Gene,97(1991)307-310
307
Elsevier
GENE
03800
Human mitochondrial factor 6 (Recombinant
DNA;
ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling
H + -translocating
ATPase;
phage 1 library;
sequencing)
Ali A. Javed, Kathleen Ogata * and D.R. Sanadi Department of Cell Physiology, Boston Biomedical Research Institute, Boston, MA 02114 (U.S.A.) Received by J.A. Engler: Revised: 30 July 1990 Accepted: 31 July 1990
Tel. (617) 742-2010
3 July 1990
SUMMARY
Coupling factor 6 (F,) is a component of mitochondrial ATP synthase which is required for the interactions of the catalytic and proton-translocating segments. A human fetal muscle cDNA clone encoding this protein was isolated by screening a lgtl0 library with oligodeoxyribonucleotide probes. The 497-bp F6 cDNA included a 96-bp segment that delineated a presequence of 32 amino acids (aa) in the precursor protein, and 140 bp of 3’-untranslated sequence. The remainder of the cDNA sequence coded for a mature human F, protein of 76 aa. The deduced primary aa sequence showed 8 17; homology to that of bovine F,, differing in 14 aa. Almost all of these aa substitutions were conservative and comparison of the hydropathy profiles revealed a similar pattern.
INTRODUCTION
Coupling factor 6 (F,) is a soluble integral component of mitochondrial (mt) ATP synthase that is necessary for respiration linked synthesis of ATP (Fang et al., 1984). The ATP synthase (also called H’ translocating ATPase. H + -ATPase or F,]-F,) is a membrane-bound enzyme complex consisting of an F, segment embedded in the membrane and an F, segment attached to the F,,. The F, seg-
C~,,,~e.\/‘r,,~clr,/r[(’ I by Southern anal) ais. The 2.6-kb Hi/ldIlI-H/ltr I fragment \\x purified by gel electrophoresis and subcloned into M I3mp 1X and M 13mp 19 (blessing. 19X3). The prcscnce of nearI! 3. l-ltb of \ ector DNA made primer li>t it impractical to use the M 13 universal sequencing. To a\ oid sequencing vector DNA. t\+o sequencing primers \verc s\ nthcsircd corresponding to the left am (5 ‘-GAGCAAGTTCAGCCTGGT) and right arm (5’-,\TGAGT./\TTTCTTCCAGG) Ilanking regions of the LYoRI site of i,gt 10 (Kuzicl and Tucker, 1987). These primers and the primers listed in Fig. 1 \vere used to sequence both strands of the IILIII~~II cDNA using the didco~\,-sequcnciiig method (Sanger ct al.. 1977; Tahoe and Rlchardron, 1987). The complctc nt sequence ofthc precursor of human fetal m~~scle E‘,, and the corresponding deduced XI seq~~cncc arc sho\\,n in Fig. 2. ;2 comparison of the human XI prescqucncc to that of the bo\,ine source (Walker ct al.. 1987) rc\,cals that of the 32 aa in the prcsequcnce. 24 wcrc identical. Most of the substitutions wcrc conscrvutivc and included nonpolar to nonpolar transitions. The t\vo cxceptions were the substitution of basic aa kg at position - 19 Ihr the u cakly basic amide. Gin. and notahl> the basic aa His at position - 13 for the neutral XI. Ser. Thcsc two nonconser\,~ltive on
substitutions
increase
the net basic charge
the prcscquencc
as compared to bovine scqucncc. The prcscqucncc contained four :\rg (\+‘alkcr ct al..
ho\,ine 500
between human and bovine sequence
by the probe.
1987). \\ hercas fi\,c kg
the deduced
and one
His rcsiduc:
residues. This obscr\,ation (for ;I review. see Schatz, for mt transport and possibly
human
king
containing
prcscqucncc
neither
an! acidic carlicr reports
contain
strongly confirms 1987) of the signal
rich in basic estendcd
XI,
ccjntainod
devoid
stretchcs
prcscqucnce of‘ acidic
aa
of hydrophobic
XI. This characteristic being
\vatcr
phospholipid Helical
makes the prcscquencc amphiphilic. of passage through the membrane bilayers (Schatz. 19X7).
soluble
whcclplot
!‘ct capable
(Schiffer
and Edmundson.
I%7) ofthc
309 -20
-30 MILORLFRFSSVIRSAVSVH 1
TCACC
ATG
ATT
CT1
CAG
ACG
CTC
TTC AGG
TTC
TCC
TCT
GTC ATT A
L
CCC
V
TCA
GCC
0
GTC
TCA
CTC
CAT S
I
l>HATURE>
-10 LRRYltVTAVAfNKELOPlO TTC
66
CGC
AGG
MC
ATT
GGT
GTT ACA
GCA
GTG
GCA
111 Ml
AAG
GAA
CT1
GAT
CCT
AlA
CAG
V
I
u
20
10 KLFVOKIREYKSKROTSGGP AAA
126
CTC
TTT
GTG
GAC MG
ATT
AGA
GM
TAC
AAA
TCT
R
1
AAG
CGA
CAG
ACA
TCT
CGA
GGA
CCT
AGG
GAG
CT1
111
MC
CTC
MC
CM
111
GM
GA1
CCC
AAA
111
40
30 VDASSEYOOELERELFKLKO CT1
186
GAT
GCT
AGT G
TCA GAG
TAT
CAG
CM
GAG
CTG
D
P
GAG
D
D
60
50 MFGNADMNTFPTFKFEDPKF 246
ATG 'TTT GGT Y
MT
GCA
GAC
ATG
MT
ACA
111
CCC
ACC
TTC
AAA 1
N
K
70 E GM
336
V
I
GTC
ATC
E GM
K
P
0
A
AAA
CCC
CAG
GCC
V
AGTTGTACM
CTAGTTAGM
GTTTCAGMT
GTGATGTTGA
AAAAAAAAMAMAAAAAMAAAM
DN.4 sequence
protem
the nt sequence.
polyadenylatton nt sequence
and the
data reported
in this paper
deduced
AAACATGCAT
aa sequence
The aa substitutions
signal. See legend to Fig.
I for seqttences corresponding
have been submitted
to GenBank
HUMAN
Fig. 3. Presequences ofbovine and human F, displayed as helical wheels. Hydrophobic aa are underlined and positively charged aa are circled = solid; weak = dotted
in the human sequence bovine sequence.
lines). Note the positively
substituted
for the weakly positive
charged
TTCATMCTG
of human
tn the bowne
bovine presequence (Walker et al.. 1987) sho\\cd that the x-helical folded structure had both h>,drophobic and positively charged faces. A similar amphipathic y-helix was also observed upon plotting the deduced human presequence on a helical wheel. Fig. 3 shows the helical wheel plot of the bovine and human F, presequences. The aa substitutions
(strong
TAAMTTMT
CTGGTMTTT
GTCACGGATT
s
mlTATTlG
Fig. 2. Complementary is gtvcn above
TGA AGAMTAAAG
Arg“’
GIu’~ in the
TCAAATGTTC
TTTTMTTCT
GAGTCCMAT
fetal muscle and bovine F,?. Sequence sequence
to the various and assigned
for the presequcnce
and mature
are given below the line. Underlined
sequcncc
probes
ofthe cDN.4
used for screening
the accession
number
and isolation
reprcscnts
the
clone. The
M37104.
did not affect the conformation of the non-polar and polar facts. The deduced aa sequence of mature human F, was 8 I ‘I(, homologous to the bovine sequence Lvith I4 substitutions in the 76 XI stretch (Fig. 2). Most of the substitutions were conservative. Of significance was the neutral to basic Thr”‘+ Lys substitution. The observed XI substitutions probably represent evolutionary differences betwecn bovine and human species, without any functional influence. A comparison of the 3rnoncoding region for the bovine and human F, reveals an extensive homology (see Fig. 3). The identification and characterization of human F,> cDNA was part of the study to characterize the cDNA and gene structure of all the subunits of the human mt ATP sqnthase. Debilitating disorders involving all the complexes of the electron transport chain have been reported implicating ATP (DiMauro et al.. 1987). Disorders synthase have been reported on the basis of rcduccd ATPase activity (Schotland et al., 1976). but to date there has been no report of attempts to characterize the dcfcct at the energy-linked level using P,-ATP cxchangc or H ’ -
3 10
pumping activity for assay, prcsumablq because of the lack of methods for identification and detection. Kcliable probes based on DNA sequence, in addition to specific antibodies to the individual subunits, should aid in the identification and characterization ofthe molecular basis ofthe disorders.
ACKNOII’LEDGEMENTS
This work was supported
by NIH grant No. GM 1364 1.
REFERENCtS .Amzcl, L.M. and Pederscn. msm. Annu. Andcraon,
S., deBruijn,
and Young, Conserved Biol.
52
M.H.L..
I.G.: Complete features
ATPases: structure and (1983)X01-824.
P.L.: Proton
Re\,. Biochem.
Coulaon, requencc
of the mammalian
A.R., Eperon.
mccha-
I.C.. Sanser,
of bovme mitochondrial mltochondrlal
F.
DN.4.
gcnome. J. Mol.
I56 (1982) 6X3-7 17.
Dihlauro.
S.. Bonilla,
Schon,
E.A.:
E.. Zevianl.
Mitochondrial
M.. Servidei. myopnthies.
S., Dcv~\o.
J. Inher.
Dib.
D.C. and IO (19X7)
113-12x. Fang. J-K.. Jacobs. J.I\‘., .4mino Acad. Hatefi.
Kanncr.
acid sequence Sci. L’S.4 XI
Y.: The
phorylatl(m
( 1984)
mitochondrial s>stcm
B.I., Racker,
of bovine
Annu.
heart
L. and Bradsha\\,
coupling
factor
R.A:
6. Proc. Natl.
6603-6607. electron Re\.
transport
Biochcm.
and ouidatlw
53 (19X5)
IO Ii-1069
phos-
307
Elsevier
GENE
03800
Human mitochondrial factor 6 (Recombinant
DNA;
ATP synthase: cloning cDNA for the nuclear-encoded precursor of coupling
H + -translocating
ATPase;
phage 1 library;
sequencing)
Ali A. Javed, Kathleen Ogata * and D.R. Sanadi Department of Cell Physiology, Boston Biomedical Research Institute, Boston, MA 02114 (U.S.A.) Received by J.A. Engler: Revised: 30 July 1990 Accepted: 31 July 1990
Tel. (617) 742-2010
3 July 1990
SUMMARY
Coupling factor 6 (F,) is a component of mitochondrial ATP synthase which is required for the interactions of the catalytic and proton-translocating segments. A human fetal muscle cDNA clone encoding this protein was isolated by screening a lgtl0 library with oligodeoxyribonucleotide probes. The 497-bp F6 cDNA included a 96-bp segment that delineated a presequence of 32 amino acids (aa) in the precursor protein, and 140 bp of 3’-untranslated sequence. The remainder of the cDNA sequence coded for a mature human F, protein of 76 aa. The deduced primary aa sequence showed 8 17; homology to that of bovine F,, differing in 14 aa. Almost all of these aa substitutions were conservative and comparison of the hydropathy profiles revealed a similar pattern.
INTRODUCTION
Coupling factor 6 (F,) is a soluble integral component of mitochondrial (mt) ATP synthase that is necessary for respiration linked synthesis of ATP (Fang et al., 1984). The ATP synthase (also called H’ translocating ATPase. H + -ATPase or F,]-F,) is a membrane-bound enzyme complex consisting of an F, segment embedded in the membrane and an F, segment attached to the F,,. The F, seg-
C~,,,~e.\/‘r,,~clr,/r[(’ I by Southern anal) ais. The 2.6-kb Hi/ldIlI-H/ltr I fragment \\x purified by gel electrophoresis and subcloned into M I3mp 1X and M 13mp 19 (blessing. 19X3). The prcscnce of nearI! 3. l-ltb of \ ector DNA made primer li>t it impractical to use the M 13 universal sequencing. To a\ oid sequencing vector DNA. t\+o sequencing primers \verc s\ nthcsircd corresponding to the left am (5 ‘-GAGCAAGTTCAGCCTGGT) and right arm (5’-,\TGAGT./\TTTCTTCCAGG) Ilanking regions of the LYoRI site of i,gt 10 (Kuzicl and Tucker, 1987). These primers and the primers listed in Fig. 1 \vere used to sequence both strands of the IILIII~~II cDNA using the didco~\,-sequcnciiig method (Sanger ct al.. 1977; Tahoe and Rlchardron, 1987). The complctc nt sequence ofthc precursor of human fetal m~~scle E‘,, and the corresponding deduced XI seq~~cncc arc sho\\,n in Fig. 2. ;2 comparison of the human XI prescqucncc to that of the bo\,ine source (Walker ct al.. 1987) rc\,cals that of the 32 aa in the prcsequcnce. 24 wcrc identical. Most of the substitutions wcrc conscrvutivc and included nonpolar to nonpolar transitions. The t\vo cxceptions were the substitution of basic aa kg at position - 19 Ihr the u cakly basic amide. Gin. and notahl> the basic aa His at position - 13 for the neutral XI. Ser. Thcsc two nonconser\,~ltive on
substitutions
increase
the net basic charge
the prcscquencc
as compared to bovine scqucncc. The prcscqucncc contained four :\rg (\+‘alkcr ct al..
ho\,ine 500
between human and bovine sequence
by the probe.
1987). \\ hercas fi\,c kg
the deduced
and one
His rcsiduc:
residues. This obscr\,ation (for ;I review. see Schatz, for mt transport and possibly
human
king
containing
prcscqucncc
neither
an! acidic carlicr reports
contain
strongly confirms 1987) of the signal
rich in basic estendcd
XI,
ccjntainod
devoid
stretchcs
prcscqucnce of‘ acidic
aa
of hydrophobic
XI. This characteristic being
\vatcr
phospholipid Helical
makes the prcscquencc amphiphilic. of passage through the membrane bilayers (Schatz. 19X7).
soluble
whcclplot
!‘ct capable
(Schiffer
and Edmundson.
I%7) ofthc
309 -20
-30 MILORLFRFSSVIRSAVSVH 1
TCACC
ATG
ATT
CT1
CAG
ACG
CTC
TTC AGG
TTC
TCC
TCT
GTC ATT A
L
CCC
V
TCA
GCC
0
GTC
TCA
CTC
CAT S
I
l>HATURE>
-10 LRRYltVTAVAfNKELOPlO TTC
66
CGC
AGG
MC
ATT
GGT
GTT ACA
GCA
GTG
GCA
111 Ml
AAG
GAA
CT1
GAT
CCT
AlA
CAG
V
I
u
20
10 KLFVOKIREYKSKROTSGGP AAA
126
CTC
TTT
GTG
GAC MG
ATT
AGA
GM
TAC
AAA
TCT
R
1
AAG
CGA
CAG
ACA
TCT
CGA
GGA
CCT
AGG
GAG
CT1
111
MC
CTC
MC
CM
111
GM
GA1
CCC
AAA
111
40
30 VDASSEYOOELERELFKLKO CT1
186
GAT
GCT
AGT G
TCA GAG
TAT
CAG
CM
GAG
CTG
D
P
GAG
D
D
60
50 MFGNADMNTFPTFKFEDPKF 246
ATG 'TTT GGT Y
MT
GCA
GAC
ATG
MT
ACA
111
CCC
ACC
TTC
AAA 1
N
K
70 E GM
336
V
I
GTC
ATC
E GM
K
P
0
A
AAA
CCC
CAG
GCC
V
AGTTGTACM
CTAGTTAGM
GTTTCAGMT
GTGATGTTGA
AAAAAAAAMAMAAAAAMAAAM
DN.4 sequence
protem
the nt sequence.
polyadenylatton nt sequence
and the
data reported
in this paper
deduced
AAACATGCAT
aa sequence
The aa substitutions
signal. See legend to Fig.
I for seqttences corresponding
have been submitted
to GenBank
HUMAN
Fig. 3. Presequences ofbovine and human F, displayed as helical wheels. Hydrophobic aa are underlined and positively charged aa are circled = solid; weak = dotted
in the human sequence bovine sequence.
lines). Note the positively
substituted
for the weakly positive
charged
TTCATMCTG
of human
tn the bowne
bovine presequence (Walker et al.. 1987) sho\\cd that the x-helical folded structure had both h>,drophobic and positively charged faces. A similar amphipathic y-helix was also observed upon plotting the deduced human presequence on a helical wheel. Fig. 3 shows the helical wheel plot of the bovine and human F, presequences. The aa substitutions
(strong
TAAMTTMT
CTGGTMTTT
GTCACGGATT
s
mlTATTlG
Fig. 2. Complementary is gtvcn above
TGA AGAMTAAAG
Arg“’
GIu’~ in the
TCAAATGTTC
TTTTMTTCT
GAGTCCMAT
fetal muscle and bovine F,?. Sequence sequence
to the various and assigned
for the presequcnce
and mature
are given below the line. Underlined
sequcncc
probes
ofthe cDN.4
used for screening
the accession
number
and isolation
reprcscnts
the
clone. The
M37104.
did not affect the conformation of the non-polar and polar facts. The deduced aa sequence of mature human F, was 8 I ‘I(, homologous to the bovine sequence Lvith I4 substitutions in the 76 XI stretch (Fig. 2). Most of the substitutions were conservative. Of significance was the neutral to basic Thr”‘+ Lys substitution. The observed XI substitutions probably represent evolutionary differences betwecn bovine and human species, without any functional influence. A comparison of the 3rnoncoding region for the bovine and human F, reveals an extensive homology (see Fig. 3). The identification and characterization of human F,> cDNA was part of the study to characterize the cDNA and gene structure of all the subunits of the human mt ATP sqnthase. Debilitating disorders involving all the complexes of the electron transport chain have been reported implicating ATP (DiMauro et al.. 1987). Disorders synthase have been reported on the basis of rcduccd ATPase activity (Schotland et al., 1976). but to date there has been no report of attempts to characterize the dcfcct at the energy-linked level using P,-ATP cxchangc or H ’ -
3 10
pumping activity for assay, prcsumablq because of the lack of methods for identification and detection. Kcliable probes based on DNA sequence, in addition to specific antibodies to the individual subunits, should aid in the identification and characterization ofthe molecular basis ofthe disorders.
ACKNOII’LEDGEMENTS
This work was supported
by NIH grant No. GM 1364 1.
REFERENCtS .Amzcl, L.M. and Pederscn. msm. Annu. Andcraon,
S., deBruijn,
and Young, Conserved Biol.
52
M.H.L..
I.G.: Complete features
ATPases: structure and (1983)X01-824.
P.L.: Proton
Re\,. Biochem.
Coulaon, requencc
of the mammalian
A.R., Eperon.
mccha-
I.C.. Sanser,
of bovme mitochondrial mltochondrlal
F.
DN.4.
gcnome. J. Mol.
I56 (1982) 6X3-7 17.
Dihlauro.
S.. Bonilla,
Schon,
E.A.:
E.. Zevianl.
Mitochondrial
M.. Servidei. myopnthies.
S., Dcv~\o.
J. Inher.
Dib.
D.C. and IO (19X7)
113-12x. Fang. J-K.. Jacobs. J.I\‘., .4mino Acad. Hatefi.
Kanncr.
acid sequence Sci. L’S.4 XI
Y.: The
phorylatl(m
( 1984)
mitochondrial s>stcm
B.I., Racker,
of bovine
Annu.
heart
L. and Bradsha\\,
coupling
factor
R.A:
6. Proc. Natl.
6603-6607. electron Re\.
transport
Biochcm.
and ouidatlw
53 (19X5)
IO Ii-1069
phos-
