Sequence of a mouse embryo cDNA clone encoding proteolipid subunit 9 (P1) of the mitochondrial H+-ATP synthase

Biochimica et Biophysica Acta, 1184 (1994) 139-141

139

© 1994 Elsevier Science B.V. All rights reserved 0005-2728/94/$07.00

Short Sequence-Paper

BBABIO 40291

Sequence of a mouse embryo c D N A clone encoding proteolipid subunit 9 (P1) of the mitochondrial H÷-ATP synthase L a j o s P i k 6 *, D o n n a E. N o f z i g e r , L i n d a M. W e s t e r n 1 a n d K e n t D. T a y l o r Developmental Biology Laboratory, VA Medical Center, 16111 Plurnrner Street, Sepulveda, CA 91343, USA (Received 14 September 1993)

Key words: ATPase; Proteolipid; Subunit 9 (P1); cDNA; Nucleotide sequence

A cDNA clone to an abundantly expressed mRNA in cleavage stage mouse embryos has been sequenced and identified as encoding subunit 9 (P1) of the mitochondrial H+-ATP synthase. The deduced amino acid sequence of the mature subunit 9 protein differs in a single residue from the corresponding rat, ovine, bovine and human subunits. The mitochondrial H+-ATP synthase complex (FIF0-ATPase; complex V of the mitochondrial oxidative phosphorylation system) is composed of two principal sectors: an integral membrane-bound segment, F0, which is concerned with proton translocation and F 1, the catalytic portion of the enzyme. In mammals, the F 0 segment consists of the proteolipid subunit 9 (dicyclohexylcarbodiimide [DCCD]-binding protein; a homologue of subunit c of bacterial ATP synthase) which is encoded by the nuclear genome and subunits 6 and 8 that are derived from mitochondrial genes [1,2]. The amino acid sequence of subunit 9 has been highly conserved in H+-ATPase complexes from a variety of sources [3]. In the bovine genome, the subunit 9 protein of ATP synthase is encoded by two different nuclear genes, designated P1 and P2. The mRNAs derived from the two genes produce identical mature protein subunits but differ in the amino acid sequence of the import signal peptide, which is removed during entry into the mitochondrion, and have divergent 5' and 3' untranslated regions (UTRs) [4,5]. Both mRNAs have been detected in all bovine tissues examined but their expression differs according to the embryonic origin of the tissue: the P I : P 2 mRNA ratio is about 1:1 in mesoderm derivatives (heart, muscle, kidney) and about 1 : 3 in endoderm derivatives (liver, intestine, lung) and * Corresponding author. Fax: + 1 (818) 8959554. i Present address: Syva Co., Palo Alto, CA 94304, USA. The sequence data reported in this paper have been submitted to the GenBank, EMBL and DDBJ Nucleotide Sequence Databanks under accession number L19737.

SSDI 0 0 0 5 - 2 7 2 8 ( 9 3 ) E 0 1 9 8 - Y

brain [4]. Two different cDNAs homologous to the bovine P1 and P2 gene transcripts have been identified also in cDNA libraries from rat [6], human [7,8] and ovine [9] tissues. Although the signal peptides of the precursor polypeptides encoded by these cDNAs vary, the sequence of the 75-amino-acid mature subunit 9 protein has been found to be identical in all of these species. Here we report the nucleotide sequence of a cDNA clone (originally designated clone A9) isolated from a two-cell mouse cDNA library and selected for study because of its high level of expression in cleavage-stage mouse embryos [10]. A GenBank data search identified this cDNA as the mouse homologue of the bovine P1 gene for subunit 9 of the mitochondrial ATP synthase. The two-cell cDNA library was originally constructed in pUC8 vector by G-C tailing [10]. The cDNA insert studied here was subcloned into pGEM4Z vector (Promega) and this clone was used for nucleotide sequencing and the production of hybridization probes. Sequencing was carried out in part by the chemical degradation method [11] and in part by thermal cycle sequencing using appropriate primers and the Vent(exo-) DNA polymerase according to the supplier's protocol (New England Biolabs). Fig. 1 shows the nucleotide sequence of the mouse subunit 9 (P1) cDNA clone in comparison to a rat subunit 9 (P1) cDNA [6]. Although the mouse cDNA is truncated at both termini, it contains an open reading frame of 408 nucleotides capable of encoding a signal peptide of 61 amino acids and a mature subunit protein of 75 amino acids (Fig. 2). The mouse and rat cDNAs exhibit a high degree of nucleotide homology

140 ~Precursor AAAATGCAGACCACC

MOUSE

MOUSE RAT

AAGGCACTGCTCATTTCTCCAGCTCTGATTCGCTCCTGTACTAGGGGTCTAATCAGGCCTGTGTC ...................... T ...... C . . . . . . . . . . . C . . . . . . . . . . . . . . . . . . . . . . .

80 130

MOUSE RAT

TGCCTCCCTCCTGAGCAGACCAGAGGCCCCATCTAAGCAGCCTTCCTGCAGCAGCTCCCCTCTCC ............... T ........... T ........ AA ........... T ...............

145 195

MOUSE

~Mature AGGTGGCCCOACGGGAATTCCAGACCAOTGTCATTTCCCOGGACATCGACACAGCAGCCAAGTTC

RAT

........

A ......................

.

.

.

.

.

.

.

.

.

.

.

.

.

210

T .................

T ..............

T

260

MOUSE RAT

ATTGGTGCTGGGGCCGCCACAGTTGGTGTGGCTGGATCAGGAGCTGGCATTGGCACAGTGTTTGG ......................................... G .......................

275

MOUSE RAT

TAGCTTGATTATTGGCTATGCCAGGAACCCATCTCTCAAGCAGCAGCTCTTCTCCTATGCCATTC .............................. G ................................

340 390

MOUSE

TGGGGTTTGCCCTGTCTGAGGCCATGGGACTCTTCTGTTTGATGGTCACCTTCCTCATCCTCTTC

405

RAT

....

455

C .......................

G ..................

A

15

GTGTGCTGTGGGCCGAGCAGGGGCTGCAGGGGAGTGGGAGTGCAGATTGA

.

G

65

RAT

325

C.

G .................

kb 12.2 9.2 7.1 5.1 4.1 :3.1 2.0

H

B E

kb

7.5 4.4

1.0

2.4 1.4

0.52

0.24

~O.75kb

$3"UTR

MOOSE

OCCATGTGAGGCTCCCTGGGGTCACCCAGCCGTCCCTGCTGCTTTGACTCCATGCCAGTCCTAOT

RAT

...............

MOUSE RAT

GCTGGAGTCTACTCAGC TTTACCATTAAA ........ G.G .................. CTACGTTTCTCT

A ...........

G ..................................

470 G..

520

499

561

Fig. 1. Nucleotide sequence of a mouse embryo cDNA clone encoding subunit 9 (P1) of the H÷-ATPase complex. The clone contains an open reading frame coding for the full-length precursor polypeptide but is truncated in the 5' and 3' UTRs. The mouse sequence is shown in comparison to a rat subunit 9 (P1) cDNA sequence [6]. In the two terminal regions where the mouse cDNA is truncated, the rat sequence is shown. For the remainder of the rat eDNA, dots indicate nucleotides identical to those in the mouse sequence. The initiation and termination codons for the precursor polypeptide are overlined; the putative polyadenylation signal ATrAAA is underlined. The arrows indicate the beginning of the sequence coding for the precursor polypeptide and the mature subunit protein, respectively.

o f a b o u t 9 4 % in t h e r e g i o n c o d i n g for t h e signal p e p t i d e a n d in t h e 3 ' U T R a n d 96.4% in t h e r e g i o n c o d i n g for t h e m a t u r e subunit. T h e a m i n o acid s e q u e n c e (as d e d u c e d f r o m t h e n u c l e o t i d e s e q u e n c e ) o f t h e full-length p r e c u r s o r p o l y p e p t i d e o f t h e m o u s e s u b u n i t 9 (P1) is shown in Fig. 2 in c o m p a r i s o n to o t h e r m a m m a l i a n p o l y p e p t i d e s d e r i v e d f r o m t h e P1 gene. T h e a m i n o acid s e q u e n c e o f

Precursor

MURINE RAT

MQTTKALLI

S PALIRSCTRGLIRPVSASLLSRPEAPSKQPS

. . . . . . . . . . .

CS S S PLQVA

V . . . . . . . . . . . . . . . . . . . . . . . . . .

K..

OVINE

.... G .......................

F .....

I..V...Y..G

BOVINE

.... G .......................

F .....

I Q . V . . .Y . .G . . . . .

HUMAN

. . .AG.

F.NS.VNS

.F .......

C ............

.....

50

.C .......

Y.NF

.....

.....

~Mature MURINE

RREFQTSVISRDIDTAAKFIGAGAATVGVAGSGAGIGTVFGSLIIGYARN

i00

RAT OVINE

........

V .........................................

BOVINE

........

V .........................................

HUMAN

........

V .........................................

M~INE

PS LK(~LFSYAII~FAI~

EAT

............................

~AMGLFCI~FLI

LFAM

136

A .......

OVINE

............................

A .......

BOVINE

............................

A .......

............................

A .......

Fig. 2. The deduced amino acid sequence of the mouse subunit 9 (P1) precursor protein is shown in comparison to the rat [6], ovine [9], bovine [4] and human [8] P1 polypeptides. The dots denote residues identical to those in the mouse protein. The arrows indicate the first amino acid residue of the precursor polypeptide and the mature subunit, respectively.

Fig. 3. (A) Southern blot analysis of the mouse H+-ATPase gene. Samples of 5 /xg mouse liver nuclear DNA were digested with EcoRI (lane E) or HindlII (lane H), electrophoresed on a 0.7% agarose gel, and blotted onto a nitrocellulose filter. The blot was hybridized with 10 ng/ml probe DNA labeled with 32p to a specific activity of about 5.10 6 dpm/ng using random priming of the mouse subunit 9 (P1) cDNA template. (B) Northern blot hybridization of a poly(A) + RNA fraction (60 ng/lane) from mouse F9 embryonal carcinoma cells with a 32p-labeled cRNA probe (about 1.6.106 dpm/ng) to mouse subunit 9 (P1) cDNA. For procedures see Refs. 14 and 15.

t h e i m p o r t signal p e p t i d e in t h e m o u s e differs in t h r e e r e s i d u e s f r o m t h e rat s e q u e n c e , seven f r o m t h e ovine, e i g h t f r o m t h e bovine a n d f o u r t e e n from t h e h u m a n . W i t h r e s p e c t to t h e m a t u r e subunit, t h e m o u s e seq u e n c e shows a single a m i n o a c i d c h a n g e ( f r o m a l a n i n e to t h r e o n i n e ) at p o s i t i o n 129 as c o m p a r e d to t h e o t h e r species. T h u s the m o u s e a p p e a r s to b e t h e first m a m m a l i a n species r e p o r t e d so far showing a v a r i a n c e in the sequence of the mature subunit 9 protein. H y b r i d i z a t i o n of E c o R I - a n d HindlII-digested m o u s e n u c l e a r D N A with a m o u s e s u b u n i t 9 (P1) c D N A p r o b e y i e l d e d t h r e e to four m a j o r a n d several m i n o r b a n d s (Fig. 3 A ) suggesting t h a t t h e m o u s e subunit 9 g e n e o f A T P synthase also occurs in m u l t i p l e copies. I n the bovine [5], ovine [9] a n d h u m a n [12] g e n o m e s , t h e s u b u n i t 9 g e n e family includes several p s e u d o g e n e s in a d d i t i o n to t h e two e x p r e s s e d g e n e s (P1 a n d P2). I n F9 m o u s e e m b r y o n a l c a r c i n o m a cells, N o r t h e r e n b l o t analysis r e v e a l e d a single p o l y ( A ) ÷ R N A b a n d o f t h e e x p e c t e d m o l e c u l a r weight ( a b o u t 750 n u c l e o t i d e s , including t h e poly[A] tail) hybridizing with t h e s u b u n i t 9 (P1) p r o b e (Fig. 3B). D o t h y b r i d i z a t i o n e x p e r i m e n t s ([10]; a n d T a y l o r a n d Pik6, u n p u b l i s h e d d a t a ) i n d i c a t e t h a t m R N A hybridizing with t h e s u b u n i t 9 (P1) p r o b e is m o d e r a t e l y p r e v a l e n t in m o u s e oocytes b u t i n c r e a s e s g r e a t l y in a b u n d a n c e in t h e early e m b r y o f r o m t h e two-cell stage o n w a r d , c o i n c i d e n t with a p r o n o u n c e d fine s t r u c t u r a l d i f f e r e n t i a t i o n a n d i n c r e a s e in r e s p i r a tory activity o f t h e m i t o c h o n d r i a [13]. W h e t h e r t h e P1

141

gene alone or both the P1 and P2 genes are expressed at these early stages of development is as yet unkown. This work was supported by Public Health Service research grant HD-19691 from the National Institute of Child Health and Human Development. References 1 Hatefi, Y. (1985) Annu. Rev. Biochem. 54, 1015-1069. 2 Futai, M., Noumi, T. and Maeda, M. (1989) Annu. Rev. Biochem. 58, 111-136. 3 Sebald, W. and Hoppe, J. (1981) Curr. Top. Bioenerg. 12, 2-64. 4 Gay, N.J. and Walker, J.E. (1985) EMBO J. 4, 3519-3524. 5 Dyer, M.R., Gay, N.J. and Walker, J.E. (1989) Biochem. J. 260, 249-258.

6 Higuti, T., Kuroiwa, K., Kawarnura, Y., Morimoto, K. and Tsujita, H. (1993) Biochim. Biophys. Acta 1172, 311-314. 7 Farrell, L.B. and Nagley, P. (1987) Biochem. Biophys. Res. Comrnun. 144, 1257-1264. 8 Higuti, T., Kawamura, Y., Kuroiwa, K., Miyazaki, S and Tsujita, H. (1993) Biochim. Biophys. Acta 1173, 87-90. 9 Medd, S.M., Walker, J.E. and Jolly, R.D. (1993) Biochem. J. 293, 65-73. 10 Taylor, K.D. and Pik6, L. (1987) Development 101, 877-892. 11 Maxam, A.M. and Gilbert, W. (1980) Methods Enzymol. 65, 499-560. 12 Dyer, M.R. and Walker, J.E. (1993) Biochem. J. 293, 51-64. 13 Pik6, L. and Chase, D.G. (1973) J, Cell Biol. 58, 357-378. 14 Taylor, K.D. and Pik6, L. (1990) Mol. Reprod. Dev. 26, 111-121. 15 Taylor, K.D. and Pik6, L. (1991) Mol. Reprod. Dev. 28, 319-324.