the Mitochondrial Ribosome - Molecular and Cellular Biology

MOLECULAR AND CELLULAR BIOLOGY, Sept. 1988, p. 3647-3660 0270-7306/88/093647-14$02.00/0 Copyright © 1988, American Society for Microbiology

Vol. 8, No. 9

Structure and Regulation of a Nuclear Gene in Saccharomyces cerevisiae That Specifies MRP13, a Protein of the Small Subunit of the Mitochondrial Ribosome JUDITH A. PARTALEDIS AND THOMAS L. MASON* Department of Biochemistry and Program in Molecular and Cellular Biology, University of Massachusetts, Amherst, Massachusetts 01003 Received 8 February 1988/Accepted 17 May 1988

MRP13 is defined by biochemical criteria as a 35-kilodalton small subunit protein of the yeast mitochondrial ribosome. The MRP13 gene was identified by immunological screening of a yeast genomic library in Agtll and a functional copy of the gene has been cloned on a 2.2-kilobase Bgl fragment. Sequencing of this fragment showed that the MRP13 coding region specifies a 324-amino-acid basic protein with a calculated M, of 37,366. Computer searches failed to reveal any significant sequence similarity to previously identified ribosomal proteins or to the sequences in the current National Biomedical Research Foundation data base. CeUls carrying disrupted copies of MRP13 lacked the MRP13 protein but were not impaired in either mitochondrial protein synthesis or assembly of 37S ribosomal subunits, indicating that, like L29 and L30 in Escherichia coli (M. Lotti, E. R. Dabbs, R. Hasenbank, M. Stoffler-Meilicke, and G. Stoffler, Mol. Gen. Genet. 192:295-300, 1983), MRP13 is not essential for ribosome synthesis or function. Analysis of the sequence in the MRP13 5'-flanking region revealed the closely linked gene for the cytoplasmic ribosomal protein rp39A. The rp39A coding region began at nucleotide -846 and ended at -325 with respect to the MRP13 translational start. The steady-state levels of the MRP13 mRNA were determined in response to carbon catabolite repression, variation in the mitochondrial genetic background, and increased gene dosage of MRP13. In [rho'] cells, transcript levels were repressed severalfold by growth in glucose compared with growth in either galactose or nonfermentable carbon sources. In respiratory-deficient strains ([rho], [mit-), however, transcription appeared to be largely derepressed even in the presence of high concentrations of glucose. Despite high levels of the MRP13 transcripts in [rhoo] cells, the MRP13 protein did not accumulate, suggesting that the protein is relatively unstable in the absence of ribosome assembly. Cells carrying the MRP13 gene on a multiple-copy plasmid overproduced the mRNA in rough proportion to the gene dosage and the protein in a significant but lesser amount. The results indicate that MRP13 expression is regulated predominantly at the transcriptional level in response to catabolite repression and the cellular capacity for respiration and, in addition, that protein levels appear to be modulated posttranscriptionally by degradation of free copies of the MRP13 protein.

Eucaryotic cells use structurally and functionally different ribosomes for protein synthesis in the cytoplasm and in the mitochondria. The cytoplasmic ribosomal proteins are different from their mitochondrial counterparts, and the two sets of proteins appear to be encoded by separate sets of genes. In Saccharomyces cerevisiae, as many as 200 of the estimated 5,500 total nuclear genes (24) may specify ribosomal proteins. Isolation and characterization of these genes will provide essential details about the structure, function, expression, and evolution of two different populations of ribosomal proteins in a eucaryotic cell. Thus far, approximately 30 genes for the 70 to 80 yeast cytoplasmic ribosomal proteins have been isolated by molecular cloning (47). Since many of these genes are duplicated in the yeast genome (2, 23, 73), their total numberestimated at 125 (70)-exceeds the number of proteins in the ribosome. The genes are scattered throughout the genome, and a few are physically linked, but each is individually transcribed. Most of the ribosomal protein genes contain a single intron, an unusual feature in S. cerevisiae, in which nuclear genes generally lack introns. The expression of the ribosomal proteins is tightly coordinated, primarily through equimolar transcription of mRNA, but also through post*

transcriptional regulation of pre-mRNA processing, translation, and protein turnover. Transcriptional regulation is mediated through conserved elements found in the 5'flanking sequences of most ribosomal protein genes (49, 75), but the posttranscriptional modulation appears to vary markedly among the different genes (70, 71). The components of the mitochondrial ribosome in yeast cells are derived from both nuclear and mitochondrial genes. The 15S and 21S rRNAs and one ribosomal protein are encoded by mitochondrial DNA (mtDNA), and 60 to 70 additional ribosomal proteins are specified by nuclear genes, translated on cytoplasmic ribosomes, and imported into the mitochondria (68). The genes for the mitochondria-encoded components have been identified and sequenced, but relatively little is known about the genes for the nucleusencoded ribosomal proteins. Recently, the first nuclear genes for yeast mitochondrial ribosomal proteins, designated MRPI and MRP2, were isolated from a plasmid library of genomic DNA by complementation of pet mutants with impaired mitochondrial protein synthesis (40). In contrast to most of the genes for cytoplasmic ribosomal proteins, MRPI and MRP2 are present in single copy and do not contain introns. MRPI codes for a basic 37-kilodalton (kDa) polypeptide with no significant relatedness to any known ribosomal protein. MRP2 specifies a 14-kDa polypeptide that is related to the Escherichia coli ribosomal protein S14 and to

Corresponding author. 3647

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PARTALEDIS AND MASON

a chloroplast-encoded

protein of chloroplast ribosomes. The transcription of both genes is catabolite repressed. To gain a better overall understanding of the structure and expression of mitochondrial ribosomal proteins in S. cerevisiae, we have begun to identify and characterize additional nuclear genes for components of the mitochondrial ribosome. Our cloning strategy is to screen Xgtl1 genomic expression libraries with monoclonal antibodies to individual ribosomal proteins. In the present study, and in our previous work, a monoclonal antibody to MRP13 has been a valuable tool for characterizing the cellular and biochemical properties of the MRP13 protein. Several criteria have been used to define MRP13 as a component of the small subunit of the yeast mitochondrial ribosome. It remains associated with the 37S subunit during centrifugation in high-salt sucrose gradients, and it appears as a single species, in roughly stoichiometric amounts, when the mitochondrial ribosomal proteins are resolved by two-dimensional gel electrophoresis or by reversed-phase high-pressure liquid chromatography (D. J. Perry, Ph.D. thesis, University of Massachusetts, Amherst, 1985). Furthermore, the accumulation of MRP13 in the cell is rRNA dependent; it is barely detectable in [rho'] and [rho-] strains that do not express either the 15S or 21S rRNA. Interestingly, it is found at significant levels in [rho-] cells when either the small or the large rRNA is expressed (E. M. Dimock, Ph.D. thesis, University of Massachusetts, Amherst, 1985). Here we report the isolation and characterization of MRP13, the gene for a 35-kDa component of the small subunit of the mitochondrial ribosome. MATERIALS AND METHODS Saccharomyces cerevisiae strains and media. The yeast strains used are as follows: YNN282 (a ura3-52 lys2-801 trpl ade2-101, from R. Davis); 22-2D (a ura3-52 leu2-3,112 trpl cyh2 can] Gal' from G. Fink); the isonuclear strains COP161 [rho'], COP161 [rhoo], and E69 [mit-] (a ade lys) were gifts from R. Butow and have been described previously (44); KY119 (a/a ura3-52 his3-A200 lys ade2 trpl, from K. Struhl); JP1 3A (trpl his3 lys2 ura3-52 ade2 leu2-3,112); and JP1-3C (trpl his3 lys2 ura3-52 ade2 leu2-3,112 mrpl3Al:: TRPJ. Cells were grown in the following media. YPD contained 1% yeast extract, 2% Bacto-peptone (Difco Laboratories), and either 2% or 5% glucose. YPGal medium contained 1% yeast extract, 2% Bacto-peptone, and 2% galactose. YPGE medium contained 1% yeast extract, 2% Bacto-peptone, 2% glycerol, and 2% ethanol. Minimal medium contained 0.67% yeast nitrogen base without amino acids (Difco) and either 2% or 5% glucose. SGE medium contained 0.67% yeast nitrogen base, 2% glycerol, and 2% ethanol. In addition, all minimal media were supplemented with the required amino acids (55). Immunological reagents. The antibody-secreting hybridoma cell lines were produced by the procedure described by Kimura et al. (30), except that either total small subunit proteins, total large subunit proteins, or single, purified ribosomal proteins were used for immunization. The monoclonal antibodies were used as hybridoma cell culture supernatants.

The specificity of the monoclonal antibodies was determined primarily by immunoblot analysis of mitochondrial ribosomal subunits resolved by centrifugation of mitochondrial lysates in sucrose gradients containing high salt concentrations. Figure 1 depicts this kind of analysis for a pool of four different monoclonal antibodies. When tested separately, each of these antibodies reacted specifically with a

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different protein associated with either the 37S or the 50S subunit (not shown). Since these proteins cosedimented with their respective subunits and were not detected in significant amounts in fractions at the top of the gradient, they are unlikely to be adventitious contaminants of the ribosomal subunits. For comparison, a nonribosomal mitochondrial protein, yeast histidine-tRNA synthetase (HTS1), was found predominantly in fractions at the top of the gradient (Fig. 1). Polyclonal antibodies to MRP13 were generated by injecting mice with a hybrid protein expressed in E. coli from a trpE-MRPJ3 gene fusion. The fused gene was constructed by ligating the 0.7-kilobase (kb) EcoRI-XbaI fragment from MRP13 (see Fig. 2) into the polycloning site of the vector pATH11 (17). The 65-kDa hybrid protein contained approximately 325 of the amino-terminal residues of the E. coli anthranilate synthetase (TrpE) followed by residues 23 to 267 of the MRP13 sequence. The recombinant plasmid was used to transform E. coli HB101, and expression of the fusion protein was induced by tryptophan starvation in the presence of indoleacrylic acid (17). Total insoluble protein was prepared from the induced cells (17), and the fusion protein was resolved by electrophoresis in 10% polyacrylamide gels prepared by the method of Laemmli (32). The hybrid protein was electroeluted, lyophilized, and used to inject mice. The procedures used for immunization and development of hyperimmune mouse ascitic fluid were as described earlier (43). The polyclonal antiserum to HTS1 was obtained by a similar strategy (T. Mason, I. Chiu, and G. Fink, unpublished). jI-Galactosidase assays. ,B-Galactosidase activity was determined in whole-cell lysates. The methods for preparation of the lysates and the enzyme assay have been described

previously (26). Screening of genomic libraries. The methods used to screen a Xgtll yeast genomic library (constructed by Michael Snyder) were those of Young and Davis (78, 79). Approximately 150,000 plaques were screened immunologically with the monoclonal antibody to MRP13. An immunopositive plaque was purified through four cycles of subcloning. Phage and phage DNA were prepared by standard procedures (37, 56). The recombinant XMRP13 contained a 2.7-kb yeast DNA insert (Fig. 2), which was used in colony hybridization (27) to identify MRP13 sequences in a yeast genomic library constructed in the shuttle vector YEp24 (8). Gene disruptions. Mutations were generated by inserting the yeast selectable marker HIS3 (61) or TRPJ (77) into selected restriction sites in the MRP13 coding region. Linear fragments containing the markers were used to transform respiratory-competent yeast strains (50). (i) Insertion at BamHI. A 3.2-kb HindIII-XbaI fragment from YEpJP8 was subcloned into a YIp5 derivative (6) to generate plasmid pJP3. The plasmid was partially digested with BamHI, and a 1.8-kb BamHI fragment containing the HIS3 gene was inserted at the three possible BamHI sites. A recombinant with HIS3 inserted at the 5' BamHI site of the MRP13 coding region was identified by restriction mapping and designated pJP4 (Fig. 3). An EcoRI-XbaI linear fragment from pJP4 was used to transform yeast strain YNN282. (ii) Insertion-deletion at BglII-EcoRI. Plasmid pJP3 was partially digested with EcoRI and cut to completion with Bglll. A 0.8-kb EcoRI-BglII TRPJ gene fragment from YRp7' (67) was used to create a deletion mutation at the 5' end of MRP13. The TRPI-MRP13 recombinant was confirmed by restriction mapping and designated pJP5 (Fig. 4). A linear BamHI fragment from pJP5 was used to transform yeast strain KY119.

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YEAST MITOCHONDRIAL RIBOSOMAL PROTEIN MRP13

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FIG. 1. Immunoreactivity of monoclonal antibodies with specific mitochrondrial ribosomal proteins. Strain 22-2D [rho'] was grown to mid-exponential phase in YPGE, mitochondrial ribosomal subunits were resolved by sucrose gradient centrifugation, and the ribosomal proteins were prepared for immunoblot analysis as described in Materials and Methods. The gradient profile is shown at the top, and immunoblot analysis of gradient fractions is shown below. The blot was incubated with monoclonal antibodies to the small subunit proteins MRP8, MRP13, and MRP19 and to the large subunit protein MRP7. For comparison, the same blot was reprobed with a murine polyclonal antiserum against HTS1. All primary antibodies were decorated with 125I-labeled anti-mouse immunoglobulin.

DNA manipulations and hybridizations. All restriction enT4 DNA ligase, E. coli DNA polymerase I, and DNA polymerase I Klenow fragment were purchased from either New England Biolabs or Promega Biotec. Radioactively labeled nucleotides were purchased from Amersham Corp. For colony hybridization, Southern blot hybridization, and Northern (RNA) blot hybridization, DNA fragments were purified after electrophoresis in 1% agarose gels (FMC Corp. Marine Colloids Division) by binding to DEAE-nitrocelluzymes,

lose paper (Schleicher & Schuell). The hybridization probes labeled by nick translation (64). For Northern analysis, total yeast cellular RNAs were prepared by a glass bead miniprep procedure (59), resolved on a 1.2% formaldehyde-agarose gel, and transferred to a nylon membrane (Schleicher & Schuell). The blots were prehybridized in buffer containing 5x SSPE (20x SSPE is 3 M NaCl, 0.2 M NaH2PO4, 20 mM EDTA, pH 7.4), 5x Denhardt solution, 50% formamide, 0.1% sodium dodecyl

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FIG. 2. Structure of the MRP13 gene. The 2.7-kb insert from the Agtll clone (XMRP13) was used as a probe in colony hybridization to isolate MRP13 from a yeast genomic library in YEp24. A 2.2-kb Bglll fragment containing the entire MRP13 gene and its flanking regions was subcloned from the 8.7-kb BamHI fragment in the YEp24 clone (YEpJP8). The large arrows indicate the position and orientation of the closely linked coding regions for MRP13 and rp39A (see text). A partial restriction map and the strategy used for sequencing are shown.

sulfate (SDS), and 100 ,ug of salmon sperm DNA per ml (5). The hybridization was performed for 20 to 24 h in the same buffer with the addition of 10% dextran sulfate plus a 32P-labeled probe. After hybridization, the blots were washed four times in 2x SSC-0.1% SDS at 25°C for 5 min and twice in 0.2x SSC-0.1% SDS at 55°C for 15 min. A.

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FIG. 3. Disruption of MRP13 by insertion of HIS3. (A) A 1.8-kb BamHI fragment containing the HIS3 gene was ligated into the 5'-most BamHI site of MRPB3. The 2.5-kb EcoRI-XbaI fragment was then used to transform yeast strain YNN282 to histidine prototrophy. (B) Southern analysis of EcoRI-digested DNA from YNN282 (lane 1) and two of the derived His' transformants (lanes 2 and 3) confirmed that integration occurred at MRP13, generating the MRPJ3::HIS3 allele. The blot was probed with the 32P-labeled 0.7-kb EcoRI-XbaI fragment from the MRP13 coding region. (C) Western blot analysis of strains carrying the MRP13::HIS3 allele shows the loss of the MRP13 protein and the appearance of a novel cross-reacting polypeptide. Total cell protein was prepared by breaking the cells with glass beads. The proteins (100 ,ug of protein per lane) were subjected to immunoblot analysis with the monoclonal antibody to MRP13 as described in Materials and Methods.

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FIG. 4. Disruption of MRP13 by deletion-replacement with TRPl. (A) A 1-kb EcoRI-BgII fragment was deleted from the MRP13 coding region and the 5'-flanking DNA and replaced by a 0.8-kb EcoRI-BglII fragment containing the TRPI gene. The 2.0-kb TRPI-containing BamHI fragment was then used to transform diploid yeast strain KY119 to tryptophan prototrophy. (B) Southern analysis of BglII-digested DNA from four spores in a tetrad derived from a sporulated Trp+ transformant confirmed the integration of the deletion-replacement to create the null allele mrpl3-AJ::TRP1. Spores A and D are mrpl3-AIl::TRPJ; spores B and C are MRP13 trpl. The hybridization probe was as described in the legend to Fig. 3B. (C) The MRP13 protein is not detectable in spores carrying the mrpl3-AI::TRP1 allele. The immunoblot analysis was performed as described in the legend to Fig. 3C except that proteins from the cell lysate and the mitochondrial fraction were analyzed and a monoclonal antibody to a different mitochondrial small subunit protein (MRP19) was included for comparison. Spores A and D are mrpl3AI::TRPI; spores B and C are MRP13 trpl.

Southern blotting was performed by the method of Southern (58). Prehybridization and hybridization were done in the same buffer described for Northern analysis except that dextran sulfate was omitted at the hybridization step. Southern blots at various levels of stringency were performed identically to the procedures described above except that the formamide was omitted or decreased to 15%. Primer extension analysis. Primer extension analysis was performed essentially as described elsewhere (3). The probe was a 5'-end-labeled double-stranded BamHI-EcoRI MRP13 fragment obtained from pJP3 (positions +67 to +158). Hybridizations were done overnight at 37°C in 30 ,ul of hybridization buffer with 100 ,ug of total RNA derived from 22-2D and 22-2D-YEpJP8. The 32P-labeled DNA-RNA hybrids were precipitated with ethanol and extended with reverse

transcriptase (3). M13 cloning and DNA sequencing. A 2.2-kb BglIH fragment from YEpJP8 (Fig. 2) was subcloned, in both orientations, into the BamHI site of the M13mpl9 RF vector (39). For DNA sequencing, a series of overlapping clones was generated by the method of Dale et al. (15). Single-stranded M13 templates were sequenced by the dideoxy chain termination procedure (51, 52). Nucleotide and amino acid sequences were analyzed with the sequence analysis software package of the Genetics Computer Group, University of Wisconsin, Madison (ver-

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sion 5.1). The GenBank data library (release no. 52.0) and the National Biomedical Research Foundation (NBRF) nucleic acid (release no. 31.0) and protein (release no. 13.0) data bases were searched with the program WORDSEARCH (72) and the align program BESTFIT (57). The RELATE program (16) was used to compare the MRP13 amino acid sequence to the amino acid sequences of 280 other ribosomal proteins contained in a library compiled by Ira Wool (9). Isolation of mitochondrial ribosomal subunits. Mitochon-

dria and mitochondrial ribosomes were prepared by the methods of Faye and Sor (20) with modifications described earlier (E. M. Dimock, Ph.D. thesis, University of Massachusetts, Amherst, 1985). Briefly, yeast cells were grown to mid-exponential phase in the appropriate medium, and spheroplasts were formed by digestion with Zymolyase 60,000 (Miles Laboratories). The mitochondrial fraction obtained after spheroplast lysis was washed extensively in 0.6 M mannitol-1 mM EDTA, pH 6.8, suspended in 50 mM NH4Cl-10 mM MgCl2-7 mM 2-mercaptoethanol-10 mM Tris chloride, pH 7.4, and solubilized by the addition of 1/10 volume of 10% deoxycholate. The resulting detergent lysate was clarified by centrifugation for 20 min at 20,000 rpm in a Sorvall SS-34 rotor. Portions (1 ml) of the clarified lysate were layered on 38-ml isokinetic sucrose gradients containing 500 mM NH4Cl, 10 mM MgCl2, 7 mM 2-mercaptoethanol, and 10 mM Tris chloride, pH 7.4. The gradients were centrifuged for 17 h at 24,000 rpm in a Spinco SW27 rotor. Fractions of approximately 1 ml were collected from the top, and the absorbance at 260 nm (OD260) was monitored with a continuous-flow cuvette. For immunoblot analysis, the proteins in alternate fractions were precipitated in ethanol with 1 OD260 of yeast tRNA as a carrier. The ethanol precipitate was solubilized in SDS-containing electrophoresis sample buffer, and the proteins were separated by electrophoresis in 12.5% polyacrylamide gels containing SDS and electrophoretically transferred to nitrocellulose filter paper for incubation with the antibodies. Polyacrylamide gel electrophoresis and immunoblotting. Protein samples were resolved by polyacrylamide gel electrophoresis in the presence of SDS (32). For immunoblotting, the polypeptides were electrophoretically transferred to nitrocellulose paper (65), and the filters were blocked, incubated with antibody, and probed with 125I-labeled rabbit anti-mouse immunoglobulins. RESULTS

Cloning the MRP13 gene. We have screened a yeast genomic expression library constructed in the phage expression vector Xgtll (78, 79) with a monoclonal antibody specific for a 35-kDa component (MRP13) of the small subunit of the mitochondrial ribosome. A single immunopositive recombinant was isolated from a screen of 1.5 x 105 phage. This recombinant, designated XMRP13, contained a 2.7-kb yeast DNA insert (Fig. 2). The orientation and approximate size of the MRP13 coding region within this insert were deduced from the analysis of the recombinant P-galactosidase-MRP13 antigen expressed in E. coli. Electrophoresis of protein extracts from AMRP13-infected E. coli Y1089 showed that a 140-kDa hybrid protein was expressed from XMRP13. In Western blot (immunoblot) analysis, this protein reacted with monoclonal antibodies to both E. coli P-galactosidase and MRP13 (not shown), confirming that the hybrid protein was expressed from an in-frame gene fusion between lacZ and MRP13. Since the lacZ sequence encodes

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a polypeptide of 117 kDa, the remaining 23 kDa of the 140-kDa fusion protein must have been specified by approximately 700 base pairs (bp) of the MRP13 coding sequence. To obtain the intact MRPB3 gene and its flanking regions, the 2.7-kb insert from XMRP13 was used in colony hybridization to screen a yeast genomic library consisting of 10- to 20-kb fragments ligated in the BamHI site of plasmid YEp24 (8). Eight plasmids with overlapping sequences were isolated, and their inserts were mapped by restriction analysis. The plasmid YEpJP8 (Fig. 2) contained the predicted location of MRPB3 and a minimum of 2 to 3 kb of flanking sequences within an 8.7-kb BamHI fragment. Yeast strains transformed with YEpJP8 overproduced the MRP13 protein, strongly indicating that this plasmid contained a functional copy of the MRPB3 gene (see Fig. 9). MRP13 is a single-copy, nonessential gene. Southern analysis of genomic DNA from yeast strain 22-2D [rho'] probed with the 32P-labeled 0.7-kb EcoRI-XbaI fragment (Fig. 2) demonstrated that the cloned DNA was present in a single copy in the yeast genome (data not shown). No other hybridizable sequences were detected, even under conditions of low stringency. To further confirm that the structural gene for the MRP13 protein had been isolated and to examine the effect of inactivating the gene, mutant alleles were created by replacing the chromosomal MRP13 gene with copies in which the coding region had been disrupted by either an insertion or a substitution. The disrupted alleles were created as outlined in Fig. 3 and 4. In the first case, a 2.5-kb EcoRI-XbaI fragment containing the HIS3 gene ligated into the BamHI site (+158) in the MRP13 coding region was excised from pJP4 and used to transform the haploid yeast strain YNN282 to His+ prototrophy. In the second case, a 0.8-kb EcoRIBglII TRPI gene fragment was ligated between an upstream BglII site (-1018) and an EcoRI site (+67) in the MRPB3 coding region, creating a 1-kb deletion. The 2.0-kb BamHI fragment from pJP5 was used to transform the diploid yeast strain KY119 [rho'] to Trp+ prototrophy. The Southern analyses shown in Fig. 3B and 4B clearly demonstrate that integration occurred at the MRPB3 locus in the His+ and Trp+ transformants. Surprisingly, however, transformants carrying either the MRP13::HIS3 or mrp13-Al::TRPJ allele grew as well as the untransformed parent strains on both fermentable and nonfermentable carbon sources at temperatures ranging from 18 to 36°C. When representative His+ and Trp+ haploid transformants were mated to his3 and trpl strains, respectively, the resulting diploids were sporulated, and at least 10 tetrads were analyzed from each cross. The His+ and Trp+ phenotypes segregated 2:2, and all of the prototrophic spores grew normally on nonfermentable carbon sources (YPGE). The absence of a respiratory-deficient phenotype associated with the disruptions of MRPB3 prompted the analysis of MRP13 gene products in the mutant strains. The normal 1.6-kb MRPB3 transcript was not detectable by Northern blot analysis of total cellular RNA from either the MRP13::HIS3 or mrpl3-AJ::TRPI strain (data not shown). Similarly, the 35-kDa MRP13 polypeptide was not detectable with the anti-MRP13 monoclonal antibody on Western blots of proteins extracted from the mutants (Fig. 3C and 4C). The immunoblot experiments presented in Fig. 4C were performed with total-cell proteins and mitochondrial proteins from each of four spores in a tetrad derived from an MRPB31 mrp13-Al:: TRPJ diploid. These blots clearly demonstrate the absence of the MRP13 protein in the two mutant spores, whereas another small subunit protein, MRP19, was present

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in all four spores. Furthermore, the sucrose gradient profiles (Fig. 5A) show that the loss of MRP13 function had no observable effect on the amounts and sedimentation properties of the mitochondrial ribosomal subunits. As shown in Fig. 5B, the MRP13 protein was not immunologically detectable in the small ribosomal subunits isolated from an mrpl3Al::TRPI mutant strain, nor was any cross-reacting polypeptide detected in other fractions of the gradient (not shown). It is also significant that no stable transcripts from the MRP13 coding region were detectable in Northern blots of RNA from the mrpl3-AJ :: TRPI mutant strain. Combined, these results make it unlikely that a functional fragment of MRP13 is expressed from the MRP13 sequences remaining in the mrpl3-AJ:::TRPJ mutant. As stated above, neither the 35-kDa MRP13 protein nor the 1.6-kb MRP13 transcript was detectable in Western and Northern blot analyses, respectively, of mutants with the MRPJ3::HIS3 allele. Surprisingly, however, these strains contained both a novel 40-kDa polypeptide that cross-reacted strongly with the monoclonal antibody to MRP13 (Fig. 3C) and a new, larger RNA that hybridized with an MRP13specific DNA probe (not shown). In contrast to MRP13, the 40-kDa polypeptide was not enriched in the mitochondrial fraction (data not shown), suggesting that it lacked mitochondrial import signals. The origin of the novel RNA and protein became clear when we examined the structure of the 1.8-kb HIS3-containing BamHI fragment used for the gene disruption. This fragment contains parts of both the PET56 and DEDI genes (61), and the orientation of the fragment in the construction we used was such that the 5' end of the DED1 coding region was fused to the 3' end of MRP13. Subsequent examination of sequence data for the two genes established that the fusion was in-frame and created an open reading frame composed of 111 codons from DEDI followed by 270 codons from MRP13. The polypeptide encoded by this open reading frame had a calculated molecular weight of 41,910, which is in good agreement with the estimated size of the polypeptide detected on the Western blots. Despite the complexity introduced by the fortuitous fusion between DEDI and MRP13, there is no ambiguity in interpreting the phenotype of strains carrying either MRPJ3:: HIS3 or mrp13-Al::TRPJ; both strains lacked the wild-type MRP13 protein and were still fully respiration competent, as determined by their growth rates on non-fermentable carbon sources. Furthermore, these mutant strains were indistinguishable from MRP13+ strains when mitochondrial protein synthesis was analyzed by labeling mitochondrial translation products in vivo with [35S]methionine in the presence of cycloheximide and comparing the profile of labeled products after electrophoresis on SDS-containing polyacrylamide gels (data not shown). Our finding that strains with the disrupted MRP13 alleles either lacked immunologically detectable MRP13 protein or contained an altered form of the protein verified the cloning of the MRP13 structural gene. The absence of any observable respiratory deficiency in strains containing the disrupted MRP13 alleles suggested that either the MRP13 protein is not an essential component of the mitochondrial ribosome or that there is another gene encoding a protein with MRP13 function. Although twin genes for cytoplasmic ribosomal proteins are common in the yeast genome, our hybridization analyses failed to reveal a twin copy of MRP13. The lack of cross-reactivity between the antiMRP13 monoclonal antibody and any other ribosomal protein also argues against the presence of an MRP13 homolog.

MOL. CELL. BIOL.

However, since a monoclonal antibody could easily fail to detect a closely related protein if it binds to a variable epitope, we generated polyclonal antisera to a TrpE-MRP13 hybrid protein that contained the carboxy-terminal threefourths of the MRP13 sequence and used these antibodies to screen for cross-reactive ribosomal proteins. As shown in Fig. SC, we again found no evidence for an MRP13 homolog. The polyclonal antibodies reacted specifically with the MRP13 protein on Western blots of gradient-purified small subunit proteins from the wild-type strain, and they did not react with any of the small subunit proteins from the mrpl3-Al::TRP1 strain. These results support the conclusion that the MRP13 protein is not an essential component of the mitochondrial translation machinery. There is precedence in E. coli for mutants lacking individual ribosomal proteins with little or no impairment in cell growth (11-13, 34). It appears that we have discovered another example of this phenomenon. Sequence features of the MRP13 gene. The nucleotide sequence of the 2.2-kb, MRPl3-containing BglII fragment was determined by the strategy depicted in Fig. 2. The complete nucleotide sequence of this region and the deduced amino acid sequence of the MRP13 protein are shown in Fig. 6. The MRP13 long open reading frame encodes a 324amino-acid basic protein (pl, 10.8) with a calculated Mr of 37,366. These properties are consistent with the mobility of MRP13 in a two-dimensional polyacrylamide gel electrophoresis system that separates ribosomal proteins on the basis of size and net charge (E. M. Dimock, Ph.D. thesis, University of Massachusetts, Amherst, 1985). The amino-terminal stretch of 20 residues had a net charge of +4 and contained three residues with hydroxyl side chains, properties that are consistent with a possible role as a leader peptide for mitochondrial targeting (69). Analysis of codons in the MRP13 coding region showed that 56 of the 61 possible codons were present, with little bias toward the codons preferred in a number of highly expressed yeast genes (4, 54). This is the expected pattern for a mitochondrial protein such as MRP13, which is expressed at low to moderate levels. The DNA sequence shown in Fig. 6 contains putative recognition sites frequently found at the 5' and 3' ends of yeast nuclear genes. Potential TATA sequences for transcriptional initiation (60) were located at positions -282, -246, -128, and -91. The upstream TATA-like sequences were approximately 200 to 240 bp upstream from the region of MRP13 transcription initiation. This distance is longer than reported for other yeast genes, which generally have TATA sequences 30 to 120 nucleotides upstream from the transcription start (60). The 5' ends of the transcripts were determined by primer extension (not shown). The putative initiation codon at + 1 was preceded by an A at position -3 and the sequence AACAA at -9. These sequence features have been found to precede other proteincoding regions in yeast cells (18, 31). The yeast transcription termination sequences TAG...TAGT... 'TTT are found at the 3' end of MRP13 (80). Computer search at the NBRF Protein Data Base failed to uncover any other protein with significant relatedness to MRP13. Comparisons performed by Ira Wool, University of Chicago, also failed to reveal significant similarity between MRP13 and any entry in a data base of 280 ribosomal protein sequences (9). A second long open reading frame beginning at -846 and ending at -325 was the coding region for the cytoplasmic ribosomal protein rp39A. This sequence was first identified through a computer search of the DNA sequences in the

VOL. 8, 1988


A.

JPI-3A [ rho t MRP13, trpl

3653

JPI-3C [ rho f4] mnp13 - A1:: TRP1

0.3

0. 3 r

E

E CD co

CD

C 0.2 LL

LUi

10.2 i

0

-

z

z

m

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cc co 0.1

0

0

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co I

I

5

10

0

0

15

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I

1

20

25

30

1

0

__I

N35

40

0

1

I

5

10

B. 11

13

17

15

19 21

23

25 27

29 31

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C.

I

I

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30

'35

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33

9

11

13

15

17

19

21

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25

27

29

31

33

Small subunit peak fractions JP1-3C JP1-3A

t4.

MRP13 _

I

25

FRACTION

FRACTION 9

20

15

*

40

f

FIG. 5. Sucrose gradient centrifugation of mitochondrial ribosomal subunits from mutant and wild-type cells. Strains JP1-3A ([rho'] MRP13 trpl) and JP1-3C ([rho']mrpI3-AI::TRPI) were grown to mid-exponential phase in rich medium containing 2% galactose. The mitochondrial fraction was prepared as described in the legend to Fig. 1. (A) Gradient profiles. (B) Western blot analysis of gradient fractions. The blot was incubated with monoclonal antibodies to MRP13 and MRP19 as described in the legend to Fig. 3. (C) Western blot of selected fractions from small subunit peak reacted with anti-TrpE-MRP13 polyclonal antibodies. Mitochondrial ribosomal subunits from JP1-3A and JP1-3C were resolved. Proteins in four alternating fractions from the small subunit peak were analyzed for each strain. The blot was reacted with murine polyclonal antibodies raised against an E. coli TrpE-MRP13 fusion protein. The incubation conditions were as described in Materials and Methods.

3654

PARTALEDIS AND MASON -960 -900 -840 -780 -720 -660 -600 -540 -480 -420 -360 -300 -240 -180 -120

MOL. CELL. BIOL.

AAGAGAGGCA CATATACAAA AAGATCGAGA GCCTCCAAGG ACTGTCAGAA GGTCCAAAGG CACAGAAACT GGTTTGAMAG GGTTTCGGTA GGTATCAAGT IjCGGTATCC GACCAGGTGC GATGTAAGGG AGACAACTAA TCAACCAAAA ATAAATAATT ACMCATCAA CTACTTAAI&.TAMGAACA AATTAAAATA CTCAT)ACAT TTGCAAGTCT AGCGCTTCGA TGCGTTGTGA GTfGTATTCA TACCCAATTT ATTGGCACTT ATTTGATACT AATGTGCTGA MMAACCTA MCCTTTCT1TAflMCAAAA

CATAACTCAT GTTTAGCATC ACTAAAGCCC GTTGGTGAAT CAMCTCCAG GAAMMTTG

CATTAAAGAA AGTTACCCTT AAMCCCTAT CTGGTGACAG TTCAATCCAA CTGTTCACGT TCAAGGAATA TTGACGAACA TCTATCTC TACTCTTGGT GTACCACGCT

GTATACTGTT GAMGCCCAA GCGTGATTTG ATTAACCAGA GGCCAGATAT TACCGTCAGA CCAAfTGAGA CATTGACTTG GTCATGAACA AACTCCCACA GATGTGCTCG

I

-60

I

I

TTCATTTCGT AATACGCGTC AATTGGTCTT TTTTAGAACA CTTTCGGTAT

GTATTATAAC CAACATGTCT GAACATCTCC ATTATCTGGT CAGAAGAAAC

CTGAAGAAAT TTTGAGA CGGTAACTTC

TCTCTGCTAC ATGACCCATC TAGAGTCACT GGAAGACACC

CATCGGTATT AGAAGAAAGA GTCTCTTGGT

TGGTCTCCGT ATAGTCAGTC TCCCAAAAAT ATCATATAT ACACTTTGTC AATGTATTTA CACCATGCAG GG&hIGMAA TGGGAACCAT AACAGTGGTG

ID

ATTAATGAGG GACCAATACT GTTGATAAGG GCATTGCACC GAGCAACGAC CAACAAGAAA

1 1

ATC TTC AGA AGT ACA GTT TGG AGA CGT TTT GCA TCT ACC GCC GAA ATT GCG Met Phe Arg Phe Thr Val Trp Arg Arg Phe Ala Ser Thr Gly Glu Ile Ala

52 18

AAA GCA AAG CTG GAT GAA TTC TTG ATA TAC CAC AAG ACA GAT GCG AAA CTA Lys Ala Lys Leu Asp Glu Phe Leu Ile Tyr His Lys Thr Asp Ala Lys Leu

102 35

AAA CCA TTC ATT TAC CGT CCC AAG AAT GCT CAG ATA TTG TTA ACT AM GAT Lys Pro Phe Ile Tyr Arg Pro Lys Asn Ala Gln Ile Leu Leu Thr Lys Asp

153 52

ATT AGG GAT CCA AAA ACA AGG GAA CCA TTA CAA CCG ACA CCT CCC GTA AAG Ile Arg Asp Pro Lys Thr Arg Glu Pro Leu Gln Pro Arg Pro Pro Val Lys

204 69

CCA CTG TCG AAG CAA ACG TTA AAT GAT TTT ATT TAT TCG GTT GAA CCC AAC Pro Leu Ser Lys Gln Thr Leu Asn Asp Phe Ile Tyr Ser Val Glu Pro Asn

255 86

TCG AGC GAA CTA CTA GAT TGG TTC AAA GAA TGG ACA GGG ACC TCC ATA AGA Ser Ser Glu Leu Leu Asp Trp Phe Lys Glu Trp Thr Gly Thr Ser Ile Arg

306 103

AAG CGC GCT ATT TGG ACG TAC ATC TCA CCA ATT CAT GTT CM MG ATG TTG Lys Arg Ala Ile Trp Thr Tyr Ile Ser Pro Ile His Val Gln Lys Met Leu

357 120

ACT GCG TCA TTT TTC AAA ATC GGT AAA TAT GCG CAC ATG CTA GGA CTC TTA Thr Ala Ser Phe Phe Lys Ile Gly Lys Tyr Ala His Met Val Gly Leu Leu

408 137

TAT GGT ATT GAG CAC MG TTT CTC AAA GCC CM MT CCA TCC GTA TTT GAT Tyr Gly Ile Glu His Lys Phe Leu Lys Ala Gln Asn Pro Ser Val Phe Asp

459 154

ATC GAG CAT TTT TTT MT ACT MT ATC ATG TGT GCT TTG CAT CGA MT AGG Ile Glu His Phe Phe Asn Thr Asn Ile Met Cys Ala Leu His Arg Asn Arg

510 171

TTG MA GAC TAT AAA CAT GCA GM ATT GCT CAG AGG MA CTG CAG GTT GCT Leu Lys Asp Tyr Lys Asp Ala Glu Ile Ala Gln Arg Lys Leu Gln Val Ala

561 188

TGG MA MG GTA TTA MT AGA AM MT MT ACT GGA CTA GCA MT ATT CTT Trp Lys Lys Val Leu Asn Arg Lys Asn Asn Thr Gly Leu Ala Asn Ile Leu

612 205

GTT GCA ACA TTA GGT AGA CAG ATT GGA TTT ACC CCA GAG CTG ACG GGC TTG Val Ala Thr Leu Gly Arg Gln Ile Gly Phe Thr Pro Clu Leu Thr Gly Leu

663 222

CM CCT GTA GAT ATT AGT TTA CCA GAT ATC CCC MT TCA TCA ACT GGC GCG Gln Pro Val Asp Ile Phe Leu Pro Asp Ile Pro Asn Ser Ser Phe Gly Ala

714 239

GM PTA MC GAC TTG CTA AGC AAA TAC GAG CGG ATA TAC CTT ATT GCA AGA Clu Leu Lys Asp Leu Leu Ser Lys Tyr Glu Gly Ile Tyr Leu Ile Ala Arg

765 256

ACA TTA TTG CAC ATC GAT CM CAC MT GCT CM TAT CTA CM TTG CM GM Thr Leu Leu Asp Ile Asp Gln His Asn Ala Gln Tyr Leu Glu Leu Gln Glu

816 273

TTT ATA CGC CM TAT CM MT CCA TTA AGT CGA ATC CAG CGA TCC ATA CGA Phe Ile Arg Gln Tyr Gln Asn Ala Leu Phe Arg Ile Gln Arg Ser Ile Arg

867 290

CAC GCA TTT GM ACC GTE CCC ACT GTT GGA MC ACA CTC TCA GGA TCA GGA His Ala Phe Glu Ser Val Gly Thr Val Gly Asn Thr Leu Ser Gly Ser Gly

918 307

AM

969

CTC TM Leu - -

AGA GGA AAA ATA CTA CM MC ACC CAT AGG MA TAT ATA MT MC ATA Lys Arg Gly Lys Ile Val Gln Asn Thr His Arg Lys Tyr Ile Asn Asn Ile

TACTAAMATGAGACAGTTC CTCACATATA TTCCTATTAT TGTATTATI

-

1024 1084 1144

AIQITA3A

CCCGGGMTC TCTTCACMT TTTCAGTACC TCTCGCTTTE TTCCAATTTT TCMATTTTT TTTTTCCGAGT TTAGTGMAA TCACTATTCT ACMCMGGC ATCACCAGCT ATAGATTMTC AGCGTMCTC CCATMCATC CCCGGGTACC CACC

VOL. 8, 1988


A.

[rho `] [rho 0] 3

MRP13

. >.

GAL X:

C:L C Hi

iJ

>-

>-~

o

C

a;

w:

J

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FIG. 9. MRP13 expression in strains with elevated MRP13 gene dosage. RNA and protein samples were prepared from 22-2D [rho'] transformed with either YEp24 or YEpJP8, a YEp24 derivative carrying the MRP13 gene. The cells were grown to mid-exponential phase in minimal medium containing amino acids but lacking uracil (to maintain selection for the plasmid) and either 5% glucose, 2% glycerol plus 2% ethanol, or 2% galactose as the carbon source. Northern blot (A) and Western blot (B) analyses were carried out as described in the legend to Fig. 7.

immunoblot analyses (Fig. 9), we conclude that the expression of MRP13 does not tightly compensate for gene dosage. The abundance of both mRNA and protein increased significantly in YEpJP8 transformants relative to that in the vector-transformed controls. The plasmid-borne copies of MRP13 were also subject to catabolite repression. Within the limits of this experiment, the increase in the MRP13 protein did not appear to be directly proportional to the increase in the mRNA, again suggesting that excess copies of MRP13 are relatively unstable. DISCUSSION We used the monoclonal antibody to MRP13 to identify XMRP13, a Xgtll clone that expresses a large part of the MRP13 protein-coding sequence. The yeast insert from the recombinant phage DNA was used as a hybridization probe to isolate a genomic DNA fragment that contains the MRP13 gene and its flanking regions. The nucleotide sequence of a 2.2-kb segment of this DNA revealed the presence of two long open reading frames. The open reading frame for MRP13 was easily identified because it included the sequence expressed by XMRP13. The second reading frame began at nucleotide -846 and ended with a TAA stop codon at position -325 with respect to the MRP13 coding sequence. A computer search of the nucleotide sequences present in the GenBank data base showed that the upstream open reading frame encoded ribosomal protein rp39A, a 19-kDA large subunit protein of the yeast cytoplasmic ribosome (74; J. Woolford, personal communication). Ironically, no significant relatedness was found between the predicted MRP13 amino acid sequence and any of the 4,750 entries in the current NBRF data base. Further sequence comparisons between MRP13 and over 280 ribosomal proteins in a data

VOL. 8, 1988


base compiled by Ira Wool (9) also failed to reveal significant relatedness to any previously identified ribosomal protein. Although it is assumed that the ribosome arose on a single occasion in evolution and there is strong evidence for homology among diverse rRNAs, including those from organelles, it may nevertheless be difficult or impossible to establish relationships between some of the mitochondrial ribosomal proteins and the protein constituents of other ribosomes. Mitochondrial ribosomes appear to have more relaxed functional constraints than those operating in procaryotic ribosomes or in the cytoplasmic ribosomes of eucaryotes (7), and indeed there is evidence suggesting that the rRNAs and ribosomal proteins in mammalian mitochondria are evolving more rapidly than their cytoplasmic counterparts (46). It is also clear that the number of proteins in ribosomes is highly variable. There are 52 ribosomal proteins in E. coli, whereas the number of mitochondrial ribosomal proteins may be as high as 70 in yeast and 85 in bovine liver (10). It is possible, therefore, that some mitochondrial ribosomal proteins may have changed enough through evolution to preclude the easy identification of their relationship to other ribosomal proteins by means of sequence comparisons. On the other hand, some mitochondrial ribosomal proteins may have arisen from nonribosomal ancestors, which were recruited during evolution to provide ancillary functions unique to mitochondrial protein synthesis. Analysis of the four available sequences for nucleusencoded mitochondrial ribosomal proteins in yeast shows that only two of the four have discernable relationships to proteins from other ribosomes. MRP1 (40), like MRP13, is not related to any of the ribosomal proteins represented in current sequence data bases. MRP2 (40) is related to the E. coli ribosomal protein S14. The sequence of a fourth protein, MRP7, was recently determined from the nucleotide sequence of the cloned gene (21). MRP7 is a 40-kDa largesubunit protein with an unusual relationship to E. coli ribosomal protein L27, a 14-kDa polypeptide. The unusual feature is that the 84 amino acid residues of L27 align with 84 residues in the amino-terminal third of MRP7. There is 49% amino acid identity within the aligned region, compared with 59% identity between L27 and its homolog in Bacillus stearothermophilus. The carboxy-terminal two-thirds of MRP7 shows no significant relatedness to other ribosomal

proteins. Despite its apparent lack of relatedness to members of the extended family of ribosomal proteins, MRP13 possesses the general features of a ribosomal protein. It is a basic protein (pl, 10.8), and although relatively large in comparison to bacterial and cytoplasmic ribosomal proteins, its size is within the 9.5- to 60-kDa range observed for the ribosomal proteins in yeast mitochondria (20). The function of the MRP13 protein remains obscure, since we have been unable to discern an effect to MRP13 mutant alleles on either the assembly or function of the mitochondrial ribosome. Although this result was unexpected, there is precedent in E. coli for mutants which lack individual ribosomal proteins and suffer little or no impairment in cell growth (11-13, 34). For example, L29 and L30 are conserved proteins, implying that they are functionally important, yet each protein is apparently completely dispensable. Furthermore, in vitro reconstitution of peptidyl transferase activity has been accomplished with only 16 of the 34 large-subunit proteins of E. coli (53). These results suggest that a subset of essential ribosomal proteins may be required for ribosome assembly and for peptide bond formation, while other ribosomal proteins might serve peripheral functions that appear

3657

nonessential in laboratory tests but provide some selective advantage in nature. Our analysis thus far indicates that MRP13 may be one of these "nonessential" proteins. It is interesting that in creating the mrpl3-AJ:: TRPJ allele, we actually introduced deletions in two ribosomal protein genes. The excision of the 1-kb BglII-EcoRI fragment from pJP3 removed not only the amino-terminal 22 codons of the MRP13 coding region, but also the entire coding region in the closely linked gene for the cytoplasmic ribosomal protein rp39A. Surprisingly, this large deletion was phenotypically silent with respect to growth rates on either fermentable or nonfermentable carbon sources. Thus, rp39A is another example of a dispensable ribosomal protein. In this case, however, there are twin genes encoding the rp39 protein, and apparently expression from the gene for rp39B can supply a full complement of rp39 in the absence of rp39A. In agreement with our results, J. Woolford and co-workers recently showed that the gene for rp39A is dispensable and that null mutants of rp39B are growth impaired (personal communication). A major objective of our work is to understand the regulatory mechanisms that coordinate the synthesis of mitochondrial ribosomal components, with particular emphasis on the expression of the nucleus-encoded ribosomal proteins. There are several interesting features in the regulation of MRP13. Our Northern analyses clearly demonstrate that transcription of MRP13 is catabolite repressed. The level of the transcripts in derepressed cells growing in the presence of nonfermentable carbon sources is significantly higher than in repressed cells growing in high concentrations of glucose. Under the same experimental conditions, we observed a sevenfold derepression of P-galactosidase expression from an MRP13-lacZ gene fusion. The magnitude of MRP13 derepression appears to be similar to that of other transcriptionally regulated genes for components of mitochondrial macromolecular synthesis, including MRPJ, MRP2 (40), and RP041, the gene for the yeast mitochondrial RNA polymerase (38). Several additional nuclear genes for mitochondrial proteins respond to catabolite repression at the level of transcription (29, 76, 81), but the molecular basis of this regulation is not clear. Coordinate transcription of gene families in yeast is typically modulated by cis-acting upstream activation sequences (UAS) and trans-acting factors that bind to the UAS elements (60). There is evidence for the involvement of upstream elements in catabolite repression (48), and recently the cis-acting elements responsible for the catabolite repression of CYCI have been identified (22). Good progress has been made in understanding the pathways that regulate catabolite repression of several nonmitochondrial proteins (35, 41), but it is not yet clear how extensively these regulatory circuits overlap with those regulating mitochondrial proteins. A curious feature in the catabolite repression of MRP13 is that the levels of MRP13 mRNA are significantly higher in glucose-grown respiration-deficient strains than in their respiration-competent counterparts. Recently, Parikh et al. (44) defined two classes of yeast nucleus-encoded transcripts on the basis of their relative abundances in strains with different mitochondrial genetic backgrounds. The abundance of class I RNAs is increased in all respiration-deficient strains relative to the isonuclear [rho'] control. Class II RNAs are more abundant in [rho-] petites than in either [mit-] or [rho'] strains. These classifications were based on Northern analysis of polyadenylated RNA from cells growing on 2% raffinose, a fermentable but weakly repressing sugar. Since

3658


the MRP13 transcript is more abundant in all respirationdeficient strains, it fits the general definition of a class I RNA. We do not know, however, whether the levels of the MRP13 mRNA would correlate with respiratory deficiency in raffinose-grown cells. Indeed, as shown in Fig. 7A, roughly equivalent levels of the MRP13 mRNA were present in [rho+] and [rho'] cells grown on galactose, a moderately repressing sugar. This suggests that the enhancement of MRP13 expression in respiration-deficient strains is most pronounced in cells growing under repressing conditions. The possible significance of this regulatory response is not obvious, since the MRP13 protein does not accumulate in excess despite the apparent overproduction of its mRNA. The tight linkage between the genes for rp39A and MRP13 suggests that MRP13 upstream regulatory elements could reside within the rp39A transcription unit. The mRNAs for rp39A and MRP13 are transcribed from the same DNA strand, and the 3' end of the upstream transcript (rp39A) maps to a site just 193 bp from the 5' end of the MRP13 mRNA (J. Woolford, personal communication). In yeast, TATA boxes are usually within 30 to 120 nucleotides upstream from the start of transcription. Four potential TATA sequences lie in the region between 86 and 282 bp upstream from the MRP13 start sites. Two of these sequences lie within the 3' noncoding region of the rp39A mRNA. UAS elements for the transcriptional regulation of MRP13 could lie further upstream, perhaps overlapping the rp39A coding region. Close spacing between genes may be a relatively common feature of the yeast genome. For example, the HIS3 transcript ends 127 bp upstream from the transcriptional start of DED1 (61), and Truehart et al. (66) have recently shown that the termination codon in the BIKI coding region ends 10 bases from the most upstream regulatory element in the 5'-flanking sequence of the HIS4 gene. Interestingly, the transcription of genes for cytoplasmic ribosomal proteins is known to be high in cells growing rapidly in rich medium containing glucose and lower in cells growing slowly in medium containing nonfermentable carbon sources (70). This pattern of regulation is the opposite of the catabolite-repressible expression of MRP13. Further analysis of this region should define the position of MRP13 promoter elements and show whether the transcriptional activity of either one of these genes affects the transcription of the other.

MOL. CELL. BIOL.

4.

5.

6.

7.

8. 9. 10. 11.

12. 13.

14.

15.

16.

17.

ACKNOWLEDGMENTS We thank Elizabeth M. Dimock and David J. Perry for their contributions in the characterization of monoclonal antibodies to mitochondrial ribosomal proteins. We are especially grateful to Gottfried Schatz for help in developing monoclonal antibodies to mitochondrial ribosomal proteins, to John Woolford for providing unpublished information about rp39A, and to Ira Wool for computer analysis of the MRP13 sequence. We also thank Rick Young for providing the Agtll library and Maurille Fournier for critical reading of the manuscript. This work was supported by National Science Foundation grant DCB-8502553 to T.L.M.

18.

19.

20. 21.

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Smith, J. G. Seidman, and K. Struhl. 1987. Current protocols in molecular biology. Greene Publishing Associates, Brooklyn, N.Y. Bennetzen, J. L., and B. D. Hall. 1982. Codon selection in yeast. J. Biol. Chem. 257:3026-3031. Berent, S. L., M. Mahmoudi, R. M. Torczynski, P. W. Braff, and A. P. Bollon. 1985. Comparison of oligonucleotide and long DNA fragments as probes in DNA and RNA dot, Southern, Northern, colony and plaque hybridizations. BioTechnol. 3: 208-220. Botstein, D., S. C. Falco, S. E. Steward, M. S. Scherer, D. T. Stinchcomb, K. Struhl, and R. W. Davis. 1979. Sterile host yeasts (SHY): a eukaryotic system of biological containment for recombinant DNA experiments. Gene 8:17-24. Cann, R. L., W. M. Brown, and A. C. Wilson. 1984. Polymorphic sites and the mechanism of evolution in human mitochondrial DNA. Genetics 106:479-499. Carlson, M., and D. Botstein. 1982. Two differentially regulated mRNAs with different 5' ends encode secreted and intracellular forms of yeast invertase. Cell 28:145-154. Chan, Y.-L., A. Lin, J. McNally, and I. G. Wool. 1987. The primary structure of rat ribosomal protein L5. J. Biol. Chem. 262:12879-12886. Curgy, J. J. 1985. The mitoribosomes. Biol. Cell 54:1-38. Dabbs, E. R. 1986. Mutant studies on the prokaryotic ribosome, p. 733-748. In B. Hardesty and G. Kramer (ed.), Structure, function, and genetics of ribosomes. Springler-Verlag, New York, Dabbs, E. R., R. Ehrlich, R. Hasenbank, B.-H. Schroeter, M. Stoffler-Meilicke, and G. Stoffler. 1981. Mutants of E. coli lacking ribosomal protein Ll. J. Mol. Biol. 149:533-578. Dabbs, E. R., R. Hasenbank, B. Kaster, K.-H. Rak, B. Wartusch, and G. Stoffler. 1983. Immunological studies of Escherichia coli mutants lacking one or two ribosomal proteins. Mol. Gen. Genet. 192:301-308. Dabeva, M. D., M. A. Post-Beittenmiller, and J. R. Warner. 1986. Autogenous regulation of splicing of the transcript of a yeast ribosomal protein gene. Proc. Natl. Acad. Sci. USA 83: 5854-5857. Dale, R. M. K., B. A. McClure, and J. P. Houchins. 1985. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18S rDNA. Plasmid 13:31-40. Dayhoff, M. 0. 1978. Survey of new material, p. 1-8. In M. 0. Dayhoff and R. V. Eck (ed.), Atlas of protein sequence and structure. National Biomedical Research Foundation, Washington, D.C. Dieckmann, C., and A. Tzagoloff. 1985. Assembly of the mitochondrial membrane system: CBP6, a yeast nuclear gene necessary for synthesis of cytochrome b. J. Biol. Chem. 260:15131520. Dobson, M. J., M. F. Tuite, N. A. Roberts, A. J. Kingsman, and S. M. Kingsman. 1982. Conservation of high efficiency promoter sequences in Saccharomyces cerevisiae. Nucleic Acids Res. 10: 2625-2630. El Baradi, T. T. A. L., C. A. F. M. van der Sande, W. H. Mager, H. A. Raue, and R. J. Planta. 1986. The cellular level of yeast ribosomal protein L25 is controlled principally by rapid degradation of excess protein. Curr. Genet. 10:733-739. Faye, G., and F. Sor. 1977. Analysis of mitochondrial ribosomal proteins of Saccharomyces cerevisiae by two dimensional polyacrylamide gel electrophoresis. Mol. Gen. Genet. 155:27-34. Fearon, K., and T. L. Mason. 1988. Structure and regulation of a nuclear gene in Saccharomyces cerevisiae that specifies MRP7, a protein of the large subunit of the mitochondrial ribosome. Mol. Cell. Biol. 8:3636-3646. Forsburg, S. L., and L. Guarente. 1988. Mutational analysis of upstream activation sequence 2 of the CYCI gene of Saccharomyces cerevisiae: a HAP2-HAP3-responsive site. Mol. Cell. Biol. 8:647-654. Fried, H. M., N. J. Pearson, C. H. Kim, and J. R. Warner. 1981. The genes for fifteen ribosomal proteins of Saccharomvces

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cerevisiae. J. Biol. Chem. 259:10176-10183.

24. Goebl, M. G., and T. D. Petes. 1986. Most of the yeast genomic sequences are not essential for cell growth and division. Cell 46:

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