Separate Genes Encode Functionally Equivalent ... - Semantic Scholar

T H EJOURNAL OF BIOLOGICAL CHEMISTRY 0 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 263, No. 29, Issue of October 15, pp. 14812-14818, 1988 Printed in U.S. A.

Separate Genes Encode Functionally Equivalent ADP/ATP Carrier Proteins in Saccharomyces cereuisiae ISOLATION AND ANALYSIS OF AAC2* (Received for publication, April 22, 1988)

Janet E. LawsonS and Michael G. Douglas$ From the Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas, Texas 75235

Genetic and biochemical analysis of Saccharomyces suggest these mammalian genes are expressed in a tissuecerevisiae containing a disruption of the nuclear gene specific manner (Schultheiss and Klingenberg, 1984; 1985). ( A A C I )encoding the mitochondrial ADP/ ATPcarrier In addition, the deduced amino acid sequences of cDNAs from has revealed a second gene for this protein. The second human fibroblasts (Battini et al., 1987) and human skeletal gene, designated AAC2, has been isolated by genetic muscle (Neckelmann et al., 1987) are only 88% identical again complementationand sequenced. AAC2 contains a 964- suggesting tissue-specific expression of multiple genes. It is base pair open reading frame coding for a protein of not known at present why a gene family for this protein is 318 amino acids which is highly homologous to the present in different organisms or if the individual members AACl gene product except that it is nine amino acids alone are capable of assembling a functional ADP/ATP carlonger at the NH2 terminus. The two yeast genes are rier. highly conserved at the level of DNA and protein and In theyeast Saccharomyces cerevisiae,a gene for the ADP/ share identity with the ADP/ATP carriers from other ATP carrier ( A A C I ) was previously isolated by complemenorganisms. Both genes complement an ADP/ATP carrier defect (opl or pet9). However, the newly isolated tation of the pet9 or opl mutation (O’Malley et al., 1982). The gene AAC2 need be present only in one or two copies deduced amino acid sequence of A A C l showed a high degree while the previously isolated AACI gene must be pre- of identity with ADP/ATP carriers from other organisms sent in multiple copies to support growth dependent on (Adrian et al., 1986). In the present study, we demonstrate a functional carrier protein. This gene dosage-depend- that the deletion of this yeast gene reveals the presence of a ent complementationcombined with the high degree of second gene for the ADP/ATP carrier in yeast. This addiconservation suggest that these two functionally equiv- tional gene, we have termed AAC2, was isolated in the same manner as A A C l and characterized. The deduced amino acid alent genes may bedifferentially expressed. degree of identity sequence of AAC2 also exhibits a substantial with other ADP/ATP carrier proteins including the previously isolated gene from yeast. Although functionally equivThe ADP/ATP carrier or adenine nucleotide translocator alent, thetwo yeast genes differ in their ability to complement is an integral protein of the inner mitochondrial membrane. an ADP/ATP carrier defect. The previously isolated gene It is the most abundant protein in mitochondria (Klingenberg, (AACI ) must be present in multiple copies, while the newly 1976) and is responsible for the exchange of ADP and ATP isolated gene is able to complement whether it is present in with a high degree of specificity across the inner mitochon- single or multiple copies. These studies are consistent with drial membrane. During periods of oxidative metabolism, the the possibility that both genes are expressed in the same ADP/ATP carrier mediates the transfer of high energy phos- manner as other nuclear-encoded mitochondrial precursors phate to other cellular locations. (Laz et al., 1980; Cumsky et al., 1987; Trueblood and Poyton, Although there appears to be only one gene for the ADP/ 1987). ATP carrier in Neurospora crassa (Arends and Sebald, 1984), there is evidence for multiple ADP/ATPcarrier genes in EXPERIMENTALPROCEDURES mammals. In adult humanliver at least two distinct genes for Strains and Media-S. cerevisk strains DC5-Al5 (MATa leu2-3 the ADP/ATPcarrierare expressed atthe mRNAlevel leu2-112 his3 pet9 canl-11, OMalley et al., 1982), W303 (MATa/a (Houldsworth andAttardi, 1988). Differences in antigenic ade2-1 leu2-3,112 his3-11,15 trpl-1urd-1canl-loo), SEY6210 properties and electrophoretic mobilities among ADP/ATP (MATa u r d - 5 2 leu2-3,112 his3-A200 trpl-AgO suc2-A9 Zys2), and carrier proteins isolated from bovine heart, liver, and kidney SEY6211 (MATa u r d - 5 2 leu2-3,112 his3-A200trpl-AgOsuc2-A9 * This work wassupported in part by National Institutes of Health Grants GM25648 and GM36536. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. The nucleotide sequence(s) reported in thispaperhas been submitted to theGenBankTM/EMBL Data Bankwith accession numbeds) 504021. 2 Supported by NationalInstitutes of Health Fellowship 5F 32GM11214 B16. Recipient of Grant 1-814 from The Robert A. Welch Foundation. To whom correspondence and reprint requests should be addressed Dept. of Biochemistry, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235.

used. SEY621-D was constructed by crossing adel-101) were SEY6210 and SEY6211. JLY-1B (MATA l e u 2 3 leu2-112 urd-1 pet9) and JLY3B (MATa leu2-3 leu 2-112 u r d - 1 pet9 aacl::LEU2) were constructed by crossing W303-2D (MATa ade2-1 leu2-3,112 his3-11,15 trpl-1 urd-1canl-100 aacl::LEU2) with DC5-A15. Unless otherwise indicated all yeast strains were grown in YPD medium, 1%yeast extract, 2% bactopeptone, 2% dextrose. Transformed yeast were plated to YNB minimal medium supplemented with the appropriate nutritional requirements (Sherman et al., 1979). Transformants with the appropriate genetic markers were plated to YPG medium, 1%yeast extract, 2% bactopeptone, 3% glycerol. For yeast genomic DNA preparations and mitochondrial preparations, strains weregrown in YPD and/or YPG. For sporulation, yeast strains were grown on presporulation medium (0.8% yeast extract, 0.3% bactopeptone, 10% dextrose) for 2 days then transferred to

14812

/

14813

Second ADP/ATP Carrier in Yeast sporulation medium (1%potassium acetate, 0.1%yeast extract, 0.05% dextrose) (Sherman et al., 1979). Dissected spores were germinated on YPD and tested for genetic markers on YNB plus supplements. Escherichia coli strain JM83 (ara Aflac-proAB) rspL 480 lacZLuMI5) was used for subcloning into pUC vectors for mapping, JMlOl (F' lac pro supE traD36) was used for propagation of M13 constructs for sequencing (Yanisch-Derron et al., 19851, and MC1066 (F' lacX74 galU dalK rspL hdsR trpC9830 leuB660 pyrF::Tn5) (Casadaban and Cohen, 1980) was used for amplification of plasmids isolated from yeast. MC1066 was grown on LB medium and JM83 and JMlOl on 2XTY medium (Maniatis et al., 1982). DNA Methods-Digestion with restriction endonucleases, nick translation, and ligation with T4 DNA ligase wereperformed according to thecommercial supplier. Genomic DNA from yeast was isolated as published (Sherman et al., 1983). Standard agarose gel electrophoresis methods were used (Maniatiset al., 1982). Southern blots (Southern, 1975) were performed using 0.4 N NaOH as a transfer buffer (Reed and Mann, 1985) to nylon filters. Hybridizations were done using sodium dodecyl sulfate/bovine serum albumin solutions (Church and Gilbert, 1984) at 42-50 "C. Autoradiography was performed using Kodak X-Omat AR GBX-2 film. Dideoxy chain termination DNA sequencing with a combination of Klenow and Sequenase (United States Biochemical) enzymes was used. Computer analysis of DNA sequence was done using the Beckman Microgenie program. E. coli transformation was done using a modification of standard procedures (Cohen et al., 1972). Transformation of yeast utilized the lithium acetate-polyethylene glycol method (Ito et al., 1983). A rapid method of preparing plasmid DNA from yeast was used (Lorincz, 1984), and standardminiplasmid DNA preparation methods from E. coli were used (Maniatis et al., 1982). Plosmids-The following plasmids were used pORFl (Weinstock et al., 1983); YEpl3 (2p LEU2) (Broach et al., 1979); pGT46 (CEN3 ARSl LEU2) pSEY8 (2p URA3) and pSEYc63 (CEN4 A R S l URAJ) (Emr et al., 1986). Protein Methods-Mitochondria were prepared from yeast as described (Daum et al., 1982). Standard polyacrylamide gel electrophoresis on 10% gels (Laemmli, 1970) was done. Western blotting was done as published (O'Malley et al., 1982). Immuno detection using the horse radish peroxidase color reaction was performed as described by the supplier. Antiserum to theADP/ATP carrier of N. crmsa was a gift from Dr. Walter Neupert, Munich (Hallermayer et al., 1977; Zimmerman and Neupert, 1980; Saltzgaber-Muller and Schatz, 1978), and antiserum to the ADP/ATP carrier of Zea mays was a gift from Dr. Chris Leaver, Edinburgh.

RESULTS

Deletion of AACI-A gene for the ADP/ATP carrier in 5'. cereuisiae was isolated previously by complementation of the pet9 or opl mutation (O'Malley et al., 1982). Since this gene encodes an ADP/ATP carrier protein (Adrian et al., 1986), we have named it AACl in order to be consistent with current terminology (Smagula and Douglas, 1988). The deduced amino acid sequence of AACl was found to have a significant degree of identity with ADP/ATP carriers from other organisms (Adrian et al., 1986). In fact, this gene has been used as a hybridization probe to select the cDNA for the ADP/ ATP carrier of 2. mays (Baker and Leaver, 1985). In this study, a null mutant in AACl was constructed by deleting the entire coding region from two yeast strains (Rothstein, 1983). A 2.6-kb' BamHI fragment of yeast genomic DNA containing AACl was subcloned in the plasmid pORFl (Fig. 1).The AACl-coding region and a portion of flanking DNA were removed by cleavage at theHindIII andBglII sites. A 2.2-kb HindIII-BamHI fragment containing the yeast LEU2 gene (Andreadis et al., 1984) was inserted in place of the AACl -coding region resulting in LEU2 bordered on both ends by AACl flanking DNA. The plasmid was digested with BamHI, and the linear fragment containing LEU2 and the AACl-flanking DNAwas transformed into both W303a/o( and SEY621-D, diploid yeast strains which were wildtype for the ADP/ATP carrier. The LEU2 gene integrated by homol-

' The abbreviation used is: kb, kilobase.

X Diploid Genome

B

Gene Replacement

4

LEU2

FIG. 1. Disruption of A A C l . The yeast LEU2 gene on a 2.2-kb HindIII-BamHI fragment was used to replace a 1.35-kbHindIII-BglII fragment of DNA containing the AACl -coding region. This resulted in LEU2 bordered on both ends by AACI-flanking DNA. This construction on a 3.45-kb BamHI fragment was transformed into diploid yeast to replace one chromosomal copy of AACI with LEU2.

ogous recombination at the AACl locus resulting in the replacement of one chromosomal copy of A A C l with a larger DNA fragment containing LEU2. A Southern blot of genomic DNA digested with BamHI and probed with the 2.6-kb Barn HI fragment containing both AACl and its flanking DNA demonstrated the presence of two hybridizing bands in the transformed ("replaced") diploid. One of the bands migrated at the position of the wild-type AACl gene. The other band exhibited the mobility expected for the replaced AACl fragment carrying the LEU2 DNA (Fig. 2). The nontransformed diploid showed only the wild-type AACl band. The replaced (AACl/aacl::LEU2) diploids were sporulated, and the resulting tetrads were analyzed by Southern blot analysis. Genomic DNA isolated from each spore of representative tetrads was digested with BamHI and resolved on an agarose gel. As shown in Fig. 3A,the larger probe containing the AACl -coding region and flankingDNA detected both the wild-type AACl gene and the larger fragment containing the LEU2 replacement at theAACl locus. Each spore exhibited the expected meiotic segregation ofAAC1 and aacl::LEU2 DNA.Fig. 3B shows the same blot probed with a DNA fragment containing only the coding DNA for A A C l . The flanking DNA of AACl had been removed at theHindIII and BglII sites in this probe. There was no hybridization to the

Second ADPIATP’ Carrier in Yeast

14814

- Replaced

3 -Wild

Type

4

I

1 i

A

B

FIG.2. Genetic disruption of AACl in the diploid. An autoradiograph of a Southern blot of genomic DNA digested with BamHZ probed with a 2.6-kb BamHZ fragment containing AACl plus its flanking DNA. Lane A, the wild-type AACl band a t 3.6 kb was detected in the nontransformed diploid. Lane B, the wild-type AACl band (3.6 kb) and the LEU2 replacement (4.7 kb) were detected in the diploid transformed with the LEU2construct (Fig. 1).

1-2 1-3 1-4 a b c d a b c d a b c d

” -

by genetic analysis of the sporulated diploids (Table I ) . Since both yeast strains, W303a/a and SEY621-D, are auxotropic for leucine, only those spores containing the copy of LEU2 carried in the gene replacement were prototrophic for leucine (designated “Leu+”in TableI ) . As expected, the spores which were “Leu-” contained the smaller hybridizing DNA fragment, i.e. the wild-type A A C l , while the spores which were leu+ contained the larger hybridizing DNA fragment. Evidence of a Second ADPIATP Carrier Gene-Since the ADP/ATP carrier in yeast is the major component of the inner mitochondrial membrane (Klingenberg, 1976) and is responsible for the exchange of ATP and ADP across that membrane, an ADP/ATP carrier deletion would be expected to result in an organelle unable to carry out oxidative phosphorylation. The yeast opl mutant is unable to grow on a nonfermentable carbon source (Kovacet al., 1967; Kolarov et al., 1972), and this phenotype has served as theselection for AACl (O’Malley et al., 1982). However, as shown in Table I , all spores, including those from whichAACl was deleted, were able to grow normally on the nonfermentable carbon source glycerol (“Gly+”). This observation thatthe uacl::LEU2 spores had functional mitochondria indicated the original opl mutation was not allelic with AACl. Thesedatafurther suggested that the oplmutation could define a second ADP/ ATP carrier gene in yeast. Support for this proposal was obtained by assessing the level of ADP/ATP carrier protein in mitochondria harboring gene replacements of AACl (Fig. 4). Mitochondrial proteins from each spore of tetrad 1in Fig. 3 andTable I were resolved by polyacrylamide gel electrophoresis, blotted to nitrocellulose, and subsequently probed with antisera to theADP/ATP carrier from N. crassa and 2. mays. As shown, all spores exhibited comparable levels of the ADP/ATP carrier protein even though there was no AACl in spores C andD (aacl::LEU2).This observation indicated that a second gene for the ADP/ATP carrier protein was the prominently expressed form under vegetative growth conditions. Furthermore, the existence of a second gene was also suggested by Southern blots of the tetrads using similar hybridization stringency but with a more restricted DNA probe.Fig. 5 shows an autoradiograph of BamHI digests of DNA from TABLEI Genetic data on sporulation of transformed diploids Tetrad Strain

BG

SEY621-D

mH

3-1

a

b

probe

C

3-2

d a b

3-3

d a b

a b c d a b c d a b c d

C

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1-2

1-3

1-4

FIG.3. Meiotic segregation of the AACl gene disruption. Autoradiographs of Southern blots of genomic DNA digested with BamHI. The LEU2-transformed diploids were sporulated. DNA from three representative tetrads (1-2, 1-3, 1-4) of W303a/a (a-d represent DNA from individual spores) is shown. The probes used are shown to the left of the figure. A, the probe detects both wild-type AACI (3.6 kb) and the LEU2 replacement (4.7 kb). B , the probe withouth AACl -flanking DNA detects only wild-type AACI.

DNA of spores in which AACl had been replaced whereas the probe detected the wild-type AACl gene in the nonreplaced spores. Similar results were obtained from the SEY621-D transformants (data notshown). The conclusions of the hybridization data were confirmed

C

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Second ADPIATP Carrier in Yeast

14815

(Fig. 3A). These data supported the presence of a second gene for the ADP/ATP carrier protein which had homology to Z WA A C l coding DNAbut not theA A C l flanking DNA. Isolation of the Second Gene for the ADPIATP Carrier-In 97.4order to isolate the second ADP/ATP carrier gene, the yeast 68 strain DC5-Al5 which contained the oplmutation was transformed with a library of yeast DNA in plasmid YEpl3 (O’Mal43 ley et al., 1982). Two sets of complementing plasmids with distinct restriction patterns were isolated. One set of plasmids was identical to the plasmid containing A A C l that was iso25.7 lated previously (O’Malley et al., 1982). The second set of plasmids, represented by pGlyl5, had a different restriction a b c d a b c d map; however,it hybridized to theA A C l probe (BglII-Hind111 FIG.4. Mitochondria fromthe AACl disruption still harbor fragment, see Fig. 3) on Southern blots. A hybridizing 3.0-kb the ADP/ATP carrier protein. Western blot of mitochondrial HindIII fragment and an overlapping 3.3-kb EcoRI fragment preparations from W303 tetrad 1-2 showing the presence of an ADP/ were subcloned and mapped by restriction enzyme analysis. ATP carrier protein in AACl deleted (aacl::LEU2) spores (a-d des- The map was used to develop the sequencing strategy shown ignate individual spores). The blots shown in A and B are identical, in Fig. 6. The nucleotide sequence and deduced amino acid and each lane of the blotted gel contained equivalent amounts of sequence of the open reading frame found on this 5.0-kb DNA protein. Blot A was probed with polyclonal antisera to theADP/ATP carrier from N. crmsa. Blot B was probed with polyclonal antisera to fragment are shown in Fig. 7 along with the amino acid the ADP/ATP carrier from 2.mays. Marker sizes (left) are given in sequence of AAC1. The gene coded for by the pGlyl5 open reading frame, which we have designated AAC2, was 954base kilodaltons. pairs in length and coded for a protein of 318 amino acids. This open reading frame is nine codons longerthan A A C l . A comparison of the two yeast sequences revealed they were 68.5% identical at the DNA level and 75.3% identical at the amino acid level. The homologybetween the amino acid sequences began at amino acid 21 of the pGlyl5 open reading frame and continued to the termination codon (Fig. 7). The NH2 termini of the two sequences (up to amino acid 20), however, showedessentially no identity. If the sequences were aligned as shown in Fig. 7 to allow for maximum identity over the entire length of the protein, the extra nine amino acids of AAC2 fell at theNH2 terminus. There was one other break in the homology, a seven-amino acid stretch beginning at amino acid 113 of AAC2 and 103 of A A C l . As expected the predicted amino acid sequence fromAAC2 has a high degree of identity with other known ADPIATP carrier sequences. The percentages of identical residues are compared in Table 11. On the whole, all of the ADP/ATP carrier proteins sequenced to date demonstrate a significant level of identity with each other, ranging from 50 to 93%. This includes carriers from fungi, plants, and mammals. There is, however,some variation in the length of these proteins. The mammalian carriers seem to be the smallest at about 297 amino acids (Battini et al., 1987; Neckelmann et dl., 1987; Aquila et aZ., 1982) while2. mays (Baker and Leaver, FIG.5. A more restricted hybridizationprobe defines a sec- 1985) and yeast AAC2 are the longest at 318 amino acids. ond AAC gene. An autoradiograph of Southern blot of BamHI- Yeast A A C l and the N. crassa (Arends and Sebald, 1984) digested genomic DNA from W303 tetrad 1-2 is shown revealing the carrier fall in between at 309 and 313 amino acids, respecA.

0.

a b c d

20 kb-

presence of hybridizing DNA in AACl deleted (aacl::LEU2) spores (a-d designate DNA from individual spores). Probe was 1.35kb BglIIHindIII fragmentcontainingAAC1 -coding region (Fig. 3A).The wildtype AACl band is a t 3.6 kb.

the same tetrad used in Fig. 4 but probed with the 1.35-kb BgZII-Hind111 fragment containing only the A A C l -coding region. In spores A and B which were wild type for A A C l , the probe hybridized to theband containing the wild-type A A C l gene but not to theaacl::LEU2 spores C and D. Under these conditions, however, hybridization to a large (approximately 20 kb) BamHI fragment was observed in all lanes. Hybridization to this larger fragment was also seen in longer exposures of the blot pictured in Fig.3B. Under the identical hybridizing and washing conditions, hybridization to this large BamHI fragment was not seen using the 2.6-kb BamHI fragment containing A A C l and its flanking DNA as a probe

1 1 E-1

HP

EH

HP

X

HPSEHHHP AHP

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FIG.6. Map and sequencing strategy forAAC2. Shown is the restriction map of the 5.0-kb fragment of DNA from pGLY15 containing AAC2. The heavy arrow marks the position of the coding region. Fragment E-I was used for complementation studies. E, EcoRI; H, HindIII; P,PstI; HP, HpaII; A, AccI; S, SaII; X - XbaI. Other AccI and HpaII sitesoccur within the 5.0-kb fragment; however, only those used for sequencing are shown. Sequencing strategy is shown below the restriction map.

14816

Second ADPIATP Carrier in Yeast TATMnTTCACGACMCCC

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ACTCAAAAAGTATATATTCA~AG~TATAC~MCATGCMTACATCTACAAGTCAAA~~~AGCCC~ATATTAT

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Thr GlyAla

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300 270 GAA GGT GiT ATC TCA TIC TGG AGA GGT AAC ACT GCT AAC GlT ATC CGT TAT TTC CCC ACT GluGlyVal Ile Ser Phs Trp ArgGly Asn Thr Ala Asn Val I l e Arg Tyr Phe ProThr

I

717 687 ACTGGT7CTTn:GAAGGTTCATIC'lTGGCTTcATICTn:~GGTGGTGlTDITACT~CT Thr Gly Sar Leu GluGlySar Phe Leu Ala Ssr Pha Leu Leu GlyTrpVal Val T h r Thr

4

I l e Leu Asp Cys Phe Lys A K Thr ~ Ala Thr His

lTC T T A CCT TCT GTC DIT GGT Leu ProSerValVal GlY

Leu Tyr Arg GlyPhs

I

657 627 A T T DIT GTC TAC AGA GGT CTA TAC TTC GGT A m TAC GAT TCT lTG AAG CCT CTA Tn: "G I l e ValValTyrArgGly Lou Tyr Phe Gly net Tyr Amp Ser Leu Lys P r o Leu Leu Leu

I

Gln

C T I TAC AGA GGT

ATAG'ITITATATTAGTAGTATTC'llTCCCG771?TAAA~TCA~ACATGA~~~ATAAAA~CAA~AT TACn'AATCACCGCCAAATAGGT~ATCAC~~CCGGCGGCAGTACmC~AAGGTTAC~GACAA~AACC

A C ~ T C T T A C ' I T I T A A C C A A C C G A G T C ~ A C A ~ T ~ C ~ G A ~ G A G A G ~ C ~ A C G A T A A G l T G

TTCATAGACGTCGACCTAGAGAAC~C~AAAGTCTATCAAAAA~GGAAACTGAA

I

A m G b Let) Leu ASP Val T Y LYE ~ LYE

FIG. 7 . Nucleotide and derived amino acid sequences of AAC2. The derived amino acid sequence of A A C l (Adrian et al., 1986) is listed below the two A A C 2 sequences. The amino acids sequences are aligned for maximum identity. A A C 2 has 10 amino acids between amino acids one and 20 that are not present in A A C l . A gap of one amino acid was allowed between amino acids 120 and 121 of A A C 2 to maintain maximum alignment of the two sequences. TABLEI1 Comparison of amino acid sequence percentage identity amongA T P / A D P carriers f r o m various sources Z. Bovine Human Human N. crassa

mays

52.464.6 48.0

52.7 64.5 74.7

fibroblast muscle

A A c2 50.568.971.075.3 AACl N . crassa 48.4 49.5 2. mays Bovine 93.0 Human fibroblast

52.0

50.3

51.6 88.3

51.2

52.4 50.0

88.3

tively. This degree of conservation suggests that different carrier proteins may be functional in yeast. Studies to test this are currently in progress. Structural Featuresof the AAC2 Gene Product-The AAC2 amino acid sequence exhibits a tripartite primary structure consisting of three similar sequences of about 100 amino acids each. This structure has been seen in the ADP/ATP carriers from beef heart (Aquila et al., 1985) and 2. mays (Baker and Leaver, 1985), the uncoupling protein from brown fat mitochondria (Aquila et al., 1987), and the bovine mitochondrial phosphate carrier (Runswick et al., 1987). The yeast A A C l amino acid sequence shows a similar repeated structure. It has been noted that a proline at the position corresponding

to amino acid 32, a glycine at position 76, twonegative charges (positions 34 and 96) and one positive charge (position 37) are conserved in each repeat of the uncoupling protein. Also, two aromatic residues (positions 54 and 74) are present in each repeat. Although the yeast AAC2 amino acid sequence has only 23% overall identity to the uncoupling protein, the proline and glycine are conserved in the first and third yeast "repeats" and the glycine in the second repeat. The proline in the second repeat is replaced by serine in both yeast genes. The aromatic residues are conserved in the first and third repeats. The three conserved charges are present in the first repeat, and the positive and one of the negative charges are presentinboth the second andthird repeats. The above elements are conserved in the A A C l amino acid sequence, and, in addition, all three conserved charges and both aromatic residues are present in the second repeat. A comparison of the hydrophobicity (Hopp and Woods, 1981) of the yeast AAC2 amino acid sequence with the other ADP/ATPcarriers listed inTable I1 and the uncoupling protein and the phosphate carrier revealed that they are very similar, each containing a pattern of six hydrophobic segments separatedby hydrophilic regions (Runswick et al., 1987; Klingenberg et al., 1987). Thepatterns, however, arenot superimposable. The N . crussa, human, bovine, and 2.mays

14817

Second ADPIATP Carrier in Yeast TABLE111 Comparison of complementation ability of AACl and AAC2 A A C l andAAC2 were subcloned into unit copy (CEN) and multiple copy (2 g m ) yeast shuttle plasmids to determine the ability of each gene to restore growth on glycerol to the respective yeast strains. Prior to transformation, theabove yeast strains were unable to grow on glycerol due to the presence of the pet9 mutation (JLY-1B and DC5-Al5) or the pet9 mutation in combination with the deletion of AACl (JLY-3B).For testing in leucine auxotrophs, AACl and AAC2 were subcloned into YEpl3 ( Z F , LEU2) and pGT46 (CEN3, LEUZ). For testing in uracil auxotrophs, AAC2 was subcloned into pSEY8 ( 2 p , URA3), and A A C l and AAC2 were subclonedintopSEYc63 (CEN4. URA3). (+) growth on alvcerol, (-) no growth on glycerol.

DISCUSSION

In this study the presence of a second gene encoding the ADP/ATP carrier protein was indicated since mitochondrial function was not impaired by deletion of AACl. Further, a mitochondrial ADP/ATP carrier proteincould bedetected by antibody in these deletion strains. In addition, genomic DNA with homology to the AAC1-coding region could be detected in these deletion strains. The second gene encoding the ADP/ATP carrier was isolated by genetic complementation of theopl mutation highly (O’Malley et al., 1982).The two S. cerevisiae genes were similar to each other at DNA and amino acid levels and are AACP AACl quite similar to theADP/ATP carriers from other organisms. 2wm CEN 2pm CEN Several of the ADP/ATP carrier proteins from different orJLY-1B + + + ganisms, the mitochondrial uncoupling protein and the phosW9) phatecarrier have atripartite primary structure, i.e. the JLY-3B + + + primary structure consists of three similar sequences or re(Aaacl,pet9) peats of approximately 100 amino acids each in length (RunDC5-Al5 + + + swick et al., 1987). Several potentially important amino acid (pets) residues, aromatic and charged, are conserved at particular positions in each repeat. The hydrophobicity plots of these ADP/ATP carriers have a hydrophilic peak near the COOH proteins are also remarkably similar. These shared characterterminus that is not present in either yeast sequence. The istics imply these proteins probably evolved from a common AAC2 sequence contains a sharp hydrophilic peak near its ancestor and have similar structures and functions. amino terminus (around amino acid 20) that is only present Since both yeast genes are highly similar to each other and in the carrier protein from 2.mays. This peak is not present to other ADP/ATP carrier genes, it is likely that both are in the phosphate carrier, the uncoupling protein, or any of expressed. The AACl gene was selected due to its ability to the other ADP/ATP carriers (including the yeast AACl). complement a defect at the opl or PET9 locus. Early yeast These similarities in primary structure are in line with the transformation protocols which were developed for complesuggestion that all of these proteins haveevolved from a mentation of pet mutants involved a two-step recovery of common ancestor and have a similar structure and function. transformants from agar following transformation with a 2 p Expression of AAC2-AAC2 on the 3.3-kb EcoRl fragment plasmid library. This method apparently favored the recovery (E-1, see Fig. 6) was placed in both orientations into yeast of plasmids harboring AACl (O’Malleyet al., 1982). From the multicopy 2 micron and single copy centromere containing present study, however, it is dear that AACl could eompleplasmids, pSEY8 and pSEYc63, respectively. Similarly, AAcl mentthis opl mutation only whenAAClwas present in on the 2.6-kb BamHI fragment was moved into a multicopy multiple copies. This data combined with the fact that muplasmid, YEpl3, and two unit copy plasmids, pSEYc63 and pGT46. These recombinant plasmids were transformed into tations inAACl and at oplsegregate independently in meiosis yeast strains containing mutations in either one or both the shows that the opl mutation is not at AACI. On the other ADP/ATP carriers. Strain DC5-Al5 contained the opl mu- hand, the AAC2 gene need be present only in unit copy to tation (O’Malley et al., 1982) and was unable to grow on the complement the opl mutation. Therefore, the opl mutation nonfermentable carbon source glycerol. Strain JLY -lB con- may be in AAC2. Mapping studies to confirm this genetically tained the opl mutation from DC5-Al5 and the wild-type are currently in progress. At this time, we have no evidence AACl gene. Strain JLY-3B contained the opl mutation and for a third ADP/ATPcarrier gene in yeast. Since the ADP/ATP carrier represents the primary route the deletion of AACl (aacl::LEU2). (As noted above strains for the compartmentation of adenine nucleotides across the without the oplmutation but with the AACl deletion are able to grow on glycerol.)Transformants were plated on appropri- inner mitochondrial membrane, there is reason to suspect ate selective media (minus leucine or uracil),and theresulting that construction of a yeast mutant which is deleted for both colonies were replica plated to glycerol to test for mitochon- AACl and AAC2 may be lethal to yeast. Additionally, since drial function. The results are summarized in Table 111. A the ADP/ATP carrier is the major protein component of the single or double ADP/ATP carrier mutation was comple- inner mitochondrial membrane, complete elimination of both mented by the pGlyl5 E-1fragment carrying AAC2 whether AAC gene products may perturb the integrity of the inner this fragment was present in single or multiple copies. Both membrane to a degree sufficient to cause cell inviability. In orientations of E-1 yield identical results indicating that the yeast, such a double mutant construction is conveniently control regions for AAC2 are present on the E-1 fragment. performed by disruption of AACZ in a diploid homozygous for The DNA upstream of the coding region ofAAC2 on E-1 the aaCl::LEU2 disruption. This gene disruption is currently exhibited no homology to AACl or other ADP/ATP carrier in progress. It is noteworthy that the pet9 or opl mutation which renders the ADP/ATP carrier nonfunctional in some genes.‘ In contrast, AACl on the 2.6-kb BamHI fragment must be present in multiple copies in order to complement a manner results in little change in growth on a fermentable single or double ADP/ATP carrier defect. No complementa- carbon source. This could be due to a partial activity of the tion was seen by control plasmids lacking yeast ADP/ATP mutant carrier or to the ability of the AACI product to carrier DNA. These results imply that AAC2 encodes the function at a level sufficient to maintain growth on glucose. ADP/ATP carrier which may bethe major gene, perhaps opl, Normal growth of the double mutant (opl aacl::LEU2) on coding for this protein in yeast. Studies to furtherresolve this glucose, however, indicates that the level of activity of the carrier containing the opl mutation is sufficient to support point are currentlyin progress, growth on a fermentablecarbon source. Growth of the double J.E. Lawson and M.G. Douglas, unpublished results. mutant was unchanged from wild type even at high glucose ~

14818

Second ADPIATP Carrier in Yeast

levels indicating that cataboliterepression of the mutant opI gene product does notinhibit growth onthefermentable carbon source. Despite the overall similarity of the two yeast genes, the NH2 termini of the genes are devoid of identity. From the initation codon to the point where identity between the two more amino acids sequences begins, AAC2 appears to have 10 of the than A A C I . Moreover, the DNA identity upstream coding regions of the two genes is much lower in comparison to the level of identitywithinthe coding regions. These observations combined with the fact that different copy numbers of the two genes appear to be required for complementation of an ADP/ATP carrier defect suggest there may be differential expression of the two genes. Differential expression of multiple genes for mitochondrial proteins has been noted for cytochrome c (Laz et al., 1984; Montgomery et al., 1980) and cytochrome oxidase subunit V (Cumsky et al., 1987; Trueblood and Poyton,1987). Alternatively, thecase in yeast may be somewhat analogous to the mammalian situation in which multiplegenesfor the ADP/ATP carrier exist and apparently areexpressed in a tissue-specific manner. In yeast, one of the two genes couldbe preferentially expressed during sporulation or under anaerobic conditions. Although the extreme amino terminus of a mitochondrial protein is often involved in the import of that protein into mitochondria,recentstudies haveshown thattheamino terminus of the ADP/ATP carrier is not necessary for its import (Pfanner et al., 1987; Smagula and Douglas,1988). These two studies have shown that sequences either part of or adjacent toa transmembrane spanning a-helix, almost 100 residues from the amino terminus, arenecessary for its binding and import.However, one of the regions of the two yeast amino acid sequences which lacked identity was within this region suggested to be potentially important for mitochondrial import (Pfanner et al., 1987; Smagula and Douglas, 1988). Studies to determine if the gene dosage-dependent complementation of these functionally equivalentgene products is a function of level of expression or import into the organelle are currently in progress. The demonstration and selection of a second ADP/ATP carrier gene in yeast now provides the genetic tools necessary of this memto initiatea detailed structure function analysis brane transporter as well as to determine its function, if any, in addition tonucleotide transport. Acknowledgments-We thank Rosa Kim for expert technical assistance and Raquel Voss in the preparation of this manuscript. REFERENCES Adrian, G. S., McCammon, M. T., Montgomery, D. L., and Douglas, M. G. (1986) Mol. Cell Biol. 6, 626-634 Andreadis, A., Hsu, Y.-P., Hermodson, M., Kohlhaw, G., and Schimmel, P. (1984) J . Biol. Chem. 259,8059-8062 Arends, H., and Sebald, W. (1984) EMBO J . 3, 377-382 Aquila, H., Link, T. A., and Klingenberg, M. (1985) Embo J. 4,23692376 Aquila, H., Link, T. A,, and Klingenberg, M. (1987) FEBS Lett. 212, 1-9 Aquila, H., Misra, D., Sulitz, M., and Klingenberg, M. (1982) HoppeSeyler's Z. Physiol. Chem. 363,345-349 Baker, A,, and Leaver, C. J. (1985) Nucleic Acids Res. 13, 5857-5866 Battini, R., Ferrari, S., Kaczmarek, L., Calabretta, B., Chen, S., and Baserga, R. (1987) J . Biol. Chem. 262, 4355-4359

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