Gene, 104 (1991) 21 l-217 0
1991 Elsevier
GENE
Science
Publishers
B.V. All rights reserved.
911
03781119~91/$03.50
05030
Characterization (Nuclear
and expression of a cDNA specifying subunit VIIc of bovine cytochrome c oxidase
gene; mitochondrial
protein;
nucleotide
sequence
Mfon S. Aqua”, Nancy J. Bachman b*, Margaret I. Lomax’
analysis)
and Lawrence 1. Grossman”
” Department qf Molecular Biology and Genetics, Wayne State University School of Medicine, Detroit, MI 48201 (U.S.A.): ” Department of Biology, Franklin and MarshaN Coitege, Lancaster, PA 17604-3003 (U.S.A.), and ‘. Department ofAnatomy and Cell Biology, ‘The University qf Michigan ~ed~cai School, Ann Arbor, Mf 4al~~-O~2~ (U.S.A.) Tel. (313)763-53% Received by J.R. Slightom: 19 November 1990 Revised/accepted: 11 March/26 March 1991 Received at publishers: 29 April 1991
SUMMARY
We have isolated a cDNA that encodes subunit VIIc of bovine cytochrome c oxidase (COX VIIc). The 325-bp cDNA contains sequences encoding the mature 47-amino acid (aa) polypeptide and a 16aa presequence. The deduced aa sequence of the processed polypeptide is identical to that of the heart protein determined by aa sequencing. Northern-blot analysis reveals a single 525-nucleotide (nt) transcript in all tissues examined, whose levels vary with the corresponding respiratory activities in different tissues; thus, no evidence for isoforms of COX VIIc is seen in adult tissues. Southern-blot analysis of bovine genomic DNA digested with three different restriction enzymes reveals several bands that hybridize with the cDNA. We present here the sequence of one genomic region that contains a processed gene encoding COX VIIc. The genomic and cDNA nt sequences are 99 y0 identical throughout the 189-bp open reading frame; the deduced aa sequences are identical. The sequence of the genomic clone suggests that the cDNh terminates prematurely at an EcoKI site in the 3’-untranslated region. We have compared COX I’ZZccDNAs from cow, human and mouse, and find the presequence similarity among them to be 100% at the aa level.
subunits subunits
Cytochrome c oxidase (COX; EC 1.9.3. l), the terminal protein complex of the electron transport chain, catalyzes the reduction of molecular oxygen to water. The protonpumping activity (Wikstrom and Krab, 1979) of COX generates energy that drives the synthesis of ATP. An intrinsic component of the mitochondrial (mt) inner membrane, COX is a multisubunit enzyme some of whose
are encoded by the mt genome, whereas other are specified by the nuclear genome. In the yeast Saccharornyces cerevisiae, COX is composed of nine distinct polypeptides (Power et al., 1984). ~amlnalian COX has been resolved into at least 13 different polypeptide subunits (Kadenbach and Merle, 1981). In both yeast and mammals, the three largest subunits (I, II, and III) are encoded by mt DNA (Poyton, 1980; Anderson et al., 1981; 1982) and constitute both the catalytic and proton-pumping
Correspondenceto: Dr. L.I. Grossman,
Abbreviations:
INTRODUCTION
and Genetics,
Wayne
Department
State University
of Molecular
School of Medicine,
Biology
Detroit,
MI
48201 (U.S.A.) Tel. (313)577-5326; man~mts.cc.wayne.edu.
Fax (313)577-5218;
* Present address: Department Biology, Northwestern University (U.S.A.)
Tel. (312)908-4075.
of Cell, Medical
Electronic
mail:
LGross-
Molecular and Structural School, Chicago, IL 60611
cytochrome
aa, amino acid(s); b, bovine; bp, base pair(s); COX Vllc, c oxidase subunit VIIc; COX Vllc gene encoding
kb, kilobase or 1000 bp; mt, mitochondrial; oligodeoxyribonucleotide; ORF, open reading sodium
dodecyl
NaCl/l
mM EDTA;
sulfate;
SSPE,
Y, C or T.
COX VIIc;
nt, nucleotide(s); oligo, frame; R, A or G; SDS,
10 mM Na.phosphate
pH 7.4!0.18 M
212 regulation to better adapt to the functional needs of particular cells. We previously presented the sequence of the bovine COX V11c cDNA (Aqua et al., 1989). To gain insights into its functional role, we have now examined the expression of this cDNA in several bovine tissues. and compared its structure to that of a processed bovine C’OX VIIcgene. Partial aa sequencing has been used to show that bovine COX VIIc dots not appear to have isoforms
domains of COX (Ludwig and Schatz, 1980; Helm et al.. 1987). The remaining subunits arc encoded by nuclear DNA and, like other nuclear-encoded proteins destined for mitochondria, most are synthesized on cytoplasmic ribosomes (Mihara and Blobel. 1980: Hare et al.. 1980) as N-terminal larger precursors carrying an additional domain, transport chondria
the presequence,
which serves as a signal for the
of cytoplasmically synthesized proteins to mito(Horwich et al., 1985). The precursors are subse-
(Yanamura et al.. 1988). We now find a single transcript of 525 nt in the tissues examined kvhose levels correspond to the respiratory levels in each tissue, consistent with the
quently cleaved (Hay et al., 1984) by mt protcascs to release the mature polypeptides, which associate with the mt subunits to form the active enzyme. The functional role of any of the nuclear-encoded subunits of cytochrome (’ oxidase remains to be elucidated. It has been suggested that these subunits may be involved in regulating the catalytic activity of the holocnzymc by binding intracellular effecters and transmitting the signal to the active center by a conformational change (Kadenbath et al., 1987). Cross-linking studies (Jarausch and Kadcnbach, 1985) suggest that in the holoenzymc COX VIIc may contact COX II, which is in the active center. Subunits that have tissue-specific isoforms may allow such
abscncc
RESULTS
120 /
l
130 *
220 *
250
290
280 *
330 I
l
340 *
.A..
350 /
,,.,...,
A
370 1
360 *
390 *
380 *
400 t
AGCATCCTAGATATGTJTGTAAATAAACTTATA~ . . . . . . . . . . ..C..AGCATCCTAGATATGTTTGTAAATAAACTTATAGCGTAAATCCTTAATGCCTC~TCTCTGAAATATGGAACATGATAATTGAACATGTG 420 *
430 f
440 /
460 f
450 *
470 *
490 *
480 *
500 *
CTTACATTTlGTlTGGGTAATATTGCATTATTAGTTTCll~TATGTATTTGATTTAGClTGTTTAAAAAAAAAAAAGCTGAGACACACAATGGGCATT
1BCOX7C.l
CTTCATGTTTAGAATCCTACACACTCATTACAACAGAAACTACAAGTATACCCCCATCCTCCACTGTTCAGGACCAAGCTGTGGCCAGGGCCTGCCAGATC
endonuclease
Vllc
mapping
extending
530 *
processed
and Southern
strongly
540 *
560 *
550 *
gene (iBCOX7c.l)
analysis
showed
arrow) translation
was carried
590
610
600 *
l
to the cDNA clones. cDNAs
510 t
l
3'
were isolated
from a library in igt IO
To isolate the processed gene, approx. 1.8 x loh plaques were screened b) Five genomic clones for bovine COX VIIc were isolated and plaque-purified.
that one of them, I.BCOX7c.1, (bCOX7c.I),
from double-stranded
was subcloned templates
contains
a 16.5-kb region.
(Hattori
and Sakaki,
A 1.35-kb EcoRI-SulI
) and sequenced.
into pBSM l3( -
1986) ofnested
The sequence
deletions
is from Aqua et al. is dashed.
( IYXY). 1 he other sequences
bCOX7c-2
designates
a circular
arc aligned
permutation
A polyadenylntion out on both strand\
signal (single underline) ofthc
with it and Identical
generated
during
site in IBCOX7c.l
library
of iBOX7c.l
was deposited
construction
oligo and
by dots. The stop (see section c). l‘hc
are shown in italics; the double arrowheads
and poly(A) tract (double underline)
cl>NA?. The sequence
nt are indicated
of
(Henikoff.
strand was obtained by subcloning regions into M 13 and priming with the universal primer or a synthetic (Genosqs. The V+‘oodlands. TX). The scquencc of the expressed cDNA, bCOX7c-1, its translation,
found at the 5’ end that match the region 3’ to the mutatedEcoR
nt in bCOX7c-2.
580 i
570 *
is compared
to the hovine c‘0.Y C’//c cDNA
from the Strll site was determined
1984). The sequence of the noncoding primer (5’-CACCTCTTAATATGCTC) site (downward
300 *
l
~BCOX7C.l
found to hybridize
dldeox! sequencing No. X%X23).
270 *
200 *
l
CAATCCCITGAATTC
I. The nt sequenceof the COX
in bCOX7c-2
190
180 *
l
260 f
l
with the oligo 5’-GTCATCATlGCIARIARlC~~CCAYTTRTTYTClAC. hybridization (Maniatis et al., 1982) with the cDNA Insert of hCOX7c-1.