Human liver cDNA clones encoding proteolipid subunit 9 of the mitochondrial ATPase complex

Vol. 144,No.

3, 1987

BlOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS Pages 1257-1264

May14.1987

HWAN LIVER cDNA CLONES ENCODING PRDTEDLIPID SUBUNIT 9 OF THE MITOCHONDRIAL ATPase COMPLEX Leigh

B. Farrell

and Phillip

Nagley

Department of Biochemistry, and Centre for Molecular Biology and Medicine, Monash University, Clayton, Victoria 3168, Australia Received

April

6,

1987

SUMMARY: Clones encoding the proteolipid subunit 9 of the mitochondrial ATPasecomplex have been isolated from a AqtlO library of human liver cDNA sequences,‘using a probe of Neurospora crassa cDNA encoding subunit 9. From nucleotide sequence analysis lt 1s concluded that the amino acid seauence of mature human iubunit 9 is identical to that of its bovine counterpart. By comparing the sequence of two cDNA clones (denoted human 1 and 2) to those of two bovine cDNA clones (denoted Pl and P2) recently described by Gay and Walker (EMB~ J. 4, 3519-3524, 1985) it is evident that there are close sequence relationships between human 1 and bovine Pl, and between human 2 and bovine P2, although both human clones are truncated at their 5'-ends. Thus, as in bovine cells there appears to be at least two human genes encoding subunit 9. o 1987 Academic PWSS, IX.

One of ATPase

the

key

complexes

is

binding

proteolipid

intrinsic

to

the

mitochondria,

with

other

two

and subunit

there of

been

a

nuclearly synthesized

the

most

other

encoded. as

protein

This

9. activity

subunit,

of

the

subunit

marked

fungi

hydrophobic

proteins,

is

subunit

F,-sector

9 proteolipid

both

in

membrane

subunit

are

These larger

the

channel

H+-ATPase

While in

subunit

divergence

proteolipid. cells,

or

hydrophobic

Although proteolipids

extremely

the

6, which

of

an

proton

mammalian

has

subunits

complexes the

and nuclearly

precursors

from

in

mammals encoded

products

positively

All

the

DCCD-

oligomer, In

in

is

fungal

the

and

F,-sector

in

between

mammals)

DCCD-binding

examined which

mitochondrial studied

N+-

(1,2).

sources

DCCD-binding

bearing

1257

an

product

compartment

encoded all

all

as

H+-ATPase.

conservation

cellular

9 is

as

assembles

gene

acid

mitochondrial

known

8 (URFA6L

mitochondrial amino

of

this

(3),

there

encodes

this

DNA of

yeast

proteolipid proteolipids

charged

is are

N-terminal

0006-291X/87 $1.50 Copyright 0 1987 by Academic Press, Inc. rights of reproduction in any form reserved.

Vol. 144, No. 3, 1987 extensions protein

BIOCHEMICAL

(leaders) into

the

Extensive corresponding

which

(4-6).

comparisons

of

subunits

(7,8)

DNA.

considerable

divergence

subunit

restricted

to

this

paper

we describe

from

a human

being

identical

MATERIALS

is

clear

those

the

the 9

of

8

genes

at On

and

these that

cDNA

in

sequence

two

parallel

encoded

of

library

which

to bovine

(6)

acid been are

these

amino

of

encoded

bovine

levels,

even

identifies

the

involving

genes cells

encoding

in show

comparisons

and

readily

subunits

acid

nuclear

the

sequences

have

by

clones

subunit

amino

of

hand,

analysis

targeting

subunits

both and

crassa

the

F,-sector

nucleotide

Neurospora

liver

the

for

proteolipids

the

and

of

other

RESEARCH COMMUNICATIONS

following

nucleotide

the It

mammals.

mitochondrial

6

because

mitochondrial

amongst

cleaved

mitochondria

to

accessible

are

AND BIOPHYSICAL

have (3,4).

subunit human

been In

9 isolated subunit

9 as

9.

AND METHODS

All methods were essentially as described (9). A 181 bp %3AI/aI fragment of plasmid pAV48 (6) coding for the major portion of mature H ATPase subunit 9 from Neurospora crassa was subcloned into M13mplO. The probe was prepared by a template copying reaction using univer331 primer and the Klenow fragment of DNA polymerase I in the presence of [aP]dATP (3000 Ci/mmole). The labelled probe sequences were excised from the reaction products by digestion with EcoRI and HindIII. After electrophoretic gel separation and isolation ofthe labelled fragment by electroelution, the probe was used to screen a library of hunan liver cDNA cloned into the bacteriophage vector XgtlO (provided by Dr.G.J. Howlett) and propagated in E. coli LE392. The DNA sequence of selected inserts from positively hybridizing clones was determined (10) after recloning into M13mplO.

RESULTS AND DISCUSSION Isolation

and characterization

Three of

1.5

x

labelled subunit

positively 105

9.

hybridizing

plaques

probe

a The

of

of

cDNA inserts

fragment from

0.4

bacteriophage

vector

M13mplO

chain

termination

dideoxy

cDNA clones

a human

approximately

by the

and

human cDNA clones

0.8

liver

from these

kb, as

were cDNA

N.

respectively),

method 1258

fragments (10).

library

crassa

clones,

EcoRI

identified in

(6)

AgtlO

encoding

denoted

human

were for

from

sequence

using the

1 and

recloned

a screen as

fungal 2 (sizes

into determination

the

Vol.

144,

No.

3, 1987

8lOCHEMlCALANDBlOPHYSlCALRESEARCHCOMMUNlCATlONS

Bovine

P2

I

km

Human2

-AIn

e

Human

1

Bovine

Pl

mvD

--...

-

II

- -ml

rm

(~1"

Fig. 1, Diagrammatic representation of the sequences encountered in human cDNA encoding subunit 9 with reference to the corresponding bovine cDNA Data for bovine clones PI and P2 are taken from ref. 4; data for clones. hunan clones 1 and 2 are from the present work. Sequences for bovine Pl and P2 are aligned according to the discontinuities (gaps) proposed by gay and Walker (4). Human 1 and 2 are aligned, in a similar manner, with respect to for reasons detailed in the text. Hatched bovine Pl and P2 respectively, and open rectangles indicate coding sequence for mature subunit 9 and Nwavy lines indicate truncation in cDNA clone terminal leader, respectively; at 5'-boundary (see text); dots indicate sequence not determined for 3'side; (A)n denotes poly(A) tail.

The appraisal nucleotide of

P2)

the

the

these

in

leaders, and

their

cDNA clones

are

Analysis The and

2

displays

were

to

compared

the

to

homology

leader

of

of

the

clones

data

1 and

amino

the

identical,

they

Furthermore,

the

are

(denoted

Pl

(4).

To

sunmarized with

9 genes. acid

(61

flanking

the

aligned

encode

length

in

9

2 are

which

of

made

subunit

the

their

level

cDNA clones

subunit

to

the is

two bovine

in

respect

predicted those

by

of

the

the

subunit

identical

of

bovine encoding

human

are

to

specify

portion

sequence,

analyses,

two

at

Whilst

sequences

of

different

N-

versus

68 amino

sequences

of

these

sequences

to

be

both

diverged.

sequences

which

both

these

sequence.

protein

found

terminal

with

acid

exact

precursors

both

of amino

were

genes,

precursors

proteolipid

also

on two

The

9

acid

data

regions

encode

subunit

terminal acids)

of 1.

analysis,

amino

distinct

and non-coding

mature

sequence

published

Fig.

genes

of

predicted

presentation

bovine

the

and

from

diagrammatically coding

results

recently

derived

clarify

the

the

sequence

context and

of

to

the

leader

bovine

P2.

22

the

bovine

open

C-terminal

9.

The remaining the

sequence Unfortunately, 1259

(Fig.

amino

25

of

frames

precursors

75

of

reading

of 2).

acids

of

bovine human

acid

but 1

lacks

Human

1

amino

residues

Pl,

1

both

25 N-terminal

amino

human

in

bovine

acids the

C-

show no homology an

N-terminal

Vol. 144, No. 3, 1987

BIOCHEMICALAND

8lOPHYSlCALRESEARCHCOMMUNlCATlONS

L20 LSO 140 LIO LlO ~CAKPVSTPSLIRRTSnLSRSL~~PETLTDgSHSS~~RPL~SLTPSRSFQTSAISRDIDT

BOV P2 HUM 2 HUM 1 BOV Pl

LSO

QKQPSY4N~PLQV~-------4~4444~444444

BQL'MiALLIS~A*JCSCTRO*~*~~*SPLS**~I~VQPSY*SGPLQV~-------*E*~*~W*~~ L20 L4G L50 110 L30 M20

1110

BOV P2 HM2 Hlml BOV Pl

1140

1130

I(50

L80

MS0

1110

AARPIGAGAATVGVAGSGAGIGTWGSLIIGYARRRSLRQQLi’SYAILGFALSBARGLPCLRVApLILPAn

RLS44444444.444*444b4.4.4444444.4444.44844;444484844444b44 ***+*************++**o*o*o********o**o*************o******o************ *L********+****Lt+***************************************************** I410 M20 MO X50 MB0 w30 Amino

acid

Ml0

predicted from nucleotide sequence of cDNA The complete sequence of the bovine subunit 9 precursor P2 (4) is shown as reference sequence, residues are numbered L. in leader and M. in mature subunit 9. Asterisks indicate residues identical to Dashes indicate discontinuities introduced to optimize those of bov+ne P2. Underlined residues in human 1 correspond homology at nucleotide level (4). to those determined analytically by microsequencing of purified hunan subunit 9 proteolipid (S. Marzuki, personal communication).

%k&encoding

methionine

and is

5'-end

lies this probe:

The to

its

of

analysis

of

personal

is

the

is

N-terminal

that

as

human subunit

truncated

amino

some

other

1 and 2); to

subunit

more

truncated

strengthened

by

of

to

bovine

case,

of

the the

there

of

the

is

human

identical amino

subunit

first bovine

human

1 as a

one was identical

direct

human

whose

We have

using

version

the

that

those

times,

has

purified

showed

sequence.

recovered,

9 indeed

exactly

the

(Figs.

N-terminal

clones

portion

the

two

subunit

which

in

leader

cDNA clone

40

1. sequence

acid

sequence

9 (S.

Marzuki,

amino

acids

of

(3).

It

processing

of

proteolipid

no C-terminal

to

9 precursor.

of

corresponding

from

human

counterpart

Analysis

bovine

that

a truncated

for

a further

was a slightly

9 corresponded

assumed

the

other

from

coding

2 additional

communication)

subunit

region

cDNA library the

conclusion bovine

to be derived

the

human

human 1 and the

9.

considered

within

rescreened labelled

sequences

subunit

human

one can

the

55 C-terminal

acids

predicted

9;

aberration

it

is

cDNA clone

possible

occurring

identify residues

by the that during 1260

human the the

2 revealed

it

to be extensively

a predicted of

bovine

amino subunit

2 sequence nucleotides cDNA

acid

have

9.

The three

no homology

encoding

synthesis

sequence

and/or

them

to

arose

cloning

Vol. 144,No.

3, 1987

procedures.

BIOCHEMICAL

Comparisons

at

identify

human 2 as being

Analysis

of

The level

nucleotide

can

hydrophilic

sequence

homology

sequences). show

22

that

for

The

be

regions

not

both).

region

of

acid

encoding

the

position

amino

mature

subunit

differences

compared human

to

P2).

1 and bovine

Pl,

an impressive

41 out

of

Human 2 was found, to

bovine

P2

sequence 8

third

for base

Significantly,

examined. changes

comparing

bovine

human

1 exactly

of

regions over

to

available

silent

codons

than

of that

human

2 and

portion

of

are

The

two breaks

in

the

human

In

human

27

base

third base

3

changes

1 sequence base

codon

substitutions

non-coding

were

segments

1 sequence,

those

be more

portions

human

of

reveals

the

two codons

of there

2 downstream

1261

and

bovine is

a

protein

total

of

displaying

Leu65), Pl.

related coding

2 and bovine

in

(Leu32

closely

of

clone

apparent of

P2

to

those

between

P2,

(but

the

to be homologous.

Pl.

and

this

respective

human

9 silent

3'

Pl

the

In the

Pl.

the

of

the

bovine

bovine

portion

of

respect

in

1 shows

6 silent

alignment

in

codon,

engendering

Pl

alignment,

match

of

a further

by nucleotide

changes

(one

bovine

bovine

comparison

proteolipids

human

to

exhibits

50 nucleotides

N-

acid

differences

divergencies

9 proteolipid

allowing

amino

divergencies

extensive

the nucleotide

predicted

sequences

sequences.

Finally,

coding

mature

sequence

there

to

the

cDNA clone

leader

compared

bovine

the

nucleotide

acid

regions

sequence

clones.

from

2),

nucleotide

identical

are

nucleotide

those

(Figure

the

clearly

recognizable

base

there

data

the

in

the

two bovine

the

but

2 first

N-terminal

from

in

coding

in

P2 at

no homology

Third,

related

differences

affecting

below)

P2.

scattered

and

sequence

apart

and

First, is

or

the

the

Pl

there

regions

has

encoding

amino

(described

to bovine

3.

divergencies

closely

proteolipids,

Fig.

little the

nucleotide

to

in

(but

Leu65,

level

related

bovine

leader,

base

3'-untranslated

clone

seen

Second, third

closely

between

be

terminal

nucleotide

RESEARCH COMMUNICATIONS

sequence

relationship

(4)

the

AND BIOPHYSICAL

In

clear

extended

sequence

which

55

only codons

first

the the

P2,

base

corresponding 3'-untranslated homology

we

have

(83%) so

far

“-I. VOI

BOV P2 HUM2 HUM 1 BOV PI

BOV P2 HUM 2 HUM 1 BOV Pl

BOV P2 HUM 2 HUM1 BOV Pl

BOV P2 HUM 2 HUM1 Bov Pl

BOV P2 HUM 2 HUH1 WV Pl

BOV P2 HUM 2 HUM1 BOV Pl

110 80 QO 100 TSTVLSRSLSAVVVRRPKTLTDBSHSSLA MCCTCTACAGTATn;AOCCGATCTOCAOfOOMOGGCA

RESEARCH COMMUNICATIONS

120

130

140

150

160

AT'TA*AC**CCTTC=TA*'='AACTTC ~TA*C*OO+GTC**'T*A*GC*TG*******CTCCT~CC*G**TAOO**AG*G'TC+MT+T*TAC**CC~C*TA****AGT*GC CTRGLIRPVSASPLSRPKIQSVQPSYSSG 90 110 120 190 140 150 160 170 100 190 220 23 170 180 200 210 240 VVPRPLTTSLTPSRSPQTSAISR 4 I D T AA GTAGMCCCCGTCCCCTOACCACCTCACTTACTCCTCCTAGCCG~~CC~C~GTGCCA~~~~CATT~CACAGCAGCCA

~~+~.~WA~~Q+.A~--------------------'WWQ&W+'.WOQ+..W'.W~O~W.~~WWWWWWWWWW..WWWWWWWWW ~~W~WQW~~~~.WWQW~~~-- ~~~~~~~~~~~~~~~~..~WW.W.Q...WWWW~W~W.~~WWWWWWWWWW..bWWWQWWWW PLQVAR RKPQTSVVSRDIDTAA 230 240 180 190 200 210 220

250 260 270 280 290 300 310 320 # 330 KPIGAGAATVGVAG8GAGIGTVFGSLIIGY AGTTCATTGGAGCTGOGGCTCCACAOTAOWOTDGCTOOCTCTCATCATTGGTTA ~Q8~~~kLW..~8.8..Q.WW..~W8~.....WWWWWW~.WWW.WWWWWWWWW ****T*****~********~********~**~********~**~*~*****~*****~***********~**~~*~********~** WW+W~WWLWW~WWWWWWWWWW*WWW*..~WW~WO*WWQ**~WW~O~.WWW.~.DWWO~W*~**.k*WWW~W8~~WQW**WW KPIGAGAATVGVAGSGAGIGTVFGSLIlGY 250 280 270 280 290 300 310 320 330 340 350 380 370 380 390 400 410 ARNPSLKQQLFSYAILGFALSBAnGLPCL TGCCAGGAACCCTTCTCTOCAGCAOCtCT~~ACGC~~C~C~CCCT~C~CCATGGGGCTC~TTGCC~ ************************~***************************W*******************************~*** ************~*****~***L****************~********~***********~**~**************~**~**~** ************~***********************b**~********************~**~*****~***********~**~** ARNPSLKQQLPSYAILQFALSBAMGLFCL 340 350 360 370 380 390 400

420

410

t

42c

430 440 450 460 470 480 490 500 HVAFLILPAU* ATGGTGGCCTTTCTCATCCTCTTCGCCATQWCcGTq'T---------CCACCTCCCATAGTTCTTCTCCCGTGTCTCATCT *****A*****************~******************~* ----------+.....*.b........~~~....~..W.~~.~~ *****C*****C***********************~~~*~~~~----------***~C~-------------------------G ****+C****WC********W**WW***WW**WQW~~~WQ~Q~~~~~~~~WWW~~W~~~W~~WWW~~WWWW~WQ~~~*~~~~~ MVAFLILFAM* 430 440 460 480 470 480 490 500 520

530

540

550

GCCCTGTATGTTTCTTTTCCTGTACCTCCCCAOOCAACCT7A

580

l WWW~W+GWWWW~WWWWWW+W~WWWWWWW~WWWW~~W l TG***GGGTG*GT*MG*T*TA~*~*~~~~~~*cQ~*TcT~T**M*~* *TG'**G*GTG*G**AAC*T*TAC*A*TAUCACA*TGT*TCTCT**AA*A

510

520 600

BOV P2

AND BIOPHYSICAL

l *****C*Q**~C**~~~*~~**~~~*T**~G*~~*****C*~TC cCAATGGATTTTTTTT*C+*CCCCT*TGCAGA*TGAAA HQTTGALLISPALIRS 30 40 50 80 70 80 10 20

510 BOV P2 HUM 2 HUM 1 BOV Pl

BIOCHEMICAL

144, No. 3, 1987

530

540

550

610

CTGTATTAATAAGAAAAA

1262

570

580

590

Vol. 144, No. 3, 1987 obtained.

It

sequence

of

From

is

these

data

two

H+-ATPase

complex.

genes

variation

Subunit

amino

acid

respectively

striking

reflection

equivalent

translocating discussion

the of

6

(equivalent

is in

not

of

subunits

6 of

generally

Nucleotide

6

the

9.

the

mammals

is

published

subunit

of

the

proton-

to

vigorous 9 (or

transmembrane

4

a key

highly

its stems

role

in

The

mitochondrial

the

(3).

subunit

these

Ht-ATPase

polypeptides

conserved

precisely

but

regions

of

those

two

to

amongst bovine and hunan cDNA sequence of the bovine P2 cDNA

numbering

system

(4).

the

sequence

Amino

acid

sequences denoted by single letter code refer only to the respective bovine cDNA clones. Arrow indicates first amino acid residue of mature subunit 9. Asterisks indicate nucleotides identical to those in bovine P2. Dashes indicate gaps in sequence to maximize homology. Gaps in bovine sequences are according to Gay and Walker (4); gaps in 3'-untranslated sequences of human 1 and 2 were introduced to optimize homology with the 3'-untranslated sequences of bovine Pl and P2, respectively. The symbol # denotes amino acid residues at which sequence polymorphisms involving first base codon changes are apparent. 1263

9 This

putative play

the

cells.

subject

that

human

correspond

at

the

of is

and

conservation,

nature

and

the

bovine

conservation

(14-16).

Two of

liver

a particularly

F,-sector

relationships The full reported the

human

throughout

proteins

sequence

subunit indicating

and

evidence

bovine

(11).

that

H+-ATPases

subunit

distributed

clusters subunit

given,

bacterial

properties

discrete

mammalian

%k?encoding clone is

to

acid

interactions

two of

the

indicate

throughout

genetic

and

amino

complexes

strong

is

mitochondrial

divergence

sequence

of

the

sequence

two

and

human

of

strong

Fo-sector

subunitc)

between

complexes

H+-ATPase

the

there

transduction

divergence

generally

the

liver,

Fo-sector

bovine of

far

F,-sector

study

in

how

tail.

of

77.9%

our

topology

of

homologue,

and

in

poly(A) bovine

the

the

9 proteins

detailed

core

subunit

51.5%

homology

the of

of

6 of

presented

data

9 proteolipid

considerable

is

subunit

to the as for

proteins

absolute

(12,13)

bacterial

the

Data

the

subunit

show

sequence

point that,

subunit

there

proteolipids

While

the

and

limited

this

hydrophobic

maintains between

our

suggested

ATPase

(11).

identity

occurs

is

encoding

level;

component

from beyond

8 (URFA6L)

mitochondrial

energy

clear

it

in

human

of

not

human 2 extends

contains

Sequence

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

8lOCHEMlCALAND8lOPHYSlCALRESEARCHCOMMUNlCATlONS

Vol. 144, No. 3, 1987 transmembrane interacting within

stems

directly

implicated

in

energy

transduction

with

subunit

9 (15,16).

By contrast,

the

mammalian

subunit

8

polypeptides

appear

(URFA6L)

and probably

regions to

of be

homology much

more

scattered.

ACKNOWLEDGMENTS: We thank Howlett for the human liver assistance with computers. Research Grants Scheme.

Dr.

W. Sebald for plasmid pAV48, Dr. G.J. cDNA library, and Mr. R.J. Maxwell for This work was supported by the Australian

REFERENCES

1. 2. 3. 4. 5. 6. 7. 8.

9.

10. 11. 12. 13. 14. 15. 16.

Linnane, A.W. et al. (1985) In: Achievements and Perspectives of Mitochondrial Research (Quagliariello, E. et al., eds.), Vol. 1, Bioenergetics, pp. 211-222, Elsevier, Amsterdam. Fearnley, I.M., and Walker, J.E. (1986) EMBO J. 5, 2003-2008. Sebald, W., and Hoppe, J. (1981) Curr. Top. Bioenerg. 12, l-64. Gay, N.J., and Walker, J.E. (1985) EMBO J. 4, 3519-3524. Schmidt, B. Hennig, B., Zimmerman, R. and Neupert, W. (1983) J. Cell Biol. 96, 248-255 Viebrock, A., Perz, A., and Sebald, W. (1982) EMBO J. 1, 565-571. Roe, B.A., Ma, D-P., Wilson, R.K., and Wong, J .F-H. (1985) J. Biol. Chem. 260, 9759-9774. Pepe, G., Gadaleta, G., and Lanave, C. (1984) In: Ht-ATPase (ATP Synthase): Structure, function, biogenesis of the F F1 complex of coupling membranes (Papa, S., Altendorf, K., Ernster, e., and Packer, L eds.), pp. 63-65, Adriatica Editrice, Bari. M&atis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory, New York. Sanger, F., Nicklen, S., and Coulson, A.Z. (1977) Proc. Natl. Acad.Sci. USA, 80, 4411-4416. Anderson, S., De Bruijn, M.H.L., Coulson, I.C., Sanger, F., and Young, I .G. (1982) J. Mol. Biol. 156, 683-717. Cox, G.B., Fimmel, A.L., Gibson, F., and Hatch, L. (1986) Biochem. Biophys. Acta 849, 62-69, Sebald, W., and Hoppe, J. (1986) Biochimie 68, 427-434. John, U.P., Willson, T.A., Linnane, A.W., and Nagley, P. (1986) Nut. Acids Res. 14, 7437-7451. John, U.P., and Nagley, P. (1986) FEBS Lett. 207, 79-83. Cain, B.D., and Simoni, R.D. (1986) J. Biol. Chem. 261, 10043-10050.

1264