MOLECULAR AND CELLULAR BIOLOGY, Aug. 1987, p. 2728-2734 Vol. 7, No. 8 0270-7306/87/082728-07$02.00/0 Copyright C) 1987, American Society for Microbiology Saccharomyces cerevisiae Positive Regulatory Gene PET]]] Encodes a Mitochondrial Protein That.. Is Translated from an mRNA with a Long 5' Leader CHRISTINE A. STRICKl* AND THOMAS D. FOX2 Section ofBiochemistry, Molecular and Cell Biology,' and Section of Genetics and Development,2 Cornell University, Ithaca, New York 14853 Received 17 February 1987/Accepted 28 April 1987

The yeast nuclear gene PETI)I is required specifically for translation of the mitochondrion-coded mRNA for cytochrome c oxidase subunit II. We have determined the nucleotide sequence of a 3-kilobase segment of DNA that carries PET))). The sequence contains a single long open reading frame that predicts a basic protein of 718 amino acids. The PETIHl gene product is a mitochondrial protein, since a hybrid protein which includes the amino-terminal 154 amino acids of PETlll fused to 0-galactosidase is specifically associated with mitochondria. PETll is translated from a 2.9-kilobase mRNA which, interestingly, has an extended 5'-leader sequence containing four short open reading frames upstream of the long open reading frame. These open reading frames exhibit an interesting pattern of overlap with each other and with the PET))) reading frame.

The yeast mitochondrial genetic system depends on a MATERIALS AND METHODS great number of nuclear gene products to express the few genes essential for respiration that are encoded in mitochon- Yeast strains and media. The wild-type Saccharomyces drial DNA (18). Many of these nuclear genes are required at cerevisiae strain was D273-1OB (ATCC 25657). PTE12 and posttranscriptional steps in the expression of specific mito- PTE14A strains both carry the pet)))-) mutation (19) and chondrial genes. These steps include 5'-end processing (14, have been described previously (50). YPEG medium con- 15, 17) and intron splicing (20, 41, 49) of mitochondrial tains 1% (wt/vol) yeast extract, 2% (wt/vol) peptone, 3% pre-mRNAs as well as mRNA translation (reviewed in (vol/vol) ethanol, and 3% (vol/vol) glycerol. SD is a minimal reference 22). For example, at least two proteins coded in medium containing glucose (60). the nucleus are required specifically to translate the mito- Plasmids and transformation procedures. DNA manipula- chondrial mRNA for cytochrome c oxidase subunit III (11, tions and transformation of Escherichia coli strains were 12, 47). Similarly, the translation of the mitochondrial performed as described elsewhere (39). Yeast were trans- mRNA for cytochrome b also depends on at least two formed by lithium acetate treatment of intact cells (32). nuclear gene products (16, 53-55). YpA35 is a yeast replicating vector carrying a 13.5-kilobase In this paper we describe the nuclear gene PETI)I (for- (kb) insert of yeast genomic DNA that complements pet))) merly PETE))), which is required for accumulation of mutations and has been described previously (50). pSP65-2.2 cytochrome c oxidase subunit II (coxII) (7, 19). CoxIl is and pSP64-XS were constructed by inserting a 2.2-kb BglII- encoded by the uninterrupted mitochondrial gene oxil (8, 10, HindIII fragment and a 1.8-kb SalI-XbaI fragment, respec- 21). Its mRNA is translated to yield a precursor protein that tively, from YpA35 into Riboprobe vectors (Promega is processed to coxIl by removal of the 15 amino-terminal Biotec; 43). YEp13-2.7L was constructed as follows. amino acid residues (51, 58). PETJ)) activity appears to be pMC1871 (9) was digested with BamHI and treated with required specifically for translation of coxll since pet))) Klenow fragment of DNA polymerase I to fill in recessed 3' mutants lack the protein but contain substantial amounts of ends. A 3-kb fragment carrying the lacZ gene was ligated its mRNA (50). Furthermore, pet))) mutations are sup- into pSP64-2.7 (a Riboprobe vector carrying a 2.76-kb pressed by mitochondrial gene rearrangements that fuse the HindIII fragment from YpA35 which contains the PET))) 5' portions of other mitochondrial genes to oxil (50). gene) which had been prepared by digestion with NdeI, Si In this paper we present the nucleotide sequence of nuclease digestion to remove 5' overhanging ends, and PETIJ) and the predicted amino acid sequence of its prod- dephosphorylation. The resulting plasmid (pSP64-2.7L) car- uct. We provide evidence that the PET) I) protein is located ried a 5.76-kb HindIll fragment containing a fusion of the in mitochondria and therefore probably acts directly to first 154 codons of PET))) in frame with codon 8 of lacZ. promote coxII translation. Interestingly, the PET I)I mRNA has an unusually long 5' leader that contains four short open YEp13-2.7L contains this Hindlll fragment ligated to reading frames. The results of recent studies on the expres- HindIII-cleaved YEp13 (6). sion of another yeast gene whose transcript also has a long 5' DNA sequence analysis. Sau3A, TaqI, AluI, and XbaI leader with short open reading frames (24, 27, 28, 46, 65) fragments of the 2.76-kb HindIII fragment that complements suggest that the expression of PET))) itself may be regu- pet))) (50) were subcloned into the M13 vectors mplO and lated at the level of translation. mpll (44). A 1.3-kb HindIII fragment from the 5' flank of the structural gene was cloned into M13 mpl8 and mpl9, and appropriate deletions were made by exonuclease III-Si * Corresponding author. digestion (26). These clones were used as templates for the 2728 VOL. 7, 1987 PETHII CODES FOR A MITOCHONDRIAL PROTEIN 2729

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N 00 bp FIG. 1. Restriction map and sequencing strategy of PETJJJ. Restriction sites shown are as follows: A, AluI; B, BglII, H, Hindlll; N, NdeI; R, EcoRI; S, Sau3A; T, TaqI; V, EcoRV; and X, XbaI. , Position and orientation of N and C termini of the open reading frame. Arrows show the extent and direction of each sequence determined by the dideoxynucleotide chain termination method (55) (-k) or by chemical degradation of end-labeled fragments (40) (-*). All restriction sites were crossed; both strands were sequenced. bp, Base pairs. dideoxynucleotide chain termination sequencing reaction Fractions were assayed for ,-galactosidase activity by the (56). Some regions were also sequenced by chemical degra- method of Miller (45) and for fumarase by the method of dation of end-labeled fragments (40). Racker (52). Isolation of RNA. Total yeast nucleic acids (primarily RNA) were prepared from cells grown on YPEG medium to RESULTS early logarithmic stage by vortexing with glass beads in the presence of phenol as described elsewhere (63). Poly(A)- Primary sequence analysis of PET])). DNA that comple- containing RNA was selected by hybridization to oligo(dT)- ments petlll mutations is carried on a 2.76-kb HindIII cellulose (3) according to the procedure of the manufacturer fragment (50). The nucleotide sequence of this fragment and (Bethesda Research Laboratories, Inc., Gaithersburg, Md.). that of a portion of a flanking HindlIl fragment was deter- Northern hybridization (RNA blot) analysis. RNA was mined by subcloning restriction fragments into M13 vectors denatured with glyoxal, fractionated by electrophoresis in and using these as templates in dideoxynucleotide chain 1.1% agarose in 10 mM phosphate buffer (pH 7.0; 42), and termination reactions (56). Some restriction fragments were transferred to a nylon membrane (Biodyne A; 66). A uni- end labeled and subjected to chemical degradation (40). The formly radioactively labeled RNA probe was prepared by exact strategy used in described in the legend to Fig. 1. SP6 polymerase transcription (43) from pSP65-2.2, a The PETJIIJ sequence (Fig. 2) contains a single long open Riboprobe vector which contained a fragment from the reading frame which extends over 2.15 kb. If translation structural gene for PET]JJ in the antisense orientation. were to begin at the first ATG in this open reading frame, Hybridization was done at 65°C in a buffer containing 50% PETIII would encode a basic protein of 718 amino acids. formamide (62). Genetic evidence indicates that this open reading frame Si nuclease protection. A probe uniquely labeled at the codes for the PET] ]I product. A DNA fragment that carries BglIH site within PETJI] (Fig. 1) was generated as follows. just the long open reading frame and 57 upstream nucleotides YpA35 was restricted with BglII, and an 8-kb band was under the transcriptional direction of the ADCJ promoter (2) isolated from an agarose gel. This fragment was treated with complemented petlII mutations (data not shown). In addi- alkaline phosphatase and labeled by using T4 polynucleotide tion, the pet]I l-I mutation was mapped to a DNA fragment kinase as described elsewhere (39). The DNA was recut with which is delineated by two XbaI sites (Fig. 1) and includes Sall, and the appropriate 4-kb fragment was isolated from an primarily open reading frame (774 nucleotides of the open agarose gel. A probe uniquely labeled at the HindIII site reading frame and 219 upstream nucleotides). Also, insertion upstream of the PETJIJI coding region (Fig. 1) was generated of foreign DNA at the BglII site in the open reading frame as follows. pSP64-XS was digested with HindIII, and a destroyed the ability of the 2.76-kb HindIII fragment to 1.3-kb fragment was isolated from an agarose gel. This complement pet]ll mutations (50). fragment was labeled as described above and recut with The predicted amino acid sequence of PET] II was used in EcoRI, and the appropriate 500-base-pair fragment was a computer search of sequences contained in the National isolated from an agarose gel. Biomedical Research Foundation (NBRF; Dayhoff) data- The labeled probes were denatured, hybridized to poly(A) base. No highly homologous proteins were found. containing RNA at 46°C (BglII-labeled probe) or 40°C The open reading frame contains the sequence TAC (HindIll-labeled probe), and digested with S1 nuclease (59). TAAC, which is essential for pre-mRNA splicing in S. Protected fragments were analyzed on a 1.5% alkaline cerevisiae (36). However, neither S1 nuclease protection agarose gel (BglII-labeled probe) or a 6% polyacrylamide-7 experiments nor RNA gel blot hybridization analysis (data M urea sequencing gel (HindIll-labeled probe). Gels were not shown) showed any evidence that PETIII encodes a dried and autoradiographed. spliced mRNA. Preparation of yeast mitochondria and enzyme assays. Transcription ofPET))) as a 2.9-kb mRNA containing long Mitochondria were prepared from spheroplasts of yeast cells 5' leader. Yeast RNA was fractionated by electrophoresis, containing the PET IJI-lacZ fusion plasmid YEp13-2.7L and transferred to a nylon membrane, and hybridized to a grown to early logarithmic stage in SD medium by the uniformly radioactively labeled RNA probe complementary method of Daum et al. (13). A sample of the resulting crude to the predicted PET]J] mRNA. This probe detected a mitochondria containing 1 mg of protein (protein concentra- single RNA species of approximately 2.9 kb (Fig. 3). This tion was measured by the method of Bradford [4]) was RNA was enriched in poly(A)+-RNA and was more abun- purified on a sucrose gradient as described elsewhere (11). dant in RNA prepared from a yeast strain carrying PETI II 2730 STRICK AND FOX MOL. CELL. BIOL.

-625 CCCAGAATTACACACTTATGAAACTATTTATGATTAAACTTGATAGTTGTGATAAACATTTCTTATGTACATTTGTTGAAGGAGTTCGTATATATACAGC -525 CAGTTGAGTAATAATTATATTTTACTATTGCATTTCATAATTATTACCCGGACCTTACGAGTTCTTCGAATAATAACCATATATTTCAGAAAGGTAGGCA -425 a TACCGAAGGAAGAGACAATGAAAsAAACTGAAATGAACAAGCTTCTGTATACGATCCAAGGATCAGAAACGGTTGTTATTTATTGTTTGGACTTCTTGTTG a b -325 b TTAGGCTTTATTTACCGTGCATAATACTATCTGATACTTTGAGAAACCCCTCCCCCCACAGAAAGGTTCAAAATTTACAATTGCTGCACTAGGTCTACAC -225 TTTCCTCTAGATTTGTAACTTGAAGACCCCATTCCAATCGTTTAACCTGTCTTTCCTTTTATTATCGTTTTATTCTCTTCCTTTTTTATAAAATCCTATC -125 c TGGTATAGAGCATTTTTATGTTGATCATAACTTTTCATGTATTCTATTAATTACACCACAACTTTGATATCGTCAAATTTTTTATCACTACCTATTAAAT c d -25 d TTAAACAATTGCTTACGAGAACTTAATGTTACAACGGAGATTTATATCCTCCAGTGGTATAAAGAGATTACTTCACAGAGAATCAAACAAGGTTATGCAC L Q R R F I S S S G I K R L L H R E S N K V M H +76 ACTGTTTTCTTTAAAGTACGATATTACTCAAC:TGAGTTGATCAAAAAAAAGCATAAGGAAGATATTGAGGATTGGGTTAAAGCGCAACTAAAAGATTCTT T V F F K V R Y Y S T E L I K K K H K E D I E D W V K A Q L K D S +176 CAACAATAAGCGGTGTGTATGAGTCTCGGAATAAACTCGATTGGATGGACTCCATTACCAAAAGCCCCAGCAGCTTAGATATTCTTAAGAATCAATATAA S T I S G V Y E S R N K L D W M D S I T K S P S S L D I L K N Q Y N +276 TATAGTGAAAGACAAAGATTTTGGAATATTGTGGAAGCAAAAATTCGAAAGTGCAGATCCAGATATTTTGATGACAATAATTAGCCTTTCCACTAACCAA I V K D K D F G I L W K Q K F E S A D P D I L M T I I S L S T N Q +376 AAGGTTTTGTTTTCAATACAGCAATTACTGATCTTAATAAATTCTCTTCACTTTTTGAAAAGGGACTACGATATTGGACAAATATACACAACATATGAGC K V L F S I Q Q L L I L I N S L H F L K R D Y D I G Q I Y T T Y E +476 AATTCACGCCCTTATTAGCAAGCCACACTGATAAAGGCACATATGGTCAGTTCATCGAAATTATGTTGGTAGTACAGCATAATTTACATCATTTTGATGT Q F T P L L A S H T D K G T Y G Q F I E I M L V V Q H N L H H F D V +576 CTGTGAAACTTTATTTGCAGAATATATCAAATATTGCAAAGTAAAGCCACAAATGATATCTTTAGGATTAAACTCTTTCATAAGGAGTAATAACACTCAA E TL FAE Y I R C K V K P Q M I S L G L N S F I R S N N T Q +676C6 L A V E F Y T Q A I T N P D T F P I T E K Q L F E F L R C M E R Y +776 TAGATATGTCCAGTATGAAACACATCTTTTATCTCTGGTTGAAAGTGAAATGTGGTGGTGAGCAATCCTCTTCAACTAACCTTCCCTCGTTCAAAACCCT L D M S S M K H I F Y L W L K V K C G G E Q S S S T N L P S F K T L +876 CGCGATAATACACAGAATGTTATTACGTTTTTCTAATACAGATGAGCTAAACGATTTTTTAACCAATCCCGTTGTTTTAAGTACGGGATATACATCAAGT A I I H G M L L R F S N T D E L N D F L T N P V V L S T G Y T S S +976 GTGCAGTTTGAGTTGATTGAATTTTGTCATTCTCTGTATTGCATCAAAGGAGACAGAACAAAAAGCATTGATGACTCAATATTGATGGAAAGAGTTGATA V Q F E L I E F C H S L Y C I K G D R T K S I D D S I L M E R V D +1076 AATTTATTACCAGACTTAATAATAATATTTCAACACGAAAAGAGCTATATATGTCAGTGGTTCAAGCGTACGTCTCTACTAACAATTTCGAGAATTTAAA K F I T R L N N N I S T R K E L Y M S V V Q A Y V S T N N F E N L K +1176 AGTAATCTTGGAAAAAATCCAGAGAGATAATGATATTAGCATAGACGGTTCGTTTCATTTGTGCATTTCTAGGTATTTTGTCAACACCAATCAATTCGAA V I L E K I Q R D N D I S I D G S F H L C I S R Y F V N T N Q F E +1276 GGACTTTTCAAATATTATCGCAGTGTAGTTAAAACCACTGATGGTAAAACGCGATTACGGCCCGCATTTATTCAACAATTATGGTCATGTGCCGTAAATG G L F K Y Y R S V V K T T D G K T R L R P A F I Q Q L W S C A V N +1376 TTTATCCAATGCTGGCAAAAGAAATTACAAATGACCTACTTGTGACTCTAAAAAGGAGCCAATACAGCAAATGTCTCACTTGGGTGTACACATTTTTACA V Y P M L A K E I T N D L L V T L K R S Q Y S K C L T W V Y T F L Q +1476 AGAAAATGCCCATATCCATAGGAGAAAGATCAATGGAGGAGAAGACTCTTCGTTATCTGGTTTCAACGCGGTTGATTTTGAAkAGGTTTGAAGAGTTCAAG E N A H I H R R K I N G G E D S S L S G F N A V D F E R F E E F K +1576 AAAAAAGTTTCTCACAATGATGTATATGGAGCAGAATTGGTGATTTCGAATAGTTTGAAGGAGGGCATTGCGCCTCAATTTTCATTTTTGTATTCCG mT K K V S H N D V Y G A E L V I S N S L K E G I A P Q F S F L Y S V +1676 TGGCATTATGCTTGAGAAACTCTTTAACACCTTTGGCACGTGTAGTTGATGTGATATTAAGGACCAGATTCCGCTATATCCCACTAAAAGTCGATATATT L A L C L R N S L T P L A R V V D V I L R T R F R Y I P L K V D I L +1776 ATGGTTGAAATGGGAGATCATTTCTAATTATAGATCTTTTGAAAAGTTGTCTGCTGAACATTTAAAAGAACTGGAATTCAAACTGAAGGAATTTGAACGA W L K W E I I S N Y R S F E K L S A E H L K E L E F K L K E F E R +1876 GTACATCAAAAGGAGCTTTCAGTTCAAAACTATTTACAACTTACTCAAATATGTTTCCACACCCGTGATTTCAAATATGCATGTTACCTAATTTCACAAG V H Q K E L S V Q N Y L Q L T Q I C F H T R D F K Y A C Y L I S Q +1976 CTAGAAAAAATTTGGATACTTCAAATAACAAACAGTGGATGATGTATTACATGACATCTTTAAAGTTGGCGTCAAGAATGCATGAAAGCGAACGGTTTAG A R K N L D T S N N K Q W M M Y Y M T S L K L A S R M H E S E R F S +2076 TAGGATTTTAAAAGACTGGAATTGTAATCATAGGGCAAGCCTGATTACCCAGGTTGCATTAGACAAATCAAAGGCTTTATGAAATATTTTGAGAAAAGGC R I L K D W N C N H R A S L I T Q V A L D K S K A L +2176 CTGCTTACATTTCAACTGCTGCTTCCATTGATAACAAGGAGATAAAGGATCGCATTGACGAGTTAGTACTTAGATATGTGGACTATAAATACCAGGGGCT +2276 TGAGAATATGAGAAAGTTAACACTTTTCTTAAAAGAATGGTTTGATGAAGAGATTTCACTATTAAAGTTGGAGCAAAATGAAAGGAAAATGAAGCTT FIG. 2. Nucleotide sequence of PET] II. Only the nontranscribed strand is shown. The sequence was determined as described in Materials and Methods and the legend to Fig. 1. The derived amino acid sequence of PET111 is shown in single-letter code below the DNA sequence. , Approximate positions of the 5' ends of the major transcripts. ATGs in the leader region are underlined, and stop codons for the short open reading frames are overlined; corresponding starts and stops are lettered a, b, c, and d. The presumed start of translation of PET111 is numbered +1. VOL. 7, 1987 PETIII CODES FOR A MITOCHONDRIAL PROTEIN 2731

PTE 12: a probe with a unique 3' label at the BglII site within the D273-10B YPA35 open reading frame and extending downstream for 560 T A T A bases. Poly(A)+-enriched RNA from a strain transformed with YpA35 protected a fragment of approximately 430 bases, which indicated that the polyadenylation site of the PETIHI mRNA is about 80 bases beyond the translation termination codon (data not shown). PET1I1-encoded mitochondrial protein. Most mitochon- drial proteins are encoded by nuclear genes and are targeted - ~~ 2.9 to the organelle by transient presequences at their amino termini (for reviews, see references 25 and 57). The prese- quences of a number of these proteins can direct the mito- chondrial import of nonmitochondrial proteins such as 3- galactosidase and dihydrofolate reductase (5, 11, 17, 30, 33). Since PETJJJ function is required for the translation of the mitochondrion-coded mRNA for coxII (50), PETJJJ might encode a protein located in the mitochondrion. On the other by activating other nuclear FIG. 3. PETII) transcript. RNA was prepared from wild-type hand, PETJJJ could function yeast cells (D273-1OB) or yeast cells transformed with a high-copy- genes, which in turn would act directly in the mitochondria. number plasmid which carried PETH)) DNA (PTE12:YpA35). Fifty To determine whether PET111 is a mitochondrial protein, micrograms of total RNA (lanes T) or poly(A)+-enriched RNA we tested the ability of the amino terminus of PET))) to (lanes A) were denatured with glyoxal, fractionated by electropho- target p-galactosidase to mitochondria. We constructed a resis on a 1.1% agarose gel, transferred to Biodyne A, and probed fusion gene composed of 389 nucleotides of the 5'-flanking with a uniformly labeled RNA probe complementary to the pre- region of the PETJJJ open reading frame and the first 156 dicted PETH)) mRNA. The transcript was visualized by autoradi- codons of PETll joined in frame to codon 8 of lacZ. Cells ography. Size was determined by comparison to RNA standards carrying this plasmid (YEpl3-2.7L) were fractionated into a transcribed in vitro. crude mitochondrial pellet and a postmitochondrial superna- tant, and fractions were assayed for 3-galactosidase activity. DNA on the high-copy-number vector YpA35 than in RNA In several mitochondrial preparations, 83 to 94% of the total prepared from a wild-type strain. Surprisingly, the PETI)) P-galactosidase activity pelleted with mitochondria. In con- transcript was approximately 750 bases longer than the trast, unmodified ,-galactosidase has no affinity for yeast predicted minimal coding sequence of the gene. We used S1 nuclease protection experiments to define the A with a unique 5' A B 5' end of the PET))I transcript. probe A T label at the BglII site within the PET I)I open reading frame 1 2 34 GGCC 1234 and extending upstream for about 4 kb was prepared and hybridized to poly(A)+-enriched RNA from a yeast strain 4.0 *4.. G transformed with YpA35. After S1 digestion, electrophoresis c revealed a major protected fragment of about 2.3 kb (Fig. I CT G 4A, lanes 1 and 2). No protected fragment was recovered A A when the same probe was hybridized to an equal amount of 2.2 T G E. coli tRNA (Fig. 4A, lanes 3 and 4). Minor protected C T C fragments of about 2.0, 1.4, and 1.3 kb were also detected A (Fig. 4A). While the major 2.3-kb fragment was consistently A the smaller minor bands were not A seen in all experiments, G reproducibly detected and were never seen after hybridiza- \C tion to an RNA probe covering the same region (data not shown). This result suggested that the PET))) transcript started about 500 bases upstream from the predicted start of 1.0 S translation. FIG. 4. Determination of the 5' end of the PET))) transcript. (A) To define more precisely the 5' end of the PET)I1 mes- A probe with a unique 5' label at the BglII site within the PET)II) sage, we used a 500-base-pair probe with a unique 5' label at structural gene was hybridized at 46°C to 100 ,ug of poly(A)+- the site 389 bases upstream of the open reading enriched RNA (lanes 1 and 2) or to 100 ,ug of E. coli tRNA (lanes 3 HindIII and 4), digested with 100 U (lanes 1 and 3) or 500 U (lanes 2 and 4) frame. Poly(A)+-enriched RNA from a yeast strain trans- of S1 nuclease, and analyzed by gel electrophoresis on a 1.5% formed with YpA35 protected two groups of fragments (Fig. alkaline agarose gel. Protected fragments were visualized by 4B). The sizes of these fragments indicated that the 5' ends autoradiography of the dried gel. Size standards are undigested of the PETI)) transcript were at bases -469 (±t1) and -459 probe (4.0 kb), Hindlll-digested probe (2.2 kb), and XbaI-digested (±1) relative to the presumed start of translation. probe (1.0 kb). (B) A probe with a unique 5' label at the Hindlll site Interestingly, the 5' ends of the PET))) transcripts lie at -389 (which was the same HindIII site used to generate the approxitnately 80 bases upstream of the 2.76-kb HindIII 2.2-kb size standard in panel A) was hybridized at 40°C to 100 ,ug of fragment that complements pet))I mutations (50). When this poly(A)+-enriched RNA (lanes 1 and 2) or to 100 ,ug of E. coli tRNA DNA is present on the high-copy-number vector (lanes 3 and 4), digested with 100 U (lanes 1 and 3) or 500 U (lanes fragment 2 and 4) of S1 nuclease, and analyzed on a 6% polyacrylamide-7 M YEp13 (6), it is transcribed as an RNA that is approximately urea sequencing gel. Protected fragments were visualized by 100 bases larger than that transcribed from wild-type PET)I) autoradiography of the dried gel. The sequence of the HindIII probe (data not shown). was determined by chemical degradation (40) to size the protected The 3' end of the PET)I) transcript was mapped by using fragments, whose approximate endpoints are indicated (*). 2732 STRICK AND FOX MOL. CELL. BIOL. mitochondria and remains in the postmitochondrial superna- L Q L T Q I C F H T R D F K Y A C Y L I S Q A R K tant (11, 17). To be sure that the PET111-p-galactosidase hybrid pro- L L T T F I M N D V P T P Y A C Y F D S A T P tein was specifically associated with mitochondria, we frac- Q Q tionated the crude mitochondrial pellet on sucrose density equilibrium gradients. Each fraction was assayed both for ,-galactosidase and for the marker enzyme fumarase, a N L D T S N N K Q W M M Y Y M T S L K L A S R M soluble mitochondrial-matrix enzyme (52). The ,B-galac- * * * * * * * tosidase and fumarase activities cosedimented within the * * * * * * * gradient (Fig. 5), showing that the amino-terminal 154 amino N Q E G I L E L H D N I M F Y L L V I L G L V S W M acids of PET111 were sufficient to direct a PET111-,- FIG. 6. Similarity between amino acid sequences of PET111 and galactosidase hybrid protein specifically to mitochondria. coxIl. The deduced amino acid sequence of PET111 is shown in the top line; the amino acid sequence of coxll is in the bottom line. The first residues shown correspond to amino acids 637 (PET111) and 7 DISCUSSION (coxII). Identical amino acids are starred; conservative amino acid replacements are underlined. PETJJJ is a nuclear gene required specifically for the translation of the mitochondrion-coded mRNA for coxll Thus, PET111 fits this previously established pattern for (50). We have determined the nucleotide sequence of a 3-kb mitochondrion-targeted nuclear gene products. segment of DNA that contains PETIJI (50). This region The predicted amino acid sequence of PET111 was used in contains a single long open reading frame that could encode a computer search of sequences present in the NBRF a protein of about 79,000 daltons. (Dayhoff) data base. While no highly homologous proteins Information in the amino-terminal 154 amino acids of were identified, an interesting region of sequence similarity PET111 was sufficient to direct a PET111-,-galactosidase (31% homology; 16 identities of 51 amino acids; another 10 fusion protein to yeast mitochondria. This result supports conservative amino acid replacements) was found between the idea that PET111 is a mitochondrial protein and func- PET111 and coxII, the mitochondrial gene product whose tions direcly to promote translation of the mitochondrion- expression depends on PETIIJ function (Fig. 6). This pos- coded coxII mRNA. Most mitochondrial proteins that are sible homology is found between the amino terminus of encoded by nuclear genes are targeted to the organelle by coxll, including its leader peptide (51, 58), and the carboxy means of information at or near their amino termini (5, 11, terminus of PET111, a region known to be essential for gene 17, 30, 33). There is, however, little primary sequence function (50). Experiments are in progress to determine if homology among the amino-terminal regions of known im- this sequence similarity could be functionally significant. ported mitochondrial proteins. Rather, these regions share The sequence TACTAAC occurs within the open reading certain overall characteristics. For example, they are rich in frame of PETJIJJ at position +1150 relative to the proposed arginine, lysine, serine, and threonine and have a paucity of start of translation. This sequence is absolutely required for acidic residues (1, 31, 67). The amino terminus of PET111 is pre-mRNA splicing in S. cerevisiae (36). The PETJJJ se- noticeably basic: of the first 36 amino acids, 8 are positively quence, however, does not contain a perfect copy of the 5' charged, while there is just a single glutamate residue. This splice site consensus sequence GTATGT (37, 64). We region also contains seven serine and threonine residues. looked for evidence of splice junctions in PET IJI RNA in S1 nuclease protection experiments and found no protected fragments that could have been generated from a splice junction. In addition, hybridization analysis of RNA gel 25 blots showed only a single transcript corresponding to PETIIJ, and this same RNA species was detected with 0 putative intron-specific and putative exon-specific hybridiza- w 20 tion probes. The sequence TACTAAC, therefore, appears to occur fortuitously within the PETJII open reading frame and 15 does not function as an RNA splicing signal. The PETIJI mRNA contains a long 5'-leader region of O 'l 10 about 470 nucleotides. There are four short open reading Im frames (labeled a, b, c, and d in Fig. 2) in this leader, with interesting patterns of overlap. Short open reading frames a co 5 and b initiate 15 bases apart, approximately 60 nucleotides downstream from the 5' end of the PETHJJ transcript. They are in different reading frames, and the stop codon of a 2 4 6 8 10 12 overlaps the start codon of b. The AUG codons of short open reading frames c and d are located 20 bases apart, FRACTUON about 340 nucleotides downstream from the 5' end of the FIG. 5. Cosedimentation of P-galactosidase and fumarase on a transcript, and these open reading frames overlap. Finally, sucrose density gradient. Mitochondria were prepared from yeast the stop codon of open reading frame d overlaps the start cells (PTE14A) carrying the plasmid YEp13-2.7L (see Materials and codon of PETJJI. While these short open reading frames Methods). Mitochondria (1 mg of protein) were centrifuged through may have regulatory significance (see below), the long 5' a linear sucrose gradient (20 to 70%). The direction of sedimentation We have deleted all was from right to left. Twelve fractions were collected and assayed leader is not required for gene activity. for P-galactosidase and fumarase. 13-Galactosidase activity is given but 57 nucleotides of this leader region, removing all four in nanomoles per minute; fumarase activity is given in micromoles short open reading frames. This DNA, under the transcrip- per minute. tional control of the ADCI promoter (2), complemented VOL. 7, 1987 PETJII CODES FOR A MITOCHONDRIAL PROTEIN 2733 petlll mutations. Also, the original 2.76-kb fragment iso- enzyme. J. Biol. Chem. 253:4396-4401. lated by Poutre and Fox (50) complements petl lI mutations 8. Cabral, F., M. Solioz, Y. Rudin, G. Schatz, L. Claviier, and P. P. despite its lack of about 80 bases of this upstream leader. Slonimiski. 1978. Identification of the structural gene for yeast II Extended 5'-leader regions which contain AUGs are un- cytochrome c oxidase subunit on mitochondrial DNA. J. Biol. usual but not without precedent for eucaryotic mRNAs. Chem. 253:297-304. 5 9. Casadaban, M. J., A. Martinez-Arias, S. K. Shapira, and J. About to 10% of those higher eucaryotic mRNAs whose Chou. 1983. 1-Galactosidase gene fusions for analyzing gene sequences are known contain one or more spurious AUG expression in Escherichia coli and yeast. Methods Enzymol. codons which start short open reading frames upstream of 100:193-308. their long open reading frames (for reviews, see references 10. Coruzzi, G., and A. Tzagoloff. 1979. Assembly of the mitochon- 29, 34, and 35). Like PETIJI, the majority of these mRNAs drial membrane system. DNA sequence of subunit 2 of yeast so far identified in higher eucaryotes encode proteins in- cytochrome oxidase. J. Biol. Chem. 254:9324-9330. volved in regulation, such as oncogenes, hormones, and 11. Costanzo, M. C., and T. D. Fox. 1986. Product of Sac- hormone receptors (for a compilation, see reference 35). In charomyces cerevisiae nuclear gene PET494 activates transla- several cases, it has been shown that these long tion of a specific mitochondrial mRNA. Mol. Cell. Biol. 6: upstream 3694-3703. leaders are not needed for gene activity (23, 61), which 12. Costanzo, M. C., E. C. Seaver, and T. D. Fox. 1986. At least two strengthens the idea that they might function in regulation. nuclear gene products are specifically required for translation of In addition to PET]JJ, several other S. cerevisiae genes a single yeast mitochondrial mRNA. EMBO J. 5:3637-3641. have 5'-leader regions that contain upstream AUGs. For 13. Daum, G., P. Boehni, and G. Schatz. 1982. Import of proteins example, the short leader region of PPRI, the positive into mitochondria: cytochrome b2 and cytochrome c peroxidase regulator of the URAl and URA3 genes, contains two AUG are located in the intermembrane space of yeast mitochondria. codons preceding the start of translation (38). Similarly, J. Biol. Chem. 257:13028-13033. there are AUGs in the 5'-leader sequence of the longer HTSJ 14. Dieckmann, C., T. J. Koerner, and A. Tzagoloff. 1984. Assembly mRNAs, encode of the mitochondrial membrane system. CBPI, a yeast nuclear which the mitochondrial form of histidine gene involved in 5' end processing of cytochrome b pre-mRNA. tRNA synthetase (48). No regulatory role has yet been J. Biol. Chem. 259:4722-4731. assigned to these 5'-leader regions. With another yeast gene, 15. Dieckmann, C. L., L. K. Pape, and A. Tzagoloff. 1982. Identifi- the transcriptional activator GCN4, the long 5' leader is cation and cloning of a yeast nuclear gene (CBPI) involved in responsible for regulation of the gene at the level of transla- expression of mitochondrial cytochrome b. Proc. Natl. Acad. tion (27, 65). The translational efficiency of its mRNA is Sci. USA 79:1805-1809. modulated in response to amino acid starvation by trans- 16. Dieckmann, C. L., and A. Tzagoloff. 1985. Assembly of the acting factors whose sites of action lie within the long 5' mitochondrial membrane system. CBP6, a yeast nuclear gene leader, specifically at the four small open reading frames (24, necessary for synthesis of cytochrome b. J. Biol. Chem. 260: 28, 46). We do not yet know if the expression of is 1513-1520. PET]J] 17. Douglas, M. G., B. L. Gelier, and S. D. Emr. 1984. Intracellular similarly controlled at the level of translation or if the level of targeting and import of an F1-ATPase f3-subunit-p-galactosidase PET111 in turn modulates coxII synthesis. However, the hybrid protein into yeast mitochondria. Proc. Natl. Acad. Sci. existence of many nuclear genes that control translation of USA 81:3983-3987. specific mitochondrial mRNAs (22) suggests the possibility 18. Dujon, B. 1981. Mitochondrial genetics and functions, p. that they may be used to modulate mitochondrial gene 505-635. In J. N. Strathern, E. W. Jones, and J. R. Broach (ed.), expression. The molecular biology of the yeast Saccharomyces: life cycle and inheritance. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. ACKNOWLEDGMENTS 19. Ebner, E., T. L. Mason, and G. Schatz. 1973. Mitochondrial We thank Jim Ingles, University of Toronto, Toronto, Canada, for assembly in respiration-deficient mutants of Saccharomyces performing computer searches with the PET]J] sequence. cerevisiae. II. Effect of nuclear and extra-chromosomal muta- This work was supported by Public Health Service training grant tions on the formation of cytochrome c oxidase. J. Biol. Chem. GM07273, research grant GM29362, and Research Career Develop- 248:5369-5378. ment Award HDO0515 from the National Institutes of Health. 20. Faye, G., and M. Simon. 1983. Analysis of a yeast nuclear gene involved in the maturation of mitochondrial pre-messenger RNA of the cytochrome oxidase subunitI. Cell 32:77-87. LITERATURE CITED 21. Fox, T. D. 1979. Five TGA "stop" codons occur within the translated sequence of the yeast mitochondrial gene for 1. Allison, D. S., and G. Schatz. 1986. Artificial mitochondrial cytochrome c oxidase subunitII. Proc. Natl. Acad. Sci. USA presequences. Proc. Natl. Acad. Sci. USA 83:9011-9015. 76:6534-6538. 2. Ammerer, G. 1983. Expression of genes in yeast using the ADCJ 22. Fox, T. D. 1986. Nuclear gene products required for translation promoter. 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