MrpL36p, a Highly Diverged L31 Ribosomal Protein Homolog With Additional Functional Domains in Saccharomyces cerevisiae Mitochondria

Corresponding author: Thomas D. Fox, 335 Biotechnology Bldg., Cornell University, Ithaca, NY 14853-2703., tdf1{at}cornell.edu (E-mail)

Communicating editor: F. WINSTON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Translation in mitochondria utilizes a large complement of ribosomal proteins. Many mitochondrial ribosomal components are clearly homologous to eubacterial ribosomal proteins, but others appear unique to the mitochondrial system. A handful of mitochondrial ribosomal proteins appear to be eubacterial in origin but to have evolved additional functional domains. MrpL36p is an essential mitochondrial ribosomal large-subunit component in Saccharomyces cerevisiae. Increased dosage of MRPL36 also has been shown to suppress certain types of translation defects encoded within the mitochondrial COX2 mRNA. A central domain of MrpL36p that is similar to eubacterial ribosomal large-subunit protein L31 is sufficient for general mitochondrial translation but not suppression, and proteins bearing this domain sediment with the ribosomal large subunit in sucrose gradients. In contrast, proteins lacking the L31 domain, but retaining a novel N-terminal sequence and a C-terminal sequence with weak similarity to the Escherichia coli signal recognition particle component Ffh, are sufficient for dosage suppression and do not sediment with the large subunit of the ribosome. Interestingly, the activity of MrpL36p as a dosage suppressor exhibits gene and allele specificity. We propose that MrpL36p represents a highly diverged L31 homolog with derived domains functioning in mRNA selection in yeast mitochondria.

MITOCHONDRIA contain a genome that is expressed using mitochondria-specific transcription and translation systems. In the budding yeast Saccharomyces cerevisiae, the mitochondrial genome encodes tRNAs, rRNAs, and eight major polypeptides—seven integral membrane proteins of the oxidative phosphorylation system and one protein of the mitochondrial ribosomal small subunit (DUJON 1981 ; GRIVELL 1987 ). All the remaining mitochondrial ribosomal proteins are encoded in the nuclear genome. Some of these are clear homologs of their bacterial ancestors, but many show no sequence similarity to ribosomal proteins in other systems (GRAACK and WITTMANN-LIEBOLD 1998 ; SAVEANU et al. 2001 ; GAN et al. 2002 ). Interestingly, several yeast mitochondrial ribosomal proteins are composed of domains that are homologous to bacterial ribosomal proteins fused to domains that are not (FEARON and MASON 1988 ; GRAACK and WITTMANN-LIEBOLD 1998 ). In addition to their roles in maintaining ribosome structure and promoting the core reactions of protein synthesis, ribosomal proteins can play more direct roles in recognizing mitochondrial mRNAs. For example, at least two proteins of the ribosomal small subunit in yeast mitochondria interact genetically with the 5'-untranslated leaders (5'-UTLs) of both the COX2 and the COX3 mRNAs (GREEN-WILLMS et al. 1998 ).

We recently found that elevated dosage of the nuclear gene encoding the mitochondrial ribosomal large-subunit protein MrpL36p (MRPL36) could suppress translation defects caused by specific mutations within the mitochondrially coded COX2 mRNA (BONNEFOY et al. 2001 ), encoding subunit II (Cox2p) of the cytochrome c oxidase complex. Cox2p is synthesized as a precursor protein with a 15-residue N-terminal leader peptide (SEVARINO and POYTON 1980 ; PRATJE et al. 1983 ). The COX2 leader peptide coding sequence encodes a positive-acting sequence element that is necessary to antagonize several inhibitory sequence elements within the downstream COX2 coding sequence (BONNEFOY et al. 2001 ; WILLIAMS and FOX 2003 ). Small deletions within the leader peptide coding sequence reduce translation dramatically, but this defect can be partially suppressed by overexpression of MRPL36 or the COX2-specific translational activator PET111 (BONNEFOY et al. 2001 ).

MRPL36 encodes a mitochondrial ribosomal large-subunit protein (GROHMANN et al. 1991 ; KITAKAWA et al. 1997 ) that is essential for mitochondrial DNA (mtDNA) maintenance and, presumably, mitochondrial translation (BONNEFOY et al. 2001 ). MrpL36p exhibits similarity to two translation-associated factors from other systems. First, an internal domain of the protein is similar and apparently orthologous to the L31 family of large-subunit ribosomal proteins conserved across eubacteria (LECOMPTE et al. 2002 ). L31 homologs in Escherichia coli and Deinococcus radiodurans have been localized to the interface of the large and small subunits of the ribosome (HARMS et al. 2001 ; VILA-SANJURJO et al. 2003 ), but little is known about the role L31 plays in translation. Second, a partially overlapping, C-terminal region of MrpL36p shows some sequence similarity to a portion of E. coli Ffh, a component of the signal recognition particle (KEENAN et al. 2001 ).

In this study we sought to investigate the functional significance of the L31- and Ffh-like domains of MrpL36p in general mitochondrial translation and in suppression of translation defects within the COX2 mRNA. Our results shed light on the puzzling observation that a gene for an essential ribosomal protein functions as a multicopy suppressor.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Yeast strains, media, and genetic methods:
S. cerevisiae strains relevant to this study are listed in Table 1. As indicated, strains are isogenic or congenic to D273-10B (ATCC no. 25627). Yeast were cultured in either complete media (1% yeast extract, 2% Bacto-peptone, 50 mg adenine/liter) or synthetic complete media (0.67% yeast nitrogen base supplemented with appropriate amino acids) containing 2% glucose, 2% galactose, or 3% ethanol/3% glycerol. Standard genetic techniques were performed as described (SHERMAN et al. 1974 ; FOX et al. 1991 ; GUTHRIE and FINK 1991 ).

Western analysis:
Total cellular protein was isolated as described previously (YAFFE 1991 ) from cells grown to mid- to late-log phase in synthetic complete galactose medium lacking leucine. Proteins were detected using the following antisera: 1:1000 mouse monoclonal anti-MYC (9E10 epitope; Roche Applied Science), 1:100 mouse monoclonal anti-Mrp7p (FEARON and MASON 1988 ), 1:100 mouse monoclonal anti-Mrp13p (PARTALEDIS and MASON 1988 ), 1:50 mouse monoclonal anti-Cox2p (PINKHAM et al. 1994 ), and 1:12,000 rabbit polyclonal anti-glucose-6-phosphate dehydrogenase (G6PDH; Sigma-Aldrich). Secondary HRP-conjugated antisera [1:5000 (Bio-Rad, Richmond, CA) or 1:10,000 (Sigma-Aldrich) goat anti-mouse IgG or 1:10,000 goat anti-rabbit IgG (Invitrogen, San Diego)] were detected using the ECL or ECL Plus chemiluminescent detection kits (Amersham Pharmacia Biotech).

Mitochondrial preparation and ribosomal sedimentation analyses:
Mitochondrial isolation was performed as described previously (DAUM et al. 1982 ; GLICK and PON 1995 ). For ribosomal sedimentation analyses, ribosomes were extracted from 2 mg of mitochondria by solubilization in 1 ml of 10 mM Mg acetate/0.1 M NaCl/20 mM HEPES-KOH, pH 7.4/1 mM phenylmethylsulfonyl fluoride (PMSF)/0.5% Triton X-100 and incubation on ice for 30 min. Insoluble debris was pelleted by centrifugation at 34,000 rpm (40,000 x g) at 4° for 20 min in a TLA100.3 ultracentrifuge rotor. The supernatant was loaded on a 36-ml continuous 15–30% (w/v) sucrose gradient with 500 mM NH₄Cl/10 mM Tris, pH 7.4/10 mM Mg acetate/7 mM ß-mercaptoethanol/0.5 mM PMSF/two miniprotease inhibitor tabs without EDTA per 40 ml (Roche Applied Science). Gradients were centrifuged for 17 hr at 20,000 rpm (72,000 x g) at 4° in a Beckman SW28 rotor, and 1-ml fractions were collected. Every other fraction was trichloroacetic acid precipitated, and 40% of each precipitate was analyzed by SDS-PAGE and Western blotting.

Phenotypic analyses:
To analyze suppression of mitochondrial mutations, appropriate strains were transformed with 2µ plasmids containing MRPL36/mrpL36 alleles or PET111-20 (pJM57; MULERO and FOX 1993A ). Transformants were patched to synthetic complete media lacking leucine (–L) or uracil (–U). Patches were printed to nonfermentable complete medium containing ethanol and glycerol (YPAEG) or to nonfermentable synthetic complete media containing ethanol and glycerol but lacking leucine [–L(EG)] or uracil [–U(EG)] to assay respiratory growth or to glucose synthetic complete medium lacking arginine and uracil (–RU) to assay arginine prototrophy.

The ability of plasmid-borne mrpL36/MRPL36 alleles to complement a chromosomal deletion was assayed in Leu⁺, Ura⁺ meiotic progeny of diploids formed by mating EHW346 (MRPL36::3xMYC, rho⁺), transformed with each 2µ, LEU2 plasmid to be tested, with NB156-5D (mrpL36::URA3, rho^–/rho°). Haploid progeny were tested for growth on –L(EG). Maintenance of mtDNA was assayed by mating haploids to a wild-type (MRPL36) rho° strain and assaying respiratory growth of the resulting diploids on nonfermentable complete medium (YPAEG).

Epitope tagging of MrpL36p:
A tagging cassette fusing 3xMYC::URA3::3xMYC to the last codon of MRPL36 was generated by PCR with Taq DNA polymerase (Invitrogen) using plasmid pMPY-3xMYC (SCHNEIDER et al. 1995 ) as template. This cassette was integrated into a wild-type MRPL36 strain, and the duplicated tag and URA3 marker were excised by selection on medium containing 5-fluoroorotic acid. A proper popout (EHW346) was confirmed by PCR and sequencing of the MRPL36-tag junction, and expression of a protein of approximately the anticipated size for mature MrpL36p-MYC was confirmed by Western blot analysis using anti-MYC antisera (data not shown).

Plasmid construction:
All mrpL36/MRPL36 plasmids are derived from a 1.2-kb SacI-XhoI genomic fragment from pNB107 (BONNEFOY et al. 2001 ) that contains wild-type MRPL36 expressed from its endogenous promoter. pEHW191 was generated by cutting pNB107 with EcoRI, blunting with T4 DNA polymerase (Invitrogen), and cutting with HindIII; the MRPL36-containing fragment then was subcloned into the SmaI-HindIII sites of YEp351 (HILL et al. 1986 ). pEHW188 was generated by replacing the MscI-BglII fragment encompassing the C-terminal coding sequence for MRPL36 in pNB107 with an MRPL36::3xMYC MscI-BglII fragment amplified by PCR using Taq DNA polymerase (Invitrogen) and template DNA from strain EHW346. pEHW192 contains the EcoRI-T4 DNA polymerase-HindIII MRPL36::3xMYC fragment from pEHW188 subcloned into the SmaI-HindIII sites of YEp351. All other plasmids contain mrpL36::3xMYC alleles expressed from the same genomic fragment as pEHW188. These plasmids were generated by fusion PCRs (HO et al. 1989 ) using Taq (Invitrogen), Pfu, or Pfu Turbo (Stratagene, La Jolla, CA) DNA polymerase and pEHW188 as template. Upstream and downstream primer pairs contained introduced EcoRI and HindIII restriction sites, respectively, allowing the PCR products generated to be cut with EcoRI, blunted with T4 DNA polymerase, cut with HindIII, and subcloned into the SmaI and HindIII sites of YEp351. To ensure proper localization, the first 25 residues of Cox4p, which encode a mitochondrial matrix targeting sequence (PINKHAM et al. 1994 ), were fused to the N terminus of mrpL36 alleles from which the endogenous N-terminal targeting sequence had been deleted. All PCR-generated regions were sequenced to confirm the absence of mutations. Further plasmid details are available upon request.

The mrpL36 alleles were designed using the original start codon assignment by the Saccharomyces Genome Database (http://www.yeastgenome.org; GOFFEAU et al. 1996 ). Recently, this assignment was revised on the basis of phylogenetic data (CLIFTEN et al. 2003 ; KELLIS et al. 2003 ), moving the predicted start 57 nucleotides downstream (and shortening the predicted pre-MrpL36p leader peptide to 14 residues). Due to this annotation change, the cox4(1-25) fusion alleles also encode an N-terminal extension of 19 residues not present in wild-type MrpL36p. Residue numbers cited to describe MRPL36 alleles are based on the new ATG annotation.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Wild-type MrpL36p and several deleted forms of the protein accumulate when overexpressed:
To facilitate detection of MrpL36p, we tagged the chromosomal copy of MRPL36 with a sequence encoding three tandem MYC epitopes. MrpL36p-MYC is fully functional at the level of mtDNA maintenance and respiratory growth (data not shown). We performed sucrose gradient sedimentation of detergent-solubilized mitochondria from a strain expressing MrpL36p-MYC and verified cosedimentation of MrpL36p-MYC with the large-subunit component Mrp7p (FEARON and MASON 1988 ) and distinct from the small-subunit component Mrp13p (PARTALEDIS and MASON 1988 ; Fig 1).

View larger version (26K): In this window In a new window Download PPT slide   Figure 1. MrpL36p-MYC cosedimentation with the mitochondrial ribosomal large subunit. Ribosomes were extracted from crude mitochondria from strain EHW346 (expressing MrpL36p-MYC) using Triton X-100 and sedimented through a continuous 15–30% sucrose gradient containing 0.5 M NH4Cl and 10 mM MgCl2. Gradient fractions were collected, precipitated, and analyzed by Western blotting with anti-Myc antibody. The small-subunit protein Mrp13p and the large-subunit protein Mrp7p were detected using protein-specific monoclonal antibodies (FEARON and MASON 1988 ; PARTALEDIS and MASON 1988 ). Ext, Triton extraction; P, insoluble pellet; SN, Triton-extractable supernatant; Top and Bottom denote the orientation of fractions in the gradient.

Overexpression of MRPL36 improves respiratory growth in strains containing mutations within the pre-Cox2p leader peptide coding sequence. One suppressible mutation, cox2-22, has an in-frame deletion of codons 7–10 and the synonymous alteration of codon 6 from AGA to CGT (BONNEFOY et al. 2001 ). We have verified that this suppression is reflected in a slight increase in the steady-state level of Cox2p in cox2-22 mutant cells without a significant increase in the level of cox2-22 mRNA (our unpublished data). MRPL36 dosage suppression also is observed in the context of cox2-22::ARG8^m (BONNEFOY et al. 2001 ), where ARG8^m is a reliable reporter of translational efficiency from the COX2 locus (BONNEFOY and FOX 2000 ; BONNEFOY et al. 2001 ; WILLIAMS and FOX 2003 ). These data suggest that, when overexpressed, MrpL36p improves translation of mutant COX2 mRNAs. In an attempt to map the MrpL36p domains necessary for its functions as a core ribosomal component and as a dosage suppressor of cox2-22, we created several partially deleted forms of MrpL36p, overexpressed them in yeast, and examined the resulting phenotypes.

MrpL36p can be divided into three regions on the basis of similarity to known proteins. The N-terminal 41 residues of MrpL36p show no apparent sequence similarity to known proteins. An internal domain from residue 42 to 104 aligns with the consensus sequence for the L31 family of large-subunit ribosomal proteins conserved across eubacteria and in some unicellular eukaryotes and plants (Fig 2A; TATUSOV et al. 1997 , TATUSOV et al. 2001 ). A partially overlapping region extending from residue 87 to the C terminus at residue 177 shows weak similarity with residues 218–327 of E. coli Ffh, a component of the signal recognition particle (Fig 2A). This region of Ffh spans the C-terminal portion of the GTPase domain and a flexible linker region (KEENAN et al. 2001 ; NAGAI et al. 2003 ). However, this region of MrpL36p lacks the residues known to be critical for GTPase activity or nucleotide binding.

View larger version (27K): In this window In a new window Download PPT slide   Figure 2. Summary of sequence similarity and functional domains of MrpL36p. (A) A schematic of wild-type MrpL36p is shown at the top. A central domain (residues 42–104) aligns with the consensus sequence for homologs of the eubacterial ribosomal large-subunit protein L31 (left; TATUSOV et al. 1997 , TATUSOV et al. 2001 ), while a partially overlapping C-terminal domain (residues 87–177) shows weak similarity with residues 218–327 of E. coli Ffh (right; BONNEFOY et al. 2001 ). Solid circles, identical residues; medium-shaded circles, highly similar residues; lightly shaded circles, weakly similar small, polar residues or similar hydrophobic residues. (B) Separation of function alleles of MRPL36 demonstrates that the internal domain of MrpL36p that partially aligns with the L31 consensus sequence is sufficient for mitochondrial translation and mtDNA maintenance (top), while the N and C termini are sufficient for dosage suppression of cox2-22 (bottom).

We created various partially deleted polypeptides to characterize four regions—residues 2–35, 36–86, 87–118, and 119–177—that are roughly based on this analysis of the MrpL36p sequence. MrpL36p residue 36 is two positions downstream of the L31 consensus N terminus while 118 is 9 residues downstream of the L31 consensus C terminus, so that the region between them corresponds to the entire bacterial L31. For alleles that deleted the N terminus of MrpL36p, 25 codons specifying the pre-Cox4p mitochondrial targeting sequence (PINKHAM et al. 1994 ) were fused to the remaining MRPL36 sequence. Due to a change in the annotation of the initiation codon during the course of this study by the Saccharomyces Genome Database (http://www.yeastgenome.org; CLIFTEN et al. 2003 ; KELLIS et al. 2003 ), constructs containing the COX4 mitochondrial targeting signal encode proteins with an N-terminal extension of 19 residues. However, the new ATG assignment does not alter the predicted mature polypeptides encoded by each allele.

Each of the alleles was subcloned into a high-copy (2µ) vector and expressed from the MRPL36 promoter. Most of these proteins bear a C-terminal 3xMYC epitope tag. Most transformants expressing the plasmid-borne alleles contained proteins that were readily detectable in whole-cell extracts using antisera directed against the MYC epitope (Fig 3A, top). Longer exposures of the Western blot were necessary to detect wild-type MrpL36p expressed from the chromosomal MRPL36::3xMYC allele present in these same strains (Fig 3A, wild-type MrpL36p-MYC), demonstrating significant overaccumulation of many of these plasmid-borne alleles relative to the chromosomally expressed allele. A weak band likely corresponding to cox4(1-25)::mrpL36(2-35, 119-177)::3xMYC also was detectable in longer exposures (data not shown), but no immunoreactive species corresponding to mrpL36(36-177)::3xMYC or cox4(1-25)::mrpL36(2-35, 87-177)::3xMYC were ever detected. For unknown reasons the Cox4p(1-25)-MrpL36p fusion protein, which contains an N-terminal extension of 19 residues due to fusion of the Cox4p residues to the formerly assigned initiation codon, is not processed efficiently to mature size (Fig 3A) despite the presence of the pre-MrpL36p processing site. Also for unknown reasons, mrpL36(36-86) accumulates as two immunoreactive species, the larger of which migrates near the anticipated molecular weight (Fig 3A). The smaller protein is likely a degradation product.

View larger version (28K): In this window In a new window Download PPT slide   Figure 3. Steady-state levels and functional activities of partially deleted forms of MrpL36p-MYC. (A) Whole-cell extracts were prepared from strain EHW419 (MRPL36::3xMYC, cox2-22) transformed with 2µ plasmids expressing the indicated forms of MrpL36p-MYC and grown to mid- to late-log phase in synthetic complete galactose medium lacking leucine. Each lane contains 50 µg of protein analyzed on a 15% SDS-PAGE gel and subjected to immunoblotting for MYC-tagged MrpL36p alleles and glucose-6-phosphate dehydrogenase (G6PDH) as a loading control. Dash denotes empty vector only; C4, Cox4p(1-25) N-terminal fusions; other alleles are mrpL36::3xMYC alleles deleted for the denoted MRPL36 residues. "Wild-type MrpL36p-MYC" represents a longer exposure of the same blot to show the level of MrpL36p-MYC expressed from the chromosomal MRPL36::3xMYC allele. For the transformant expressing plasmid-borne MRPL36::3xMYC (WT, under the MYC heading), this signal reflects expression of both the chromosomal and the plasmid-borne alleles; for the transformant expressing plasmid-borne cox4(1-25)::MRPL36 (C4::WT), accumulation reflects expression of the chromosomal MRPL36::3xMYC allele and accumulation of any MYC-tagged cleavage products of the fusion protein that comigrate with wild-type MrpL36p-MYC. The smaller immunoreactive species detectable for mrpL36(36-86)::3xMYC (36-86) likely is a degradation product. (B) Summary of the functional activities for the MrpL36p derivatives shown in A. Suppression refers to restoration of respiratory growth to a cox2-22 mutant in the presence of the designated 2µ mrpL36/MRPL36 construct and a wild-type chromosomal gene. Complementation refers to respiratory growth (Resp) and maintenance of mtDNA (rho+) in the presence of a chromosomal mrpL36 mutation. Columns are aligned to the allele labels in A. +++, growth observed when 2µ untagged wild-type MRPL36 is present.

It is noteworthy that wild-type MrpL36p and many of the partially deleted proteins overaccumulate under overexpression conditions, rather than being maintained at a steady-state level consistent with the level of chromosomally encoded MrpL36p. This is in contrast to what has been observed for several other yeast mitochondrial and cytoplasmic ribosomal components (reviewed in GRAACK and WITTMANN-LIEBOLD 1998 ; WARNER 1989 ). Interestingly, the steady-state level of chromosomally expressed MrpL36p-MYC decreased when untagged wild-type MRPL36 was overexpressed, suggesting that protein turnover nevertheless does limit the total amount of protein that can accumulate in the matrix (Fig 3A, wild-type MrpL36p-MYC). Overexpression of mrpL36(87-177)::3xMYC may similarly trigger a dosage control mechanism (Fig 3A, wild-type MrpL36p-MYC). Stable accumulation of many of these altered proteins allowed us to assay their activities in vivo as core ribosomal components and as dosage suppressors of cox2-22. Unless otherwise noted, all results presented hereafter refer to these MYC-tagged alleles.

The essential ribosomal function of MrpL36p requires only the domain of the protein similar to the L31 family:
We assayed the ability of the plasmid-borne mrpL36 alleles to complement a chromosomal deletion (mrpL36::URA3) in strains carrying wild-type mtDNA, to test their ability to function in translation. Both wild-type MRPL36 and the wild-type gene preceded by codons for the pre-Cox4p mitochondrial targeting sequence, cox4(1-25)::MRPL36, fully complemented the deletion at the level of growth on nonfermentable carbon sources and mtDNA maintenance (Fig 3B). The cox4(1-25)::mrpL36(2-35), mrpL36(87-177), and cox4(1-25)::mrpL36(2-35, 119-177) gene products also were capable of partially complementing the chromosomal deletion (Fig 3B and Fig 4A). Some colonies expressing mrpL36(87-177) were able to respire efficiently, while others contained intact rho⁺ mtDNA, as judged by their ability to yield respiring diploids when mated to a rho° tester strain, but were not able to respire. This suggests that mrpL36(87-177) in some cells supports sufficient translation to ensure mtDNA maintenance, but not enough to allow respiratory growth. The observed clonal differences among cells expressing mrpL36(87-177) could be due to differences in plasmid copy number.

View larger version (60K): In this window In a new window Download PPT slide   Figure 4. Phenotypes of MRPL36 alleles. (A) Complementation of mrpL36 by plasmid-borne alleles of MRPL36. Haploid meiotic progeny with chromosomal MRPL36::3xMYC or mrpL36::URA3 (WT and , respectively) containing either a 2µ plasmid bearing cox4(1-25)::mrpL36(2-35, 119-177) or an empty vector (dash; YEp351; MATERIALS AND METHODS) were streaked to synthetic complete glucose medium lacking leucine (–L), printed to –L and to nonfermentable synthetic complete medium lacking leucine [–L(EG)], and incubated for 1 day at 30°. (B) Suppression of cox2-22 by plasmid-borne alleles of MRPL36. Transformants of the cox2-22 mutant EHW419 with the indicated 2µ mrpL36/MRPL36 constructs were patched to synthetic complete glucose medium lacking leucine (–L) and then printed to –L and to complete medium containing ethanol and glycerol (YPAEG) and incubated at 30° for 1 and 3 days, respectively. Whole-cell extracts were prepared from transformants of strain NB64 grown to mid- to late-log phase in synthetic complete glucose medium lacking leucine, and 100 µg was analyzed for each strain on a 12% SDS-PAGE gel, followed by immunoblotting. (Phenotypes for EHW419 transformants are indistinguishable from those of strain NB64.) The blot was probed for Cox2p and for glucose-6-phosphate dehydrogenase (G6PDH) as a loading control. Allele abbreviations are described in Fig 3.

Taken together, these data suggest that internal residues 36–118 are sufficient for translation, while both the N- and the C-terminal regions are dispensable for ribosomal function (Fig 2B). MrpL36p residue 36 corresponds approximately to the second position downstream of the L31 consensus N terminus, while MrpL36p position 118 would be 9 residues beyond the L31 consensus C terminus (Fig 2A). Thus the double deletion cox4(1-25)::mrpL36(2-35, 119-177) produces a protein that corresponds to the entire L31. MrpL36p residue 86 corresponds approximately to residue 48 of the 67-residue consensus L31 sequence. Thus mrpL36(87-177) would produce a protein lacking most of the C-terminal residues of the globular domain but with the ß-sheet structure of L31 (HARMS et al. 2001 ) intact, if MrpL36 has a similar structure. Thus our data on the ability of deleted forms of MrpL36p to function in translation support the idea that this domain of the protein is in fact orthologous to its L31 eubacterial counterparts.

Variants of MrpL36p lacking the L31-like domain function as dosage suppressors in the presence of wild-type MrpL36p:
We also tested the plasmid-borne mrpL36 alleles for activity as dosage suppressors of the respiratory defect of a cox2-22 strain. We found that two partial deletion alleles, mrpL36(36-86) and mrpL36(36-118), were stronger suppressors of cox2-22 than wild-type MRPL36 in terms of both respiratory growth and restoration of Cox2p accumulation (Fig 4B). Thus, the L31-like domain is not required for dosage-dependent suppression.

These variants suggest that the Ffh-like region may play a role in suppression. The first 13 residues of the Ffh-like region, missing in the product of mrpL36(36-118), are not required for suppression, but both suppressing forms contain the C-terminal portion of the aligned domain. The C-terminal region of MrpL36p appears to be necessary for suppressor activity, since the mrpL36(87-177) allele failed to suppress cox2-22 (Fig 4B) despite the fact that its product can accumulate in the matrix (Fig 3A). However, we cannot rule out that this variant fails to suppress because it is less abundant than the other suppressing forms and because it induces degradation of the chromosomally expressed wild-type form coexpressed in the cell (Fig 3A). We attempted to determine whether the N-terminal region of MrpL36p was necessary for suppression by targeting proteins deleted for this region [i.e., cox4(1-25)::mrpL36(2-86)] to mitochondria using the pre-Cox4p targeting sequence. In no case did we observe suppression with such deletions (Fig 3B and Fig 4B). However, since the corresponding "wild-type" gene, cox4(1-25)::MRPL36, did not suppress when overexpressed (Fig 3B and Fig 4B), we are unable to draw conclusions about suppression by the other cox4(1-25) fusions. In any event, it is striking that those deletion alleles that complement a chromosomal deletion for translation in mitochondria containing wild-type mtDNA do not function as dosage suppressors of cox2-22 and vice versa. Thus, there appears to be a clear separation of these two functions of MrpL36p (Fig 2B and Fig 3B).

Translationally competent forms of MrpL36p differ in ribosomal association from those with suppressor activity:
We next investigated whether forms of MrpL36p capable of supporting translation differ from the suppressing forms of MrpL36p in their association with the large ribosomal subunit during sucrose gradient sedimentation. Detergent-solubilized mitochondrial extracts from strains expressing two of the translationally competent polypeptides were fractionated by sedimentation and analyzed by Western blot (Fig 5). MrpL36p(87-177) sedimented entirely with the large ribosomal subunit. Some Cox4p(1-25)-MrpL36p(2-35) was associated with the large subunit, while the remainder was in the upper fractions of the gradient (Fig 5). In contrast, the suppressing forms MrpL36p(36-86) and MrpL36p(36-118) exhibited no detectable ribosomal association; instead, these proteins accumulated entirely in the upper fractions of the gradient (Fig 5). Overexpression of wild-type MrpL36p resulted in both strong ribosomal association and accumulation at the top of the gradient. Therefore, translational activity of the MrpL36p variants clearly correlates with ribosomal association under these sedimentation conditions, while suppression correlates with the presence of slowly sedimenting MrpL36p. Whether the suppressing forms of MrpL36p are in fact dissociated from the ribosome in vivo or are simply more labile in this assay is not known.

View larger version (32K): In this window In a new window Download PPT slide   Figure 5. Sedimentation of variant forms of MrpL36p-Myc with the mitochondrial ribosomal large subunit. Mitochondrial ribosomes were prepared from strain EHW419 (MRPL36::3xMYC, cox2-22) transformed with 2µ plasmids expressing the MYC-tagged MRPL36/mrpL36 alleles shown and analyzed as described in Fig 1. A table summarizing the observed activities of the alleles in complementation of mrpL36::URA3 (Compl.) and in suppression of cox2-22 (Suppr.) is shown on the right. Fractions containing the large ribosomal subunit, as defined by the sedimentation of Mrp7p (data not shown), are marked (Large). Allele abbreviations are described in Fig 3.

Dosage suppression by MRPL36 exhibits gene and allele specificity:
We next investigated whether MRPL36 overexpression suppresses other mitochondrial mutations. We transformed the high-copy plasmid-borne, untagged MRPL36 gene into strains carrying numerous mitochondrial mutations and found that only a small subset of cox2 alleles could be detectably suppressed. In addition to suppression of cox2 mutations affecting the leader peptide coding region (BONNEFOY et al. 2001 ), overexpression of MRPL36 weakly suppressed two cox2 initiation codon mutations (Fig 6). Suppression was detectable for an AUG AUU mutation in the cox2(1-91)::ARG8^m reporter construct, which encodes a translational fusion of the first 91 residues of pre-Cox2p to the Arg8p reporter protein discussed above, and for an AUG AUA mutation in otherwise wild-type COX2. However, this suppression was not nearly as strong as that caused by overexpression of a gain-of-function allele of the COX2 mRNA-specific translational activator, PET111-20 (Fig 6; MULERO and FOX 1993A ; BONNEFOY and FOX 2000 ). Interestingly, overexpression of MRPL36 did not detectably improve translation of an AUG AUA mutation in the COX3 mRNA (Fig 6), suggesting that suppression could be specific for the COX2 mRNA. In addition, MRPL36 overexpression did not detectably suppress mutations in the COX2 5'-UTL (data not shown) that are suppressible by overexpression of PET111-20 (MULERO and FOX 1993A , MULERO and FOX 1993B ; DUNSTAN et al. 1997 ). Other classes of mitochondrially encoded alleles for which no suppression by MRPL36 overexpression was detectable include those with a missense or nonsense mutation in COX2 or with a cytochrome c oxidase assembly defect (data not shown). Therefore, dosage suppression by MRPL36 appears specific for cox2 alleles with translation defects due to mutation of the initiation codon or of the leader peptide coding sequence.

View larger version (55K): In this window In a new window Download PPT slide   Figure 6. Gene specificity in dosage suppression of initiation codon mutations by MRPL36. 2µ plasmids expressing untagged MRPL36 (pNB107; BONNEFOY et al. 2001 ) or the dominant allele PET111-20 (pJM57; MULERO and FOX 1993A ) of the COX2-specific translational activator Pet111p were transformed into strains containing a wild-type (AUG) or mutant (AUA or AUU) initiation codon in the context of COX2, cox2(1-91)::ARG8m, or COX3. For cox2(1-91)::ARG8m strains (top), transformants were replica plated to synthetic complete glucose media lacking uracil (–U) and lacking uracil and arginine (–RU) to select for plasmid retention and arginine prototrophy (–RU), respectively. For COX2 and COX3 strains (bottom), transformants were replica plated to synthetic complete medium containing ethanol and glycerol to select for respiratory growth [–U(EG)] and to synthetic complete medium lacking uracil (–U) to select for plasmid retention. Plates were incubated at 30° for 2 days for –U and –U(EG) and 4 days for –RU. Strains used, from top to bottom: NB43, NB110, NB134, NB80, NB60, NB164, DUL1, and LSF74.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

We have shown here that the yeast mitochondrial ribosomal large-subunit protein MrpL36p has at least two functional domains. The domain essential for large-subunit function is centrally located and is homologous to L31, a ribosomal protein strictly conserved in eubacteria (LECOMPTE et al. 2002 ). E. coli L31 is a 70-residue protein (BROSIUS 1978 ; EISTETTER et al. 1999 ) that is loosely associated with the large subunit (EISTETTER et al. 1999 ) and is not essential for peptidyl transferase activity in vitro (FANNING and TRAUT 1981 ; HAMPL et al. 1981 ; SCHULZE and NIERHAUS 1982 ). X-ray crystallography studies of both E. coli (VILA-SANJURJO et al. 2003 ) and homologous eubacterial D. radiodurans (HARMS et al. 2001 ) ribosomes localized L31 near the subunit interface and the peptidyl transferase site, consistent with early biochemical experiments (GIMAUTDINOVA et al. 1981 ; HANAS and SIMPSON 1985 ; TRAUT et al. 1986 ; GRAIFER et al. 1989 ).

When present at elevated levels, MrpL36p also functions as a suppressor of translation defects due to mutations in the leader peptide coding region of the COX2 mRNA (BONNEFOY et al. 2001 ). Two variant forms of MrpL36p, both lacking the central L31-like domain, also function as dosage-dependent suppressors. Thus, the N- and/or C-terminal regions of MrpL36p have an activity revealed by the suppression assay that is distinct from that of the L31-like domain. Further, these suppressing variants, if bound at all, are less firmly bound to ribosomes than those containing the L31-like domain. We were unable to determine whether the N-terminal region, which exhibits no sequence similarity to known proteins, is required for suppression, but our analysis did suggest that the C-terminal region is important for suppression. This C-terminal region (residues 119–177) shows weak but detectable sequence similarity to E. coli Ffh (Fig 2). Ffh facilitates the insertion of integral membrane proteins into the bacterial plasma membrane (KEENAN et al. 2001 ), a process suggestive of the insertion of Cox2p into the mitochondrial inner membrane. However, MrpL36p is unlikely to be a component of an analogous pathway in yeast mitochondria. Sequence similarity is limited to a small region of Ffh containing part of its GTPase domain and an adjacent short helix and flexible linker (KEENAN et al. 1998 ). The highly conserved GTPase motif IV, "DARGG" motif, and closing loop in this region of Ffh (FREYMANN et al. 1997 , FREYMANN et al. 1999 ) are not conserved in MrpL36p, however. Thus, while this region may be necessary for the dosage suppression activity of MrpL36p, its biochemical function remains unclear. Our data nevertheless establish the existence of a functional domain of MrpL36p that is distinct from the L31-like domain.

Like MrpL36p, several other yeast mitochondrial ribosomal proteins have domains with recognizable homology to eubacterial ribosomal proteins fused to protein sequences whose origins and functions are unclear (GRAACK and WITTMANN-LIEBOLD 1998 ). For example, MrpS28p has an N-terminal domain of unknown origin fused to a eubacterial S15-like domain (HUFF et al. 1993 ), which can function in vivo in E. coli (LI et al. 1993 ). Both domains are essential for translation in yeast mitochondria, and these domains can function in trans if they are expressed as separate polypeptides (HUFF et al. 1993 ). We attempted to reconstitute either translation or suppression function by expressing pairs of nonfunctional fragments of MrpL36p in trans but observed no activity in either assay (our unpublished results).

A fascinating but unanswered question is how MrpL36p functions as a dosage suppressor of certain translation-defective cox2 mutations. As expected, suppression appears to be due to increased translation of mutant mRNAs. We observed no increase in cox2-22 mRNA levels in the presence of increased MrpL36p, while steady-state Cox2p protein levels increased slightly (our unpublished data). Furthermore, increased MrpL36p elevates expression of the ARG8^m reporter fused to the mutant cox2 reading frame (BONNEFOY et al. 2001 ). The suppressible cox2 mutations specify mRNAs with short deletions in the pre-Cox2p leader peptide coding region, downstream of the initiation codon, that reduce translational efficiency (BONNEFOY et al. 2001 ; WILLIAMS and FOX 2003 ). Whether these mutations interfere with initiation or elongation (or both) is not established, although translational inhibition in the absence of the leader peptide coding sequence is caused by distinct sequence elements embedded in downstream coding sequence (WILLIAMS and FOX 2003 ). The fact that overexpression of MrpL36p also weakly suppresses cox2 initiation codon mutations implicates this ribosomal protein in initiation, while its failure to suppress a cox3 initiation codon mutation suggests that this function could be mRNA specific.

COX2 mRNA translation depends upon interaction of its 5'-UTL with the rate-limiting mRNA-specific translational activator Pet111p (FOX 1996 ; GREEN-WILLMS et al. 2001 ). Increased activity of Pet111p strongly suppresses the leaky cox2 mutations affecting both the leader peptide coding region (BONNEFOY et al. 2001 ) and the initiation codon (MULERO and FOX 1994 ; BONNEFOY and FOX 2000 ). One interpretation of these results is that excess MrpL36p bearing the suppressing domain could work independently, or together with Pet111p, to enhance COX2 mRNA translation initiation. Alternatively, excess suppressing MrpL36p could improve the efficiency of COX2 mRNA translation elongation by reducing the effect of inhibitory elements within the coding sequence, thereby increasing expression of these leaky cox2 alleles.

We have previously reported that two mitochondrial ribosomal small-subunit proteins, Mrp21p and Mrp51p, can be mutated to suppress 5'-UTL mutations in both the COX2 and the COX3 mRNAs in an allele-specific manner (GREEN-WILLMS et al. 1998 ). These interactions strongly suggest that these ribosomal proteins play a role in selection of mRNAs by ribosomes. In addition, the same cox2 alleles that are suppressed by overproduced MrpL36p are also suppressed by alteration of a novel mitochondrial ribosomal small-subunit protein, Rsm28p. In this case, however, both cox2 and cox3 initiation codon mutations are suppressed (E. H. WILLIAMS, N. BSAT, N. BONNEFOY, C. A. BUTLER and T. D. FOX, unpublished results). Taken together with these findings, the data are consistent with the idea that mitochondrial ribosomes take an active role in mRNA selection. Evidence pointing to similar conclusions regarding cytoplasmic ribosomes is also highly suggestive (MAURO and EDELMAN 2002 ). Recently, mRNA-specific interaction of the cytoplasmic ribosomal protein L13a was observed in the translational regulation of the ceruloplasmin mRNA (MAZUMDER et al. 2003 ). Release of phosphorylated L13a from ribosomes allows it to bind to a target in the 3'-untranslated region of ceruloplasmin mRNA and repress its translation. The possible parallels between the behavior of L13a and overproduced MrpL36p are intriguing.

No homologs of L31 (or MrpL36p) have been identified in metazoan genomes or purified ribosomal subunits sequenced to date (KOC et al. 2001 ), and cryoEM studies of bovine mitochondrial ribosomes revealed a hole in the electron density where L31 is located in the D. radiodurans structure (HARMS et al. 2001 ; SHARMA et al. 2003 ). It nevertheless would appear possible that a divergent L31-like protein could be weakly associated with mammalian mitochondrial ribosomes and thus far have escaped identification. Some unicellular eukaryote L31 homologs are encoded in organellar genomes, including a mitochondrial gene in Reclinomonas americana (LANG et al. 1997 ) and a chloroplast gene in the alga Porphyra purpurea (REITH and MUNHOLLAND 1995 ). Thus, nuclear genes encoding L31-like proteins in other eukaryotic species are likely to code for mitochondrial and/or chloroplast ribosomal proteins as well. The product of a Neurospora crassa gene (GenBank accession no. CAC28768) has an L31-like domain centrally located in a larger protein, much like MrpL36p, although Clustal W alignment (HIGGINS et al. 1996 ) of these proteins (Saccharomyces Genome Database; http://www.yeastgenome.org) does not reveal strong similarities outside the L31-like domain. Intriguingly, an Arabidopsis thaliana nuclear gene (GenBank accession no. NP_565109) encodes a predicted chloroplast L31-like protein with a C-terminal domain that aligns weakly with that of MrpL36p (Saccharomyces Genome Database; http://www.yeastgenome.org). Whether this and other higher plant homologs might partition to both mitochondria and chloroplasts (PEETERS and SMALL 2001 ) is not known.

	FOOTNOTES

¹ Present address: Johns Hopkins University School of Medicine, 725 N. Wolfe St., 714 PCTB, Baltimore, MD 21205.

	ACKNOWLEDGMENTS

We thank T. L. Mason and J. M. Herrmann for antisera. E. H. Williams was a Howard Hughes Medical Institute Predoctoral Fellow. X. Perez-Martinez is a Pew Charitable Trust Fellow. This work was supported by the National Institutes of Health (grant GM-29362 to T.D.F.).

Manuscript received November 21, 2003; Accepted for publication January 15, 2004.

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