Vol. 171, No. 3, 1990 September

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1168-1174

28, 1990

PRIMARY

STRUCTURE

SUBUNIT

AND

OF COMPLEX

SPECIFIED

Arnaldo

Videirai,

Institut

fiir

Goethestr.

Received

BY

I

THE

Maximilian

D-8000

OF

A

NUCLEAR-CODED

HOMOLOGOUS

TO

PROTEINS

CHLOROPLAST

GENOME

Tropschug

Physiologische

33,

August

EXPRESSION

and

Chemie

Miinchen

2,

der

Sigurd

Werner

Universitit

Federal

Miinchen,

Republic

of

Germany

8, 1990

A 31-kDa subunit of complex I from Neurospora crassa, of nuclear origin, was cloned. The precursor polypeptide (33 KDa) could be efficiently expressed in an in vitro system for transcription and translation. The processing of the precursor to mature protein was also vitro. the obtained in An open reading frame coding for a precursor protein of 283 amino acids (32247 Da) was found by DNA sequencing. The predicted primary structure shows significant homology with proteins made This in chloroplast. supports the hypothesis that an enzyme similar to respiratory chain NADH dehydrogenase might exist in 01990 Academic Press, Inc. these organelles.

chain

Respiratory is

localized

more

in

than

review

see

1).

is

are unit

polypeptides. (7).

complex In

(1-6). This

I

carrying

addition,

and

subunits in

in

into

and besides

and

other

the less

information

is

known

in

complex

should Largo

be

$1.50

1168

in

The

order

I

sent at: Professor

to

of

in

the

assemble the

most

a

latter subunits

nuclear

coded

increase

upon

Instituto de Abel Salazar

Ci&‘Icias 2, 4000

might

be

these

majority

to

since

seem

a this

for

regarding

required, groups

its (for

synthesized

in

1.6.5.3) contains

synthesized

species.

nucleus,

is

prosthetic

0 1990 by Academic Press, Inc. of reproduction in any form reserved.

membrane

information

mitochondria

Much

interest

encoded

and

EC

clusters)

sequence

human

To whom correspondence Biombdicas de Abel Salazar, Porto, Portugal.

Copyright All rights

are

I,

subunits

iron-sulphur

DNA

1

0006-291X/90

mitochondrial

encoded

imported

(Complex

polypeptide

and

available and

functional of

Seven

subunits

cytoplasm

inner (f lavin

organelle,

proteins

dehydrogenase

different

groups

cellular the

the

25

prosthetic

NADH

Vol.

171,

No.

recent

in

3, 1990

BIOCHEMICAL

either

unit this

RESEARCH

human

COMMUNICATIONS

diseases

to

deficiencies

enzymatic

activity

or

subunit

content

(6, 9). paper, we

present

the

primary

structure

the

membrane In

BIOPHYSICAL

particular

relating

reports

AND

cytoplasmically-synthesized

subunit of report and

cDNA sequence data) vitro. Furthermore, processing in this protein are encoded in the

complex I its efficient we

found

chloroplast

(as

of

this of

deduced synthesis

that

from and

homologues

genome of

a

of

different

species. MATERIALS AND METHODS The full-length clone encoding the 31-KDa subunit of complex crassa I was isolated from a N. cDNA library (IO), by with a hybridization previously obtained probe (11) (12). Subcloning (13), in vitro transcription (14) and translation (6), immunoprecipitation and protein electrophoretic techniques (15) have been described. DNA sequence determination was carried out as detailed before Specific oligonucleotide primers for (12). sequencing were provided by Drs. J. Arnold and 1. Leitner from the Genzentrum Muenchen. Homologues of the 31-KDa protein were found in the protein data bank (MIPSY) of the Max-Planck Institut Muenchen.

RESULTS AND DISCUSSION a) Isolation and in vitro expression the 31-KDa subunit of complex I We

have

previously

corresponding screening

a

nuclear-coded the library largest

to

the

cDNA

isolated 31-WDa

expression

transcription

insert

found

vector

pGEM4.606 polymerase and

(No.

generating transcribed

a

a

cDNA

subunit

library

cDNA

of with

protein (6). This cDNA by hybridization and to

cDNA

of

insert isolate

clone

complex antiserum

in

(no

62) I (121, by against the to rescreen

vitro

into

and

the

material and

specifically 31-KDa protein

the

gels

using

4).

pGEM4

SP6

RNA

in

the

treated

directed mass of and

the

SDS analyzed by gel fluorographed (Fig. I). A (lane 1) could be synthesized

were were

polypeptide immunoprecipitated (lane

The

the

in a rabbit reticulocyte lysate was separately presence of [35S]methionine. The mixture with an antiserum to the 31-KDa protein and an antiserum to a different subunit of complex I (apparent molecular translation products 22-kDa), as a control. Total immunoprecipitated electrophoresis radioactively-labeled

encoding

was used related inserts.

606) was subcloned pGEM4.606.

was translated

clone

The 1169

with the polypeptide

antiserum has an

against apparent

Vol.

171, No. 3, 1990

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

kDa

,Pst

43

I

INSERT

606

30 20 A .

.

0 .

14

I +

02

-

1POP

-

u In vitro expression and processing of the precursor of the 31-LDa protein. Plasmid pGEM4.606 was transcribed in vitro with SP6 RNA polymerase and translated in rabbit reticulocyte lysates in the presence of [35S]methionine. The mixture was treated with purified mitochondrial processing peptidase or incubated with specific antisera against subunits of complex I. The material was then analysed by SDS gel electrophoresis and the gel was fluorographed. Total translation products incubated in the absence or presence of the processing enzyme are depicted in lanes i and 2, respectively. Analysis of immunoprecitates obtained with a control antiserum (lane 3) and with antiserum against the Jl-kDa protein (lane 4) is shown. Flg.2. Sequencing clone pGEM4.606. insert. Arrows determination. primers (arrows

mass

molecular identified

purified

removes

an

polypetide.

In

mass of indicate

I, of

Nucleotide Fig.

amino The

and

2

acid cDNA

contains

of the

displays

sequence insert

peptidase

the

(an

same

all

enzyme

that

proteins

(16)) of

apparent

the

the been lysates

behaviour

migration the

with has

the

molecular

information

results subunit for

the

precursor.

outlines 3

of

mitochondrial

displays

it

sequencing

Fig.

treatment

imported the

shown) which

These protein (fig. 1, lane 2). 606 indeed specifies the 31-BDa

31-KDa insert that

the

it

not

cDNA insert of region of the of sequence specific DNA

protein,

processing of

case,

mature cDNA

complex

insert.

alteration this

the that

synthesis

from

the coding extent using

(data

Ji-kDa

Furthermore,

mitochondrial

in

comigrates the

(6).

presequences

results

It

of

previously

with

of

33 KDa.

of precursor

cytoplasmic

b)

strategy and structure of The box corresponds to the show the direction and Some sequence was obtained with open circles).

of includes

the

cDNA

strategy

used

the

nucleotide

the

precursor 1153

insert

base

1170

of for

pairs

pGEM4.606

sequencing

sequence of

clone

the (we

and 31-KDa have

the the

cDNA deduced

polypeptide. estimated

a

Vol.

-60

61 21s

TCGCCAGCTATCAGGTGCCTCGCGACCACCAGCCGTAACCTTATCAACATGCCCGAACGC PA I R C L AT T S R N

L

I

N

M

P

E

R

121 41P

CCC:AACCCGCGGCAGTTCCCCCGTGAGCCCCTGCCGGGCGCCCTGAATGCAGCCGTGGTC N P R Q F P R E P L P G

A

L

N

A

A

V

V

181 61

AACCCGGCCGACAAGTACCAGTCCAAGGCCGACAATCTCCACAAGTACGGGTCGTGGCTC N P A D K Y QSKADNLHKYGSWL

241 81

ATGGGCTGTCTCCCCAAGTACATCCAGCAATTCTCGGTTTGGAAGGACGAGTTGACCATT II G C L P K Y I QQFSVWKDELTI

301 IOlY

TACATTTCTCCCGCCGGAGTCATCCCTGTCTTTTCGTTCCTCAAGTACAATACGGCGGCC I SPA G V I P V F S F

L

K

Y

N

T

A

A

361 121

GARTACACCCAAGTGAGTGACATCACTGCGGTTGATTTCCCCACCAAGGACCAGCGCTTC E Y T Q V S D I T A V D F

P

T

K

D

Q

R

F

421 141E

GAGGTCGTCTACAATCTGCTGAGCGTGCGCCACAACTCGAGAATCCGCGTCAAGACGTAC V V Y N L L S V R li N S

R

I

R

V

K

T

Y

481 161A

GCCGACGAGGTGTCCCCCGTGCCCAGCATCACCCCCCTCTACGATGGCGCCAACTGGTAC D E V S P V P S I T PLY

D

G

AN

W

Y

541 181

GAGCGCGAGGTCTACGATCTCTTTGGCGTCTTCTTCACCGGCCACCCGGACCTGCGCCGC E R E V Y D L F G V F F T

G

H

P

D

L

R

R

6Oi 201

ATCATGACCGACTACGGCTTCGACGGCCACCCGCTGCGCAAGGACTTCCCCATGACCGGC I M T D Y G F D G H P L R

KDFPWTG

661 221

TACACCGAGATCCGCTACGACGAGGAGAAGAAGCGCATCGTGACGGAGCCTCTGGAGATG Y T E I R Y D E E K K R I

V

T

E

P,

L

E

M

721 241

ACACAGGCCTTCCGCAACTTTGAGGGTGGGTCCAGCGCCTGGGAGCAGGTCGGAGCCGGT 1’ Q A F R N F E G G S S A W

E

Q

V

G

A

G

781 261

ATCGACCGCAAGCCCGAATCTTTCAAGCTCCCAACGCCGAAGCCGGAGACGAAGCCGGAG IDRKPESFKLPTPKPETKPE

841 281

GAGAAGAAGTAGACGGAAAGAAACGGCACACCAAACACCACTTCAAACACGAAATTTGGG E K K

of triplet

pGEM4.606. is of the

GGAGTGGAGAAAGGTTGTAGATATTTGCTGGGTATGGCAAGCCTTCTGCATCCAGCTGGT TGGTTCTGTGGGTTTGCGCTTTGTAGATAGTTTACTCGGAGCACAGAACGAGAGCTACTA CTTAGATGGGGACGGCAATACAGAACGTCCACCACAAAAAA

F&g

Nucleotide

stop

codon

The protein

predicted is

si2e

of

Fig.

sequence of the preceding in the frame primary structure of shown in the one-letter

kilobases

1.3

contains

this

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

-92 CGAGGCTGCTGGCTGTTGTTGTTCCGTGATCC ATTGGCGCCTCTTCCCCAGTCTCGCCCATCTCGTTCAGGCCATCGCCAGCAACCTACAAG Pst I 1 ATGGCCAGCAAGCTCTGCAGAAGCAGGGCCCTGGCCTCTGCCCTGCGCTCCGCGAAGCCG 1IlASKLCRSR A L A S A LRSAKP

901 961 1021

A

BIOCHEMICAL

171, No. 3, 1990

an

TGA

open

stop 2)

codon

preceds

represents

polypeptide.

for

reading frame

calculated

corresponding for

(nucleotides in

the We

the

frame

-66 the

first

initiation a

insert cDNA first ATG the precursor abreviation.

a to

-64 ATG codon

molecular 1171

lURNA

protein

mass

of

283

indicated triplet, for of

A marked. 31 LDa

and

(la), amino

by

a

acids. bar

indicating precursor

the 32247

in that

Da

for

the

Vol. 171, No. 3, 1990

protein for

which the

The sequence,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fairly

precursor 31-KDa as

agrees

with

polypeptide subunit is most

particular protease

sequence (see above).

different consensus

precursors

imported

did

sequence,

this

were that,

is

given after

not

precursor (positions in a

ref. import

occurs 17 and mature

reveal

besides

always in

vitro in of cleavage

can be removed The comparison

charged amino acid residue this residue is an arginine and

the value of 33 BDa estimated by SDS gel electrophoresis. synthesized with a cleavable signal proteins This mitochondrial (17).

a

the at

-2.

21 out

of

into between 18

of

mitochondria, amino the fig.

polypetide

3, with

a

positively

been

noted

propose

cleavage of acids serine

the and

33-kDa alanine

This

aminoacid

Da in agreement electrophoresis.

with

c)

acid

sequence

homology

the

would mass

complex-1

of

We chloroplast

found significant proteins of

homology of the 31-kDa polypeptide several species (Fig. 4), namely

157

Synechocystis

sp.

protein

31 KDa

subunit

proteins

and

result and a

residues the

chloroplast

PCC6603

that

we

of 30446 SDS gel

of

38 strict

concept

mass by

between

a

cases (IT), (7 examples

respectively). 266

within

“typical” crdssa

true in the case of N. 17). According to this

molecular estimated Amino

has

22

specific

for of

It

the

sites

requirement

importance

position in

by

159

and with protein

of

Zea

ways protein 158 of Nicotiana tabacuw and (la), (191, protein 169 of Harchantia polyworpha (20). In chloroplasts, the existence of genes that potentially encode homologues of mitochondrially-synthesized subunits of complex I has been already noticed (19-21). More recently, homology has been found between two chloroplast proteins (protein 392 of M. polyworpha (20) and protein 393 of N. tabacum (21)) and a cytoplasmically-synthesized 49-KDa subunit of complex I (22). These results lead to the suggestion that, in chloroplasts, a cluster of mitochondrial

genes

encode

components of an enzyme similar to complex I, possibly NADH:pJastoquinone reductase (22, 23). Our results, showing a second example of homology between chloroplast proteins and a nuclear-coded subunit of complex I (from fungi), further support this hypothesis. Assuming a common ancestor to both mitochondria and chloroplasts initially

(20), belonged

it to

is

possible mitochondria 1173

that and

the then

Jl-KDa-protein migrated

to

gene the

Vol.

171, No. 3, 1990

BIOCHEMICAL

11

a) b) cl d) e)

154

a) b) c) d) e)

181

VRFT

110

IVYE

111

ISYD

122

IHYE

112

ISYD

a) b) c) d) e)

209

F*

KT KV KV

82 83

EVCI

RESEARCH COMMUNICATIONS

DEV

PLYDGANWY

PRE

WIWKAADWQ

Ill

SRR

KIPILRKNPKI

94 84

YAn FL

AND BIOPHYSICAL

EVCI

KVFHGKDNPRI

u

GHPD

1

RIMTDYGFD

NHPR I

138 139 150 140

Comparison of the amino acid sequence of the subunit of I from Neurospora with chloroplast proteins from species. The figure shows the most conserved region the N. crassa mitochondrial protein and (a) proteins from S. 6803 tabacum (b), A’. (c)s polymorpha and Z. mays Cd) (e).

complex different between chloroplast M.

nucleus

it

,where

targeting

has

acquired

information

for

a

mitochondrial

signal. ACKNOWLEDGMENTS

A. V. would like to CientLf ica e Tecnol6gica and Abel Salazar da Universidade support. This research Forschungsgemeinschaft (SFB

thank Junta National Instituto de Ciencias do Porto, Portugal, was supported by 184).

de

Investigag%o Biomedicas de for financial the Deutsche

REFERENCES 1 . 2.

3.

4. 5.

Ragan, Anderson,

C.I.

(1987) Curr. Top. Bioenerg. 15, I-36. S. , Bankier, A.T., Barrell, B.G., de Bruijn, n.H.L., COulSon, A.R., Drouin, J., Eperon, I.C., Nierlich, D.D., Roe, B.A., Sanger, P., Schreier, P.H., Smith, A.J.H., Staden, R. and Young, I.J. (1981) Nature 290, 457-465. Chomyn, A., MariOttini, P., Cleeter, n.W.J., Ragan, C.I., and Attardi, Matsuno-Yagl, A., Hatefi, Y., Doolittle, R.P. G. (1985) Nature 314, 592-597. m.W.J. I Ragan, C.I., Riley, II., Chomyn, A. , Cleeter, Doolittle, R.P. and Attardi, 0. (1986) Science 234, 614-618. lse, W., Haiker, H. and Weiss, H. (1985) EMBO J. 4, 2075-2080.

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(1988)

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Chomyn, A., Patel, S.D., Cleeter, N.W.J., Ragan, C.I. and Attardi, G. (1988) J. Biol. Chem. 263, 16395-16400. Halt, I.J., Harding, A.E. and Norgan-Hughes, J.A. (1988) Nature 331, 717-719. Wallace, D.C. (1989) Trends Genet. 5, 9-13. Videira, A., Tropschug, M. and Werner, S. (4990) Biochem. Biophys. Res. Commun. 166, 280-285. Benton, W.D. and Davis, R.W. (1977) Science 196, 180-182. Videira, A., Tropschug, M., Wachter, E., Schneider, H. and Werner, S. (1989) J. Biol. Chem., in press. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Melton, D.A., Krieg, P.A., Rebagliati, M.R., Maniatis, T., Zinn, K. and Green, M.R. (1984) Nucl. Acids Res. 12, 7035-

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Borchart, U., Zauner, R., Christner, J., Jung, G., Eur. J. Machleidt, W., Videira, A. and Werner, S. (1985) Biochem. 150, 447-454. Hawlitschek, G., Schneider, H., Schmidt, B., Tropschug, M., Hartl, P.-U. and Neupert, W. (1988) Cell 53, 795-806. Hartl, F.-U., Pfanner, N., Nicholson, D.W. and Neupert, W. (1989) Biochim. Biophys. Acta 988, l-45. Sayre, R.T. Steinmueller, K., Ley, A.C., Steinmetz, A.A., and Bogorad, L. (1989) Mol. Gen. Genet. 216, 60-69. Wakasugi , T. , Shinoraki, K., Ohme, M., Tanaka, N., Hayashida, N., Matsubayashi, T., Zaita, N., Chunwongse, J., Obokata, J., Yamaguchi-Shinozaki, K., Ohto, C., Torazawa, B.Y., Sugita, Il., Deno, H., Kamogashira, T., K. e Meng, Yamada, K., Kusuda, J., Takaiwa, P., Kato, A., Tohdoh, N., Shimada, H. and Sugiura, N. (1986) EMBO J. 5, 2043-2049. Sano, S., Ohyama, K., Fukuzawa, H., Kohchi, T., Sano, T., Shirai, H., Umesono, K., Shiki, Y., Takeuchi, H., Chang, Z., Aota, S.-i., Inokuchi, H. and Ozeki, H. (1988) J. Mol. Biol. Yamaguchi Ohto, C., M. (1987)

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