Biochimica et Biophysica Acta, 1172 (1993) 205-208 205 © 1993 Elsevier Science Publishers B.V. All rights reserved 0167-4781/93/$06.00

BBAEXP 90464 Short Sequence-Paper

An adenine nucleotide translocator gene from Arabidopsis thaliana

Wolfgang Schuster, Sebastian Kloska and Axel Brennicke Institut fiir Genbiologische Forschung, Berlin (Germany)

(Received 16 November 1992)

Key words: Adenine nucleotide translocator; Mitochondrion; (Arabidopsis)

The sequence of an adenine nucleotide translocator (ANT) gene of Arabidopsis contains three introns, the first of which is located upstream of the assumed initiation codon. The presequence characteristic for plant ANTs is processed also in Arabidopsis as suggested by Western blot analysis, most likely at the conserved cleavage site.

The adenine nucleotide translocator (ANT) medi- ates the ADP and ATP transport between the mito- chondrial matrix and the cytosol [1]. In yeast ceils, however, the functional A D P / A T P translocator is only required for anaerobic growth, but not under dere- pressed conditions [2]. It is as yet unclear how the ANT function can be substituted, particularly since ANT is one of the most abundant proteins in the mitochon- drial membrane. In plants nuclear encoded ANT cD- NAs and the corresponding genes have been identified first in maize [3,4]. These and protein sequence data show that in this plant ANT is synthesized with an amino terminal extension which is processed upon im- port into mitochondria [1]. The potato ANT open reading frame is also extended by a long amino-termi- nal sequence [5] that was found in detailed DNA and protein analyses to be processed in vivo and also in vitro in import experiments [1]. This mode of ANT import appears to be unique to higher plants, since in mammalia and fungi the ANT proteins are targetted with protein internal signals that require no processing [6].

Here, we describe an Arabidopsis gene that encodes ANT. Genomic and cDNA clones for ANT from Ara- bidopsis thaliana (ssp. Columbia) were isolated with the maize pANT-1 probe [3]. Sequence alignment of cDNA and genomic sequences reveals two introns of 89 and 102 nucleotides, respectively, both located in positions

Correspondence to: W. Schuster, Institut ffir Genbiologische Forschung, Ihnestrage 63, D-1000 Berlin 33, Germany. The nucleotide sequence data reported in this paper have been submitted to the EMBL and GenBank databases under the accession number X68592.

identical to maize (Fig. 1). An additional intron is present in the 5' region of the gene. This intron with 458 nucleotides is probably situated outside of the ANT coding sequence, since this region is not con- served between the different plants analysed to date. Sequence conservation of the Arabidopsis cDNA with maize and potato extends only as far as the first in frame methionine codon suggesting this triplet to be the in vivo initiator of translation, although in all plants examined to date the reading frame is open up to the 5' end of the cDNA clone. This assignment positions the first intron in the non-translated leader.

Northern blot experiments (not shown) identify a single transcript of 1600 nucleotides, corresponding roughly in size to the cDNA clones identified. This observation suggests little if any further 5' extension of the ANT transcripts beyond the cDNA end, depending on the in vivo length of the poly(A) tail.

The encoded amino acid sequence of the Arabidop- sis ANT is highly conserved with the respective maize and potato proteins (Fig. 2). The Arabidopsis deduced protein has 83.9% of all amino acids identical with the maize ANT, 83.3% with potato [1], 76% with Neu- rospora [7], 67.9% with yeast [6] and 55.1% with a human ANT [8]. Although the leader sequence of the plant proteins is less conserved than the mature pro- tein sequence, the amino acids surrounding the pro- cessing site of potato [1] are identical with Arabidopsis. The first two amino acids of the mature protein are also conserved be tween the two dicots and maize. These similarities suggest that the mature ANT protein in Arabidopsis mitochondria is also processed from the larger precursor predicted from the cDNA sequence. Western blot experiments with antibodies against the

206

1 AT•TA•cGGT••TA•AGTccccGAGTGT•TAGAATAGTG•GTTAcA•TTGATTcCTTcTAGTGTGAc••TATTT•TGGAG•C•ATAAA•GGcT•GTGT•A

> cDNA

i01 AAACTCGCCTCTTCCCCACAAAGAGAATTCTCTCATTTCTTCTTCTTCGCTCAGCTCCGACAACGAAGGIGC G~ ~%A T~T r T~C 1 n CGC CTC RAT

f a 1 p s i f 1

193 TTC GCA TTG CCA TCG ATT TTC TTA G~LT~AATTCTGGTTACTCTGAGATCTCTATCGCTTT~TCTCGTTGTTTATCTCTGTTCTTCATTTG

284 ATTcTATTGATCTGCTTCACAGTCTTATATATCATCRACRAGGTAAAATGATTCGTTTCGTcTTGATGATCTcATGGTTTCTCTGCGTAATCCTAGATTG

384 RATGTAGTGTAGATCTCGTGTGGAGTGTGAATGT•TCGAGATCCTGGATTATTCGGCTCTGTTACTCTTATTTTCCGCcATTRAGTTACACTGTTGTATG

484 GATCTGTTTA~CCTAR~TTTTCTGCGTCTGRATGTGTTCATGGTTTAGTTTCTGATTGCATCGTTTCTACATGTAGCCTcTCTTATGCTTRATGGTGGAT -

584 RAGTGTTCTATTCcACTGGAGATGCACAATTGCTTTTACTGcACTTTATAGCATATTcCATTTCTTACACTACGGGTTGTTTAATTTTGTAG G GTT

k M V E Q T Q H P T I L Q K V S G Q L L S S S V S

681 AAA ATG GTT GAA CAG ACT CAG CAC CCC ACG ATT CTT CAG AAG GTT TCT GGC CAA CTC TTG AGC TCG AGT GTT TCT

Q D I R G Y A S A S K R P A T ¥ Q K H A A ¥ G N Y

756 CAG GAC ATT CGT GGT TAT GCT TCG GCT TCC ~KAAGG CCT GCT ACA TAC CAG A~A CAT GCA GCT TAC GGA ARC TAC

S N A A F Q Y P L V A A S Q I A T T T S P V F V Q

831 TCC RAT GCT GCA TTT CAA TAT CCT CTT GTG GCT GCA TCT CAG ATT GCA ACT ACT ACT TCT CCT GTG TTT GTC CAA

A P G E K G F T N F A I D F M M G G V S A A V S K

906 GCT CCA GGA GAG RAG GGA TTT ACT RAC TTT GCT ATT GAT TTC A~G ATG GGT GGT GTT TCT GCT GCT GTG TCA AAG

T A A A P I E R V K L L I Q N Q D E M L K A G R L

981 ACT GCT GCT GCT CCC ATT GAG CGT GTC RAG CTT TTG ATT CAA RAC CAG GAT GAG ATG CTT RAG GCT GGA AGG CTT

T E P Y K G I R D C F G R T I R D E G I G S L W R

1056 ACT GAG CCA TAC RAG GGT ATT CGT GAC TGT TTC GGC AGG ACA ATT AGG GAT GRA GGT ATT GGT TCG TTG TGG AGA

G N T A N V I R Y F P T Q - -

1131 GGA RAC ACC GCT RAT GTT ATC CGT TAC TTC CCC ACT CAG GTTGGTTTAGTTTCACTTCTTTCGGCCTTTTTTAAAATGTCTTAGTTA

A L N F A F K D Y F K R L F

1218 TATATGATGTATTGGTTACTGATCTATCTCTCACTTGCTAG GCC TTG RAC TTT GCA TTC AAA GAT TAC TTC RAG AGG CTT TTC

N F K K D K D G Y W K W F A G N L A S G G A A G A

1301 AAC TTC RAG AAA GAC RAG GAT GGC TAC TGG AAG TGG TTT GCT GGT AAC TTG GCA TCT GGA GGT GCT GCT GGT GCC

S S L L F V Y S L D Y A R T R L A N D S K S A K K

1376 TCT TCC CTT CTC TTT GTG TAC TCT CTT GAC TAT GCA CGT ACC CGT CTT GCC RAT GAC TCC RAG TCT GCC RAG RAG

G R G E R Q F N G L V D V Y K K T L K S D G I A G

1451 GGG AGA GGC GRA AGG CAG TTC RAT GGT CTT GTT GAT GTC TAC RAG RAG ACC CTC RAG TCA GAT GGTATT GCT GGA

L Y R G F N I S C A G I I V Y R G L Y F G L Y D S

1526 CTT TAC CGT GGT TTC RAC ATC TCG TGT GCT GGT ATC ATT GTC TAC CGT GGT CTC TAC TTT GGA CTG TAC GAC TCT

V K P V L L T G D L Q 1601 GTG AAG CCT GTC CTC CTC ACT GGA GAC CTG CAG GTTTGTTTGAGCTCTATTCTTATATATCTGTAAGGTGACAGCTTRAGAAATCAGGG

D S F F A S F A L G W L I

1690 ATCCTGATTCTAATGTTGTTATTTCTTTCCTTGCTGGATATTTCAG GAC AGT TTC TTC GCT AGT TTT GCT CTT GGA TGG CTC ATC

T N G A G L A S Y P I D T V R R R M M M T S G E A

1775 ACC RAT GGT GCG GGC CTT GCG TCG TAC CCG ATT GAC ACG GTT AGA AGA AGA ATG ATG ATG ACA TCA GGT GAA GCG

V K Y K S S F D A F S Q I V K K E G A K S L F K G 1850 GTG AAG TAC ~G AGC TCA TTT GAT GCG TTC TCA CAG ATC GTG RAG R~%G GAA GGA GCC RAG TCT CTG TTC AAG GGT

A G A N I L R A V A G A G V L A G Y D K L Q L I V

1925 GCG GGT GCC RAC ATC CTG CGT GCC GTT GCA GGT GCT GGT GTG CTT GCT GGC TAC GAC A~G CTG CAG TTA ATC GTC

F G K K Y G S G G A * 2000 TTC GGC AAG AAG TAT GGA TCT GGA GGT GCC TAA GTACCACCAAATCCCGCAGTCATTAAGCTTCGCCTTTTGTTTTTTTCTCCATTTTT

2089 TTTGCAGTTT•AGATTT•TTTTATAAAAAAGATGTTGATGAAAT•ATTT•••CATAAAATTAGAGAGG•GC/%AGGGGA/&ATATTATCTGTT

Fig. 1. Nucleotide sequence of the Arabidopsis thaliana ANT2 gene. The open reading frame is shown in translated triplets up to the beginning of the cDNA clone (arrow). The amino acid sequence encoded upstream of the first in frame methionine codon (shown in small letters) is probably not translated since conservation with other plant ANTs is observed only with the downstream region. Positions of the three introns

were identified from the cDNA-genomic DNA comparison. The conserved G T / A G dinucleotides at the intron borders are underlined.

207

Arabidopsis ANT2 Potato ANTI Maize ANT-G1 Maize ANT-G2 Yeast PET9 Neurospora ANTI Human ANTI

Ara ANTI Pot ANTI Mai ANT-G1 Mai ANT-G2 Yea PET9 Neu ANTI Hum ANTI

Ara Pot Mai Mai Yea Neu Hum

Ara Pot Mai Mai Yea Neu Hum

ANTI ANTI ANT-G1 ANT-G2 PET9 ANTI ANTI

Y A S A S K R ~ T ~ ~ A ~ - - ~ T T T - - ~ YGGVQ -BI ~ ~ G L~RGQATQDL~L~TS NA - - ~ ~ A ~ c ~ s v ~ ~ s ~ w ~ ~ - ~ 0 ~ ~ N I C P S F S P Y E ~ R - ~ T Q S L W ~ M ~ - ~ P ~ P ~ K

MSHTETQTQQS

~o0~ MGDHA~

ANTI ANTI ANT-G1 ANT-G2 PET9 ANTI ANTI

Ara ANTI Pot ANTI Mai ANT-G1 Mai ANT-G2 Yea PET9 Neu ANTI Hum ANTI

l vo..Ko i

Fig. 2. Amino acid comparison of the Arabidopsis thaliana deduced adenosine nucleotide translocator protein with ANTs of other species. The higher plant ANTs (Arabidopsis thaliana ANT2, this work; potato, Solanum tuberosum [1]; maize, Zea mays [1,4,9]) all show a long presequence that was shown to be cleaved in potato and maize before the amino acid indicated by an arrow. The resulting proteins correspond in length with the fungal (Yeast, Saccharomyces ceret,isiae [6]; Neurospora, Neurospora crassa) [7] and mammalian (human, Homo sapiens [8]) proteins. Amino

acids conserved with the Arabidopsis sequence are shown white in black.

Neurospora ANT confirm this deduction, since only a single polypeptide of about 30 kDa in the membrane fraction of purified mit0chondria is detected (Fig. 3). The size of the Arabidopsis protein correlates with the processed plant ANT proteins [1].

The ANT gene identified here may not be the only gene encoding an adenine nucleotide translocator in Arabidopsis. A recently sequenced cDNA from Ara- bidopsis, termed ANT1 ( E M B L / G e n b a n k accession number X65549), shows considerable similarity with the ANT sequence analysed here, which is thus named

ANT2. The two genes may encode two distinct ANT proteins, analogous to the two ANT genes also en- coded in maize [4]. Three different ANT genes have been identified in the human and yeast genomes, but their individual functions are as yet unclear [1].

We are grateful to C.J. Leaver and his collaborators (Oxford) for the kind gift of the maize ANT probe and to W. Neupert (Mfinchen) for the generous gift of the antibody against the Neurospora ANT. We also thank U. Halfter and L. Willmitzer (Berlin) for the Arabidop- sis cosmid library.

208

30kd

21.5kd

X e~

~ ® E E

References

1 Winning, B.M., Sarah, C.J., Purdue, P.E., Day, C.D, and Leaver, C.J. (1992) Plant J. 2, 763-773.

2 Drgon, T., Sabova, L., Nelson, N. and Kolarov, J. (1991) FEBS Lett. 289, 159-162.

3 Baker, A. and Leaver, C.J. (1985) Nucleic Acids Res. 13, 5857- 5867.

4 Bathgate, B., Baker, A. and Leaver, C.J. (1989) Eur. J. Biochem. 183, 303-310.

5 Emmermann, M., Braun, H.P. and Schmitz, U.K. (1991) Curr. Genet. 20, 405-410.

6 Adrian, G.S., McCammon, M.T., Montgomery, D.L. and Douglas, M.G. (1986) Mol. Cell. Biol. 6, 626-634.

7 Arends, H. and Sebald, W. (1984) EMBO J. 4, 2369-2367. 8 Cozens, A.L., Runswick, M.J. and Walker, J.E. (1989) J. Mol. Biol.

206, 261-280. 9 Winning, B.M., Day, C.D., Sarah, C.J. and Leaver, C.J. (1991)

Plant Mol. Biol. 1% 305-307.

Fig. 3. The ANT protein is detected only in the membrane fraction of purified mitochondria. Antibodies against the Neurospora ANT show sufficient specificity to detect a single polypeptide in the mitochondrial membrane fraction of Arabidopsis. The size of this protein of about 30 kDa corresponds to the size of the processed protein predicted from the DNA sequences. This result correlates with the data from maize and potato, where processing of the ANT

has been shown to result in a protein of this size [1].