubilized in the SDS-
sample buffer and analyzed by SDS-PAGE and Western blotting.
Ni-NTA purification of Oxa1Hisand cross-linked adducts. For the Ni-nitrilo-
triacetic acid (NTA) purification of Oxa1Hisand cross-linked adducts, mitochon-
dria (200 ?g total protein) were solubilized in 200 ?l TNT buffer (1% Triton
X-100, 300 mM NaCl, 60 mM Tris-HCl, pH 7.4) for 30 min on ice. Where
indicated, mitochondria were initially solubilized in 0.1% SDS prior to the
addition of the TNT buffer. After a clarifying spin (20,860 ? g; 15 min at 4°C),
the supernatants were incubated for 1 h at 4°C with the Ni-NTA beads (equili-
brated in the TNT buffer containing 30 mM imidazole). The beads were washed
three times with TNT-imidazole buffer, and bound proteins were eluted with
SDS-sample buffer containing 5% (vol/vol) ?-mercaptoethanol and 0.5 M im-
Mrp20 and cross-linked adduct immunoprecipitation. For the immunopre-
cipitation of Mrp20 and its cross-linked adducts, mitochondria (200 ?g total
protein) following cross-linking were solubilized in SDS (1%) buffer and cooked
(5 min at 95°C), followed by dilution into immunoprecipitation (IP) buffer (1%
Triton X-100, 300 mM NaCl, 10 mM Tris-HCl, pH 7.4) for 30 min on ice. After
a clarifying spin (20,860 ? g; 15 min at 4°C), the supernatants were incubated
overnight at 4°C with protein A-Sepharose beads and 30 ?l of culture superna-
tant containing the Mrp20 monoclonal antibody. The beads were washed three
times with IP buffer and twice with IP buffer without Triton X-100. Bound
proteins were eluted with SDS-sample buffer containing 5% (vol/vol) ?-mercap-
toethanol. The immunoprecipitated Mrp20 and cross-linked adducts were ana-
lyzed by SDS-PAGE, Western blotting, and immunodecoration with MrpL40
MrpL40 antiserum generation. The region of the MRPL40 ORF encompass-
ing codons 1 to 284 was amplified by PCR and cloned in frame with an N-
terminal His tag into the pET-28a(?) vector. The resulting His-tagged protein
(35 kDa) was expressed in an isopropyl-?-D-thiogalactopyranoside-inducible
manner in Escherichia coli. Following sonication to disrupt the bacterial cells, the
soluble His-tagged MrpL40(1-284) was purified by Ni-NTA chromatography and
subjected to thrombin cleavage to remove the His tag. The resulting MrpL40(1-
284) 33-kDa fragment was injected into rabbits to generate a polyclonal anti-
Triton X-100 solubilization of mitochondria and sucrose gradient centrifuga-
tion. Sucrose gradient analysis of detergent-solubilized mitochondrial ribosomes
was performed essentially as previously published (36, 37). Mitochondria (300 ?g
protein) were solubilized with 300 ?l of lysis buffer (0.5% Triton X-100, 10 mM
Mg-acetate, 0.1 M NaCl, 20 mM HEPES-KOH, pH 7.4, 1 mM phenylmethyl-
sulfonyl fluoride) for 30 min on ice. The lysate was clarified by centrifugation at
30,000 ? g for 30 min at 4°C, and the supernatant was layered onto an 11-ml
continuous sucrose gradient (15 to 30%) containing 500 mM NH4Cl, 10 mM
Tris-HCl, pH 7.4, 10 mM Mg-acetate, 7 mM ?-mercaptoethanol, and 0.5 mM
phenylmethylsulfonyl fluoride. Gradients were centrifuged at 20,500 rpm for 17 h
at 4°C in a Beckman SW41 Ti rotor. Fractions (750 ?l) were collected, trichlo-
roacetic acid precipitated, and subjected to SDS-PAGE and Western blot anal-
Antisera used in this study. Antisera against Cox2, Su e, MrpL40 were gen-
erated as described above and as published previously (2, 18). The following
antisera were generously obtained from the following sources: MrpL36 (J. M.
Herrmann, University of Kaiserlautern), MrpL32 (T. Langer, University of Co-
logne), Mrp47 (M. Boguta, Polish Academy of Sciences, Warsaw, Poland),
Mrp10 (A. Tzagoloff, Columbia University, New York, NY), Mrp20 monoclonal
(T. Mason, University of Massachusetts, Amherst), Tim17 (W. Neupert, Uni-
versity of Munich), Atp6 (J. Velours, Bordeaux, France), and the F1sector (D.
Mueller, Rosalind Franklin Medical School, Chicago, IL).
Miscellaneous. Mitochondria were isolated from cultures grown at 30°C in
yeast extract-peptone (YP)–0.5% lactate, 2% galactose medium. Isolation of
mitochondria and in organello labeling with [35S]methionine were performed
essentially as described earlier (17, 19). Standard procedures were used for
SDS-PAGE, Blue native-PAGE, and Western blotting (20). The enzyme activity
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