exposed to x-ray film (Kodak X-OMAT AR GBX-2) at -70 "C with intensifying screen. Digesxion with restriction endonucleases and random priming to label DNA ...
THEJOURNAL OF BIOLOGICAL CHEMISTRY
Val. 264, No. 26, Issue of September 15, pp. 15628-15633.1989
0 1989 by The American Society for Biochemistry and Molecular Biology, Inc
Printed in L I S A .
Energy-linked Anion Transport CLONING, SEQUENCING,AND CHARACTERIZATION OF A FULL LENGTH cDNA ENCODING THE RAT LIVER MITOCHONDRIAL PROTON/PHOSPHATE SYMPORTER* (Received for publication, April 6, 1989)
Gloria C. FerreiraS, Raymond D. Pratts, and Peter L. Pedersenll From the Laboratory for Molecular and Cellular Bioenergetics, Department of Biological Chemistry, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
A full length cDNA clone encoding the precursor of ble for the translocationof Pi across the inner mitochondrial the rat livermitochondrial phosphate transporter (H+/ membrane (1-3). The major transport system, the Pi carrier Pi symporter) has been isolated from a cDNA library (Pic),’ catalyzes an N-ethylmaleimide-sensitive PJH’ symusing a bovine heart partial length phosphate trans- port, whereas the other system catalyzes an N-ethylmaleimporter clone as a hybridizationprobe. The entireclone ide-insensitive electroneutralexchange of Pi and/or dicarboxis 1263 base pairs in length with 5’- and 3”untrans- ylate (3, 4). lated regions of 16 and 168 base pairs, respectively. Pic has been purified to homogeneity from bovine heart and The open reading frameencodes for the mature proteinrat liver mitochondria (5,6). Also, the ratliver mitochondrial (312 amino acids) preceded by a presequence of 44 transporter has been successfully reconstituted into phosphoamino acids enriched in basic residues. The polypeptide sequence predicted from the DNA sequence was con- lipid vesicles, retaining a high degree of function (7). The firmed by analyzing thefirst 17 amino-terminal amino sequence of an amino-terminal fragment (8), as well as the partial sequence of the bovine heart Pic, has been determined acids of the purephosphate transporter protein. The rat liver phosphate transporter differs from by thechemical protein sequence analysis (9). More recently, the bovine heart transporter in 32 amino acids(i.e. 10%). amino acid sequence has been deduced from the nucleotide It contains a region from amino acid 139 to 159 which sequence of several overlapping clonesencoding for the bovine is 37% identical with the @-subunitof the liver mito- heart Pic (10). However, a full length clone encoding for the chondrial ATP synthase. Amino acid sequence compar- Pic is a necessary prerequisite for both the expression of a isons of the Pi transporter with Pi binding proteins, functional transporter and the study, by site-directed mutaother H+-linked symporters, and the human glucose genesis, of the role of individual amino acids in the transport transporter did not reveal significant sequencehomol- mechanism. Inthispaper, we reporttheisolation, sequencing, and ogy. Analysis of genomic DNA from both rat andS. cere- characterization of a full length cDNA clone for the rat liver uisiae by Southern blots using therat liver mitochon- mitochondrial Pic. In addition, we have examined the capacity drial Pi carrier cDNA as a probe revealed remarkably of this clone to hybridize with genomic DNA sequences from similar restriction patterns, a finding consistent with both rat and the yeast, Saccharomyces cereuisiae. the presence in lower and higher eukaryotes of homologous Pi carrier proteins. EXPERIMENTALPROCEDURES This is the first report of the isolation, sequencing, Materials and characterization of a full length cDNA coding for a protein involved in energy-coupled Pi transport. A rat liver Xgtll cDNA library was purchased from Clontech
-
The supply of substratesrequired foroxidative phosphorylation in eukaryotic cells involves the exchange of cytoplasmicADPfor mitochondrial ATP and the uptake of inorganic phosphate (Pi)from the cytoplasm into the mitochondrial matrix. Nucleotide exchange is maintained by the ADP/ATP carrier, while two transport systems areresponsi-
* This work was supported in partby National Science Foundation Grant DMB-8606759 (to P. L. P.). 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 solely to indicate this fact. The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) M23984. 3 Supported by Fellowship DRG-986 from the Damon RunyonWalter Winchell Cancer Fund. Supporkd by National Institutes of Health Physician Scientist Award K12-AM01298. V To whom correspondence and reprint requestsshould be addressed.
Laboratories. Restriction enzymes were obtained from New England Biolabs, United States Biochemicals, Bethesda Research Laboratories (BRL),PharmaciaLKB Biotechnology Inc., and Boehringer Mannheim Biochemicals and were used according to the suppliers’ instructions.Sequenase and Klenow fragment were from United States Biochemicals, and T4DNA ligase was from New England Biolabs. Universal sequencing 17-merprimers,M13mp18RF and -19RF, pUC18 and -19, and J M 101 strain were purchased from New England Biolabs. pUC-9, deoxy- and dideoxynucleotide triphosphates, and random hexamers were from Pharmacia. 13%]dATPwas from Amersham Corp., and ”P-labeled nucleotides were from ICN. Acrylamide and gel reagents were products of Bio-Rad, and Seakem GTG agarose was from FMC Corp. Bromochloroindoyl galactoside, isopropyl thiogalactoside, and lysozyme were from Sigma. NA45 nitrocellulose (DEAE) membrane was obtained from Schleicher & Schuell. All other chemicals were of the highestpurity available. Escherichia coli strain Y1090 was a gift of Dr. D. W. Cleveland (The John Hopkins School of Medicine). S. cerevisiae 2180-1Awas obtained from the Yeast Genetic Stock Center, Berkeley, CA. A partial bovine heart P,c cDNA was kindly provided by Dr. J. E. Walker (Medical Research Council, Cambridge). The abbreviations used are: P,c, phosphate carrier/or phosphate transporter; SDS, sodium dodecyl sulfate; bp, base pair(s); kb, kilobase pair(s).
15628
Mitochondrial Liver Phosphate Transporter Rat Methods
15629 RI
0
SP
0
RI
HD
HD
RI
Library Screening-A rat liver cDNA library was screened with a ['"PIdATP-labeled DNA fragment according to the method of Benton and Davis (11).The DNA fragment was obtained after EcoRI and HindIII restriction of partial bovine heart Pic cDNA clone, followed by separation by agarose gel electrophoresis (12) and random primer labeling of the electroelutedPic DNA (13). The first round of screening of lo6 phages (plated a t a density of 300-400 plaques/cm2) yielded 19 potential positive clones. These were rescreened a t a lower density (30,000 plaques/l5-cm dish), and the remaining 12 potential positive FIG. 1. Sequencing strategy for the Pic cDNA clone. The clones were subjected to one more roundof screening a t a density of of sequence obtained from less than 100 plaques/l6-cm dish. This led to the isolation of 10 arrows represent the direction and length each fragment cloned into M13mp19 and -mp18. The protein coding cDNA clones. Isolation and Analysis of Recombinants-The plaques giving the region is represented by the heauy closed box. The single-stranded strongest signal were amplified, and the phage DNA was purified templates were sequenced by the dideoxy chain termination method as described under "Methods." RI = EcoRI; HD = HindIII; SP = essentially as described by Maniatis et al. (12). The recombinant DNA was released by digestion with KpnI and SacI, and the cDNA SphI; 0 = Eco0109I. clone thatyielded the longest insert was further restricted withEcoRI. The products were separated through a 1%agarose gel, electroeluted from thegel with a DEAE-membrane according to the manufacturer's instructions. DNA was recovered by phenol extraction and ligated into theEcoRI site of M13mp18 and -mp19vectors. The recombinant -40 -30 DNA wassequencedaccording to the dideoxy chaintermination 1 M l l C C G G C U l C l l A G W G W l G l l C l C G l C C G l A G C C C * t C ~etPhcScrSerValAlaUi~LMla*rgllMsnP~oPhMsMl~ProHioL~lnLwVsluls method (14),using E. coli .JM 101 as host. Preliminary sequence data enabled further restrictions of the recombinant DNA: 1) EcoRI and SphI; 2) EcoRI and HindIII; 3)EcoRI and Eco01091, that were used to create new cloned fragments and complete thesequence. The DNA sequences generated were compiled and analyzed with the help of the BIONET Data Base (Intelligenetics, Inc.) and the DPSA program obtainedfromDr.Ch.Marck(CentreD'Etudes Nucleaires de Saclay, France). Alignment of amino acid sequences was performed using the ALIGN Program (Scientific and Educational Software,SilverSpring, IMD). This programused themethod of Hirschenberg(15)tofindthebest overall alignment between all residues in two sequences. Hybridizations with Genomic DNA-High molecularweight genomic DNA was isolated from rat liver (16) and S. cereuisiae 21801A (17). The restriction enzyme digestions were performed overnight a t 37 "C, and the resulting fragments were separated on a 0.7% agarose gel, depurinated, denatured, and transferred to nylon membranes (16). Hybridizations were performed using sodium dodecyl sulfate/bovine serum albumin solutions with 50% deionized formamide a t 68 "C (18).After hybridization, the blotswere washed twice with 2 X SSC (0.15 M NaCI, 0.015 M sodium citrate, p H 7.0), 0.1% SDS at 25 "C, once with 1 X SSC, 0.1% SDS, and then at final stringency of 0.2 X SSC, 0.1 SDS at 68 "C for 45 min. Filters were exposed to x-ray film (Kodak X-OMAT AR GBX-2) a t -70 "C with 160 rm 180 intensifyingscreen. Digesxion with restriction endonucleases and 631 GMCAACGCllullGCGllClACMGGGlGllGClCClGlGlGWlWWUWlCCUlACACCAlWl~GllCGCClGClllGM random priming to label DNA fragments were performed according GluClUGlvLeul~n)ilaPhelvrL~GlyYalAllaPr~VaIfr~efArgGlnlleProlyrlh~letlletLysPhMl~CysPheCLu to instructions provided by the commercial suppliers. Protein Sequence Analysis-Amino-terminal sequence analysis of the purified rat liver mitochondrial P,c, through17 residues, was performed on a 470A Applied Biosystems sequenator. Purified rat liver mitochondrial carrier was prepared as described by Kaplan et al. (6), except the fast flow DEAE-Sepharose was used in place of Sepharose CL-GB, and the cardiolipin in all the column buffers was replaced with a mixture of asolectin (80%)/cardiolipin (20%) a t 1.0 mg/ml. An aliquot (16 pg) of the purified protein was precipitated with 50 volumes of 100% ethanola t -20 "C. The resulting precipitate was resuspended in aqueous 100 mM 2-mercaptoethanol/acetonitrile/ dimethyl sulfoxide (1:l:l). RESULTS
Isolation and Sequence of a Full Length Rat LiverPiccDNA Clone-After the third roundof screening of a rat liver cDNA library in X g t l l (-lo6 plaque-formingunits), 10 positives reacted with a "P-labele'd hybridization probe, derived from a bovine heart clone (10). The 10 recombinant clones were isolated and amplified, and each DNA was purified for further analysis. The longest clone, predictedto be of sufficient length to encode the complete Pic, was sequenced. Fig. 1 illustrates the sequencing strategy, while Fig. 2 summarizes both the nucleotide and aminoacid sequences. The entireclone is 1263 bp in length with 5'- and 3"untranslated regions of 16 and 168 bp, respectively. The open reading frameencodes for the
1261 G M
FIG. 2. Nucleotide and predicted amino acid sequence of a full length cDNA clone encoding the precursor of the rat liver mitochondrial Pic. Numbering of the nucleotides starts at the second base of the EcoRI linker introduced during library construction.Thefirstamino acid of the Pic is designated+1, andthe presequence runs from amino acid -44 to amino acid -1.
15630
Rat LiverMitochondrial Phosphate Transporter TABLEI cDNA sequence versus protein sequence The P, transporter protein was prepared as described under"Experimental sequence was determined using a 470A Applied Biosystems sequenator. Pi transportercDNA Chemical protein sequence
Procedures." The amino acid
NH2-A-V-E-G-Y-S-C-E-F-G-S-M-K-Y-Y-A-L-C NH2-A-V-E-G-Y-S-C---F-G-S-M-K-Y-Y-A-L-C (E)
TABLE I1 Amino acid differences between rat liver and bovine heart mitochondrial P, transporters Residue No." Rat liver Bovine heart"
Another computer search revealed a number of segments in other proteins which are similar to various regions in the sequence of the ratliver mitochondrial Pic.Among them, and as previously reported (9, lo), the bovine heart Pic, ADP/ ATP carrier,andtheuncouplerprotein yielded thebest 4 GlY Glu 5 Gln matches throughout the entiresequence. Although the align9 Glu ASP ment of the three proteins gives an overall sequencesimilarity 10 Phe Tyr in the the same range (i.e. 20-25%) as in thecase of other Pi13 Met GlY binding proteins and H'-linked symporters (Table 111), the 14 LYs three mitochondrial proteins contain three highly conserved 15 Trr Phe Phe 16 Tyr internal repeats of approximately 100 amino acids in length. 17 Ala Ile In fact, the sequence alignment of the nine repeats suggests 21 Phe Leu that the three internal repeats of the three proteins are related 24 Val Ile to each other. 25 Leu Ile Secondary Structure Comparison withOther Energy-Trans29 Leu Thr fer and P,-binding Proteins-The rat liver mitochondrial Pic 34 Val Leu 53 sequence was further analyzed by computing a hydropathy GlY Ser 60 Ile Val plot (19). As shownin Fig. 3, thisanalysispredictsthe 67 Val Phe presence of six membrane-spanning segments,a characteristic 78 Leu Phe in common with the bovine heart ADP/ATP carrier and the 83 Met Leu uncoupler protein. Thepolypeptide chain of Pic possibly spans 97 Ala V a1 the membranesix times and, assuming that Cys-41 the residue 102 Ile Met 108 Thr Ala is on the cytoplasmic side of the membrane (20), both the 153 Glu ASP NH, and COOH termini would be on the matrix side of the Ala 155 Val inner mitochondrial membrane, as illustrated in Fig. 4. In a 165 Asn LYs side by side comparison, hydropathy plots of the mitochonVal Leu 174 drial energy transfer proteins withE. coli H+-linked symportSer 209 Thr ers and human glucose transporter show a major difference. Pro 211 Ala 252 Gln Glu 12 a-helices Allof the latter transporters appear to have 255 Gln LYs associated with the membrane. These data suggest that at a Leu 311 TYr secondary structure level the mitochondrial transport pro313 Glu Gln teins belong to a class of transport proteins distinct from ' Sequence numbering and sequence from Runswick et al. (IO). those of E. coli and erythrocytes. Analysis of Genomic DNA of Rat and S. cereuisiae with mature protein (312 amino acids),preceded by a presequence Probes Derived from Rat PiTransporter cDNA-In order to of 44 amino acids. The molecular weight of the mature protein determine the extent of homology in the gene structure and is calculated to be 34,740, whereas the precursor is 39,450. sequences for Pic proteins in the rat and lower eukaryotic The sequence of the first 17 amino acids of the purified rat genomes, digests of rat and yeast (S. cereuisiae) DNA were liver mitochondrial Pic agrees with that predicted from the hybridized with probes derived from the rat liver Pic cDNA. nucleotide sequence (Table I). These genomic DNAs were digested with three different reSignificantly, the rat liver Pic sequence differs in 32 amino striction enzymes: EcoRI, BamHI, and HindIII. The fragacids (10%) from that of bovine heart (Table 11). Moreover, a ments were separated byelectrophoresis and examined by glutamine residue is present at the fifthposition of the bovine Southern blot hybridization. heart Pic withouta corresponding amino acid in the rat liver Cleavage of the rat and yeast genomic DNA with EcoRI transporter, leading consequently to 313 and 312 amino acid and HindIII, enzymes that have respectively one and two proteins, respectively. All these differences are conservative, restriction sites in the coding region of the Pic cDNA, gave a and although four of them represent charged residue differ- unique bandwhen probed withan 814-bp SphIIBclI fragment ences, the overall net charge is the same in both sequences. of thePic cDNA (Fig. 5 A ) . Incontrast,treatmentwith H'Sequence Homology with P,-binding Proteins and Other BamHI, anenzyme that does not act in theP,c cDNA region, Linked Symporters-A computer searchof the BIONET pro- yielded one band with a higher molecular weight (4.2 kb) for tein data bankwas used to compare the amino acid sequences rat genomic DNA andtwo bands (4.3 kb and8.0 kb) for yeast of the mitochondrial Pic with E. coli H'-linked symporters genomic DNA, whenprobed with the SphIIBclI fragment. (e.g. lactose permease,xylose proton symporter, andmelibiose Hybridization of yeast genomic DNA with a probe that incarrier), E. coli Pi-binding protein, and humanglucose trans- cludes the amino terminus of the Pic cDNA (i.e. extending 80 to the PstI sitenucleotide at from the PstI sitenucleotide at porter. This search did not reveal any striking similarities 914) gave a pattern similar to the SphIIBclI fragment, except (Table 111). The greatest overall identity in amino acid sefor an additional band present6.6 atkb (Fig. 5B). Ratgenomic quence (24%) was with the E. coli xylose carrier (Table 111).
15631
Rat Liver Mitochondrial Phosphate Transporter TABLEI11 Summary of amino acid sequence alignments for various transporters and Pi-binding proteins Amino acid sequenceswere aligned by computer, and identical residues werecounted. The values in the vertical columns were calculated by using the formula: % PA1 =
number of identical amino acids x 100 number of total amino acids
where the total number of amino acids refers to the carrier heading the column. P,c, rat liver P, carrier; AAC, ADP/ATP carrier; UCP, uncoupler protein; LACP,E. coli lactose permease; XYLC, E. coli xylose carrier; MELC, E. coli, melibiose carrier; PBP, E. coli Pi-binding protein;and HMGT, human glucose transporter. Percentage amino acid identity (PAI)
P,c
Pic 17.0 AAC
16.0
27.0 22.0 23.0
UCP LACP XYLC 25.0 MELC 24.0 PBP HGTR 23.0
100 15.022.0 21.0 23.0 24.0 22.0 19.0 23.0
UCP
LACP
XYLC
MELC
23.0 15.0 100 26.0 21.0
21.0 25.0
18.0
15.0
16.0
17.0
19.0
15.0
17.0 18.0 100 24.0 -
16.0
17.0
14.0 19.0 30.0
100 20.0 21.0
100 23.0 -
20.0 100 22.0 -
19.0
19.0
17.0 16.0
16.0 31.0
PBP
HGTR
AAC
23.0
24.0 100 22.0
16.0 100 -
roplast triose phosphate/phosphate carrier (23) showed no common structural features with the mitochondrial Pic. In (i.e.Pic, the ADP/ATP contrast, three mitochondrial carriers carrier, and the uncoupler protein) seem to form ahomologous protein family (9, 10, 24). The three proteins are thought to be derived from a common ancestor, by gene duplication of an internal repeat corresponding to 100 amino acids (9, 10). Over a 19-amino acid stretch, rat liver Pic is 37% similar to the @-subunitof the ATP synthase. This particular stretch falls outside thosehomologous regions thought tobe involved in ATP binding(22). The significance of this finding, if any, DISCUSSION remains to be established. The hydropathyprofile of the mitochondrialPic is consistIn studies described here, we report the isolation and sequencing of a full length cDNA clone encoding the rat liver ent with six transmembrane segments, which suggests that mitochondrial Pi transporter. The identity of the cloned DNA the amino and carboxyl termini are both on the matrix side was confirmed by determining the sequence of the 17 amino- of the membrane. However, this orientation of the Pic in the be substantiated terminal aminoacids of the purified transporter isolated from inner mitochondrial membrane has yet to inner mitochondrial membraneof rat liver. Three differences with direct experimental data, asfor example reactivity with to on the outside from thebovine heart Picwere found. First, and perhaps mostantibodies directed to peptides predicted be significant, 32 amino ac-iddifferences exist between the bovine or inside membrane surfaces. Interestingly, the bacterial carheart and the rat liver Pi carriers. Second, the rat liver Pic riers (E. coli lactosepermease, xylose/H+ symporter,and consists of 312 amino t3cids and the bovine heart carrier of melibiose carrier) and human erythrocyte glucose symporter 313 amino acids. Third, a glutamine residue comprises the are much more hydrophobic than the mitochondrial carriers 5th residue of the mature bovine heart Pic, while no corre- and have 12 transmembrane segments instead of six, as in the case of the mitochondrialcarriers. Although the secondary sponding amino acid is present in the ratliver carrier. It is interestingtocomparethenumber of amino acid structure of these carriersis quite distinct, only three-dimendifferences reported here and those noted earlier for the ATP sionalstructuralstudies will reveal whetherthe“rules of synthase @-subunitof these same tissues. @-Subunitsof the transport mechanism” are set in the same manner for the if the rat liver and bovine heart ATP synthases contain 479 and differentcarriers. It istemptingtospeculatethat mitochondrial carriers turn out to be dimers (as in the case 480 amino acid residues., respectively,of which only 11 (-2%) are different(21,22).It lippears that more amino acid changes for ADP/ATP carrier (25)), perhaps the mitochondrial car12 membranedomain are tolerated by the Pi carriers than by the @-subunitof ATP riers will functionsimilarlytothe synthases. Perhaps the Pic has undergone a greater frequency bacterial carriers. of mutations. The significance of these changes in the two The protein predicted from the nucleotide sequence indicrucial proteins involved in oxidative phosphorylation has yet cates that the mature rat liver Pic ispreceded by a peptide of to be determined. 44 amino acids. Most nuclear-encoded mitochondrial proteins The only reactive cysteine of the rat liver Pic, previously aretranslatedasprecursors with amino-terminalpreseshown to be located at. the cytoplasmicside of the inner quences which target the protein to its location in the mitomitochondrial membrane (20), is present at position 41, sur- chondria. The presequence is removed by proteolysis during rounded by lysine and arginine residues (Fig. 2). Comparison the import (26). Curiously, the ADP/ATP translocase (27, of the sequences of the mitochondrial Pic with prokaryotic Pi28), as well as the hamsterbrown fat uncoupling protein (29), binding proteins and H’-linked symporters and the human do nothave a presequence. However,it hasbeen demonstrated erythrocyte glucose transporter failed to reveal sequence sim- that the “information” for the importof the ADP/ATP carrier ilarity. Moreover, the recently reported sequence of the chlo- resides in the first 115 amino acids (27, 28). Since recently it DNA digestedwith EcoRI and probed with the PstIIPstI fragment gave a slightl!~ different pattern from the one with 2.8 kb and the BclI/SphI fragment, with an extra band at absence of the 0.6-kb band. Similarly, theHind111 restriction digest yielded a high molecular weight band at 6.6 kb and no band at 0.9 kb (Fig. 5 B ) . The above results indicate that higher and lower eukaryotes contain homologous phosphate carriers. They alsosuggest that more than one region of genomic DNA may exhibit significant homology with the mitochondrial Pic.
Mitochondrial Liver Phosphate Transporter Rat
15632
1
E. coli LACXVSE PERMEASE
"
u
-a-
n.
1.
m.
Iv.
v.
VI.
-3B
49
-._. . . . . .u1 ......... lzm'
1
.....................
"
sa'
Z4d
d
cP
30-
lJn--
8.'
- 1
50* 40-
-
4-
Ea 40
!E.coli -
tu1
b .
2-
I(ELIB1OSE CARRIER
"
1-
ala e-
-le -2ll
-
484 l a 1m 2u ad .I.* 7 . . . . . . . . . . . . . . .-m -30
-41)-51)
JII
m'
Y'
4Sa1
FIG. 3. Comparison of the hydropathy profiles of the Pi transporter, ADP/ATP translocase, uncoupler protein, and E. coli H+-linked symporters. Consecutive hydropathy averages are plotted for a window of 11 amino acids advancing from the NH3 to the COOH terminus. (Z,ZZ = internal repeat 1; III, IV = internal repeat 2; V, VI = internal repeat 3). Uncoupler = uncoupler binding protein.
has been shown that the uncoupling protein has no presequence as well (29, 30), probably its import process in the mitochondrialmembrane is similartothat of ADP/ATP carrier. These findings raise the question of how Pic is imported into mitochondria. Is the presequence involved or is the import process similar to that of the ADPjATP carrier? Analysis of genomic DNA from both rat and S. cereuisiue by Southern blots, using probes to almost the entire coding region of the rat mitochondrial Pic,reveals a similar restrictionpattern.This suggests thatmitochondrialPicasthe mitochondrial ADP/ATP carrier shares significant homology among species both at the aminoacid and thegene structural level. Comparison of published nucleotide sequencesof human and yeast ADP/ATP carriers reveals a 53.5% overall homology, with many stretches of DNA having 80-90% homology. The hybridization data suggest also that the5' region of the FIG. 4. Secondary structure modelof the rat liver mito- rat liver Pjc cDNA shows homology with more genomic fragchondrial Pi transporter based on the hydropathy profile of ments than the most distalregion of the Pic. Whether these the protein. Hydrophobic segments are shown in bones representing transmembrane cu-helical domains. Hydrophilic loops are shown con- are simply repeated sequenceswhich share homologies or represent multiple genes for the same transporter cannot be necting the transmembraneregions. determined at this time. The existence of multiple genes for
Rat Liver Mitochondrial Phosphate
Transporter
15633
3. Pedersen, P. L., andWehrle, J. P. (1982) in Membranes and Transport (Martonosi, A. N., ed) Vol. 1, pp. 645-663, Plenum Publishing Co., New York H B E H B E H B E H B E 4. Kaplan, R. S., and Pedersen, P. L. (1983) Biochem. J. 212, 27923.1288 Pst-Pst > 23.15. Kolhe, H. V. J., Costello, D., Wong, A., Lu, R. C., and Wohlrah, 9 4m H. (1984) J . Biol. Chem. 259,9115-9120 .:6.6.? 6. Kaplan, R. S., Pratt, R. D., and Pedersen, P. L. (1986) J. Bid. 2.32.0. Chem. 261, 12767-12773 7. Kaplan, R. S., Pratt, R. D., and Pedersen, P. L. (1989) Methods 1.oEnzymol., in press 8. Kolbe, H. V. J., and Wohlrab, H. (1985) J . Bid. Chem. 2 6 0 , 0.615899-15906 Sph-Bcll 9. Aquila, H., Link, T. A., and Klingenberg, M. (1987) FEBS Lett. Hlndl I I 212,l-9 EcoRI Hlndlll PstlPstl I Bcll P f tI Soh I 10. Runswick,M. J., Powell, S. J., Nyren, P., andWalker, J. E. (1987) EMBO J . 6, 1367-1373 11. Benton, W. P., and Davis, R. W.(1977) Science (Washington, D.C.)196, 180-184 Presequence PI Carrrer 12. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Cold Spring Harbor Laboratory, Cloning:A Laboratory Manual, 188 bp Cold Spring Harbor, NY FIG. 5. Hybridization of genomic DNA of rat and the yeast S. cereuisiae with probes derived from rat Pi transporter 13. Feinberg, A. P., and Vogelstein, B. (1983) Anal. Biochem. 132, 6-13 cDNA. Genomic DNA from rat and S. cerevisiae was digested with: 14. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. 1) HindIII ( H ) ; 2)BamHI ( B ) ; and3) EcoRI ( E ) , subjected to Acad. Sci. U. S. A. 74, 5463-5467 electrophoresis, transferred to Hybond-N membranes, and hybridized 15. Hirschenberg, D. S. (1975) Commun. Assoc. Comput. Mach. 18, with the probes as described under“Methods.” A , Southern blot 341-343 probed with 814-bp SphI-BclI fragment. R, Southern blotprobed with 16. Davis, L. G., Dibner, M. D., and Battey, J. F. (1986) in Basic 834-hp PstI-PstI fragment. The markers are the positions of fragMethods in Molecular Biology, Elsevier Science Publishing Co., ments of DNA from bacteriophage X generated by digestion with the Inc., New York restriction enzyme HindIII. 17. Sherman, F., Fink, G. R., and Hicks, J. B. (1986) in Methods in Yeast Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY mitochondrial transporters has beenshown for both yeast 18. Hames, B. D., and Higgens, S. S. (1986) Nucleic Acid Hybridizaand mammalian systems(31). tion: A Practical Approach, pp. 113-133, IRC Press, WashingKnowledge of the molecular structure and topology of the ton, D.C. P, carrier in the membrane is a prerequisite to the understand-19. Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 157, 105-132 ing of the mechanism of Pi transport across the membrane. 20. Houstek, d., and Pedersen, P. L. (1985) J. Biol. Chem. 260,62886295 With a full length cDNA clone encoding the Pi transporter, we can now answer questionspertinenttothe molecular 21. Breen, G. A. M., Holmans, P. L., andGarnett,K. E. (1988) Biochemistry 27, 3955-3961 mechanism of Pi transport across the mitochondrial mem22. Garboczi, D. N., Fox, A. H., Gerring, S. L., and Pedersen, P. L. brane as well as the role of individual amino acids in the (1988) Biochemistry 27,553-560 transport process. 23. Flugge, U. I., Fisher, K., Gross, A,, Sehald, W., Lottspeich, F., and Eckerskorn, C. (1989) EMBO J. 8,39-46 Acknowledgments-We wish to thank Dr. P. Shenbagamurthi for 24. Saraste, M., and Walker, J. E. (1982) FEBS Lett. 144, 250-254 25. Aquila, H., Link, T. A., and Klingenberg, M. (1985) E M B O J . 4, performing the amino-terminal amino acid sequencing of the mito2369-2376 chondrial phosphate carrier. We are grateful to David Garboczi for 26. Hay, R., Bohni, P., and Gasser, S. (1984) Biochim. Biophys. Acta carefully reading the manuscript and for his advice throughout the 779,6547 course of this work. We also thank Dr. Xavier Ysern for discussions throughout the study.Finally, we thank Starlene Murrayfor process- 27. Smagula, C., and Douglas, M. G. (1988) J . Biol. Chem. 263, 6783-6790 ing this manuscriptfor publication. 28. Adrian, G. S., McCammon, M. T.,Montgomery, D. L., and Douglas, M. G. (1986) Mol. Cell. Bid. 6, 626-634 REFERENCES 29. Kozak, L. P., Britton, J. H., Kozak, U. C., and Wells, J. M. (1988) 1. LaNoue, K.F., andSchoolwerth, A. C.(1979) Annu.Rev. J. Biol. Chem. 263, 12274-12277 Riochem. 48,871-922 30. Ridley, R.G., Patel, H. V., Gerher, G. E., Morton, R. C., and 2. Klingenherg, M. (1985) in The Enzymes of Biological Membranes Freeman, K. B. (1986) NucleicAcid.7 Res. 14, 4025-4035 (Martonosi, A. N., ed) Vol. 1, pp. 511-553, Plenum Publishing 31. Lawson, J . E., and Douglas, M.G. (1988) J . Biol. Chem. 2 6 3 , Co., New York 14812-14818 B
A
Rat Yeast
Yeast Rat
.
