Biochimica et Biophysica Acta, 1131 (1992) 203-206 © 1992 Elsevier Science Publishers B.V. All rights reserved 0167-4781/92/$05.00
BBAEXP 90360
203
Short Sequence-Paper
Sequence of the feline cardiac sarcoplasmic reticulum C a 2 +-ATPase A n n e M. G a m b e l a, Thomas N. Gallien a, Terrie Dantzler-Whitworth a, Michael Bowes a,1 and D o n a l d R. Menick a,b Cardiology Dicision, Department of Medicine, and the Gazes Cardiac Research Institute; and b Department of Biochemistry and Molecular Biology Medical Unicersity of South Carolina, Charleston, SC (USA) (Received l0 March 1992)
Key words: Sarcoplasmic reticulum; ATPase, (Ca 2 ++ Mg 2+ )-; cDNA; Cardiac; Nucleotide sequence
The complementary DNA for the feline cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase has been cloned and sequenced. The deduced amino acid sequence consists of 997 amino acid residues which shows greater than 98% identity with the pig, rabbit and human SR CaZ+-ATPase. The 5' and 3' untranslated regions are also strikingly similar to the published rabbit sequence. Calcium ions play a pivotal role in the contractionrelaxation cycle in the heart. Therefore, the control and regulation of intracellular [Ca 2+] is a crucial factor of cardiac function. Intracellular calcium in eucaryotic cells is regulated by the plasma membrane, and by intracellular organelles. In cardiac and skeletal muscle cells the sarcoplasmic reticulum (SR) plays the primary role in the regulation of cytoplasmic calcium [1]. Furthermore, the Ca2+-ATPase of cardiac and skeletal muscle SR is the sole protein responsible for active transport of calcium into the SR. Because of the central role it plays in calcium homeostasis it has been the focus of extensive physiological, biochemical, biophysical and molecular biological studies. The SR Ca 2+ATPase is structurally and functionally related to other ion motive P-type ATPases such as the N a + / K + [2] and H + / K + [3] ATPases of animal cells, the plasma membrane H+-ATPase of Neurospora and yeast [4,5] and the K+-ATPase of Streptococcus and Escherichia coli [6]. We have cloned a full-length cDNA of the feline cardiac SR Ca2+-ATPase. Four oligonucleotides were designed from the known amino acid sequence of the rabbit slow twitch SR Ca2+-ATPase [7,8]. Three of the oligonucleotides cor-
Correspondence to: D.R. Menick, Cardiology Division, Department of Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2221, USA. 1 Present address: Michael Bowes, Department of Biology, Humboldt State University, Arcata, CA, 95521, USA. The sequence data in this paper have been submitted to the EMBL/Genbank Data libraries under the accession number Z11500.
respond to regions of the Ca2+-ATPase which exhibit a high degree of homology with the Na+/K+-ATPase and the H+-ATPase of Neurospora and yeast [4,9]. These include (i) a region surrounding the phosphorylation site, (ii) a region which appears to contribute to the ATP binding containing the FITC-reactive lysine, and (iii) a region of unknown function corresponding to residues 143-156 of the rabbit SR Ca 2+ATPase. The remaining oligonucleotide codes for a portion of the putative calcium binding region near the N-terminus which should be unique to the SR Ca 2+ATPase. These oligonucleotides were used to screen a cat cardiac cDNA library construction Agtl0. An initial screening of approx. 250000 independent plaques yielded five positive clones. One of these, H1, is 1.4 kilobases long and hybridizes to probes from the putative calcium binding domain, phosphorylation site, and the putative ATP binding site. HI was then used to rescreen the cat cardiac cDNA library. The screening of 150000 independent plaques in the cat library yielded 73 positive clones. These clones were rescreened with the RC oligonucleotide (calcium binding domain). One of these clones, pll.4, contained a 3.8 kb insert. Nucleotide sequencing of both strands by dideoxy chain termination method [10] revealed a single ORF for the full-length SR Ca2+-ATPase (Fig. 1). The cDNA clone contained a 158 bp 5' UTR and the single termination codon was followed a 807 bp 3' UTR with a poly(A) tail of 15 bases. There are two canonical polyadenylation signals and one out of the five independent clones we sequenced utilized the first signal with the remaining four using the second. The
204 quence amount out the of the
amino acid sequence of the feline cardiac Ca 2 +-ATPase exhibited a striking 98% homology with that of the rabbit [7,8], human [11], rat [12] and pig [13] cardiac/slow twitch SR Ca2+-ATPase sequences. Se-
GCGCCCGGCGAGGCGGAACGGGCCGCAGGAGGAGGAGGAGGGGGGAGAGGCCAGTCAGCGCCTGGC
-156 90
analysis reveals that there is a remarkable of homology at the nucleic acid level throughO R F and in the 5' and 3' UTRs between each cardiac/slow twitch SR Ca:+-ATPase clones
GCG•AGGGTGG•ACGAG•C•GCGGC••GAGTGCGAGG•GGAGGCGAGGAGGC•G•GGGGACTGGAGG•GAGGCCG••GGG•CC•GCAGCC
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GCCCTGG•TGTGG•GG••ATTCCTGAAGGT•TGCCTG•TGT•AT•A••AC•TG•CTGGCT•TTGGAA•T•GcAGAATGGcAAAGAAAAAT A
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AAACAGAAGGT CAT GT CT GTCAT CAGGGAAT GGGGTAGT GGCAGCGACACACTGCGGTGCCTGGCTCTGGCCACTCATGACAACCEACTG K
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GCGATTTACAACAATATGAAGCAGTTTATCCGCTACCTCATCTCTTCGAATGTGGGGGAAGTTGTCTGTATTTTC•TGACTGCAGCCCTT A
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GGATTTC•TGAGGCTTTAATTCCTGTC•AG•TGCTCTGGGTCAATCTGGTGACAGACGGCCTGCCTG•CACCGCACTGGGGTTTAAC•CA G
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TGTTACGTTGGCGCTGCTACTGTGGGTGCTGCCG•GTGGTGG•TTTATTGCTGCCGACGGGGGTCCAAGGGTGTCCTTCTACCAACTGAGC C
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CA•TTCCTACAGTGTAAAGATGACAACCCGGACTTTGAAGGAGTGGATTGTGCAATCTTTGAGTCCCCGTACC•GATGACAATGGCGCTG H
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CACATCTACCAGCCCTCTGCATGATTGATGTTGGGGGAAAAAGAAAAGTAGAAAAAAGTCCCCA
ACTCATTGTGTGTTATGTGGAGGAAA
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CGTGTATTACCAACGGGTTTAGCTTTTAAATCAAAATAATAATTACAAATGTACAATTTAGCTT
AACGAATCAGAAAGCCTCTCCAGCGA
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TAGCTAGATTTCTCTATGGTGTCATT
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TAAACATGCTACTTTTTAATTGGCTCTGTACAGTTTGCTTATTTATAAATTTATTGAAAACACT
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3691
CTTTAGACTGTAAATAGAGATCAGTTGTTTTATTTCTGTGCTGGTATAGCAACAAGCATTGCAC
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ATCTACGATAAAAAAAAAAAAAAA
CGTCAATTTTCACAACGCATGTTTGA AGACATGGTTTCAGGTAAATAAATCT
Fig. 1. Nucleotide sequence and deduced amino acid sequence of the full-length feline cardiac SR Ca2+-ATPase cDNA. Nucleotides are numbered with nucleotide '1' being the A of the A T G start codon. The two polyadenylation sequences in the 3' U T R are underlined. The sequence has been submitted to the E M B L / G e n b a n k Data libraries under the accession n u m b e r Z11500.
despite the species differences. Kyte and Doolittle hydropathy profile [14] reveals 10 putative transmembrane helices and two large cytoplasmic domains containing the phosphorylation site (Asp-351) and the nucleotide binding fold as described for the rabbit SR Ca2 +-ATPase [8]. This work was supported in part by grant NHLBI RO1 44202 from the National Institutes of Health
(DRM) and a grant from the American Heart Association, SC Affiliate, 1988-90 (DRM). References 1 Carafoli, E. (1987) Annu. Rev. Biochem. 56, 395-433. 2 Shull, G.E., Schwartz, A. and Lingrel, J.B. (1985) Nature 316, 691-695.
206 3 Shull, G.E. and Lingrel, J.B. (1986) J. Biol. Chem. 261, 1678816791. 4 Hager, K.M., Mandala, S.M., Davenport, J.W., Speicher, D.W., Benz, E.J. Jr. and Slayman, C.W. (1986) Proc. Natl. Acad. Sci. USA 83, 7693-7697. 5 Serrano, R., Kielland-Brandt, M.C. and Fink, G.R. (1986) Nature 319, 689-693. 6 Solioz, M., Mathews, S. and Furst, P. (1987) J. Biol. Chem. 262, 7358-7362. 7 MacLennan, D.H., Brandl, C.J., Korczak, B. and Green, N.M. (1985) Nature 316, 696-700. 8 Brandi, C.J., Green, N.M, Korczak, B. and MacLennan, D.H. (1986) Cell 44, 597-607.
9 Green, N.M., Taylor, W.R. and MacLennan, D.H. (1988) in The Ion Pumps: Structure, Function and Regulation (Stein, W.D., ed.), pp. 15-24, Alan R. Liss, New York. 10 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 11 Lytton, J. and MacLennan, D.H. (1988) J. Biol. Chem. 263, 15024-15031. 12 Komuro, I., Kurabayashi, M., Shibazaki, Y., Takaku, F. and Yazaki, Y. (1989) J. Clin. Invest. 83, 1102-I 108. 13 Eggermont, J.A., Wuytack, F., De Jaegere, S., Nelles, L. and Casteels, R. (1989) Biochem. J. 260, 757-761. 14 Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132.
BBAEXP 90360
203
Short Sequence-Paper
Sequence of the feline cardiac sarcoplasmic reticulum C a 2 +-ATPase A n n e M. G a m b e l a, Thomas N. Gallien a, Terrie Dantzler-Whitworth a, Michael Bowes a,1 and D o n a l d R. Menick a,b Cardiology Dicision, Department of Medicine, and the Gazes Cardiac Research Institute; and b Department of Biochemistry and Molecular Biology Medical Unicersity of South Carolina, Charleston, SC (USA) (Received l0 March 1992)
Key words: Sarcoplasmic reticulum; ATPase, (Ca 2 ++ Mg 2+ )-; cDNA; Cardiac; Nucleotide sequence
The complementary DNA for the feline cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase has been cloned and sequenced. The deduced amino acid sequence consists of 997 amino acid residues which shows greater than 98% identity with the pig, rabbit and human SR CaZ+-ATPase. The 5' and 3' untranslated regions are also strikingly similar to the published rabbit sequence. Calcium ions play a pivotal role in the contractionrelaxation cycle in the heart. Therefore, the control and regulation of intracellular [Ca 2+] is a crucial factor of cardiac function. Intracellular calcium in eucaryotic cells is regulated by the plasma membrane, and by intracellular organelles. In cardiac and skeletal muscle cells the sarcoplasmic reticulum (SR) plays the primary role in the regulation of cytoplasmic calcium [1]. Furthermore, the Ca2+-ATPase of cardiac and skeletal muscle SR is the sole protein responsible for active transport of calcium into the SR. Because of the central role it plays in calcium homeostasis it has been the focus of extensive physiological, biochemical, biophysical and molecular biological studies. The SR Ca 2+ATPase is structurally and functionally related to other ion motive P-type ATPases such as the N a + / K + [2] and H + / K + [3] ATPases of animal cells, the plasma membrane H+-ATPase of Neurospora and yeast [4,5] and the K+-ATPase of Streptococcus and Escherichia coli [6]. We have cloned a full-length cDNA of the feline cardiac SR Ca2+-ATPase. Four oligonucleotides were designed from the known amino acid sequence of the rabbit slow twitch SR Ca2+-ATPase [7,8]. Three of the oligonucleotides cor-
Correspondence to: D.R. Menick, Cardiology Division, Department of Medicine, Medical University of South Carolina, 171 Ashley Avenue, Charleston, SC 29425-2221, USA. 1 Present address: Michael Bowes, Department of Biology, Humboldt State University, Arcata, CA, 95521, USA. The sequence data in this paper have been submitted to the EMBL/Genbank Data libraries under the accession number Z11500.
respond to regions of the Ca2+-ATPase which exhibit a high degree of homology with the Na+/K+-ATPase and the H+-ATPase of Neurospora and yeast [4,9]. These include (i) a region surrounding the phosphorylation site, (ii) a region which appears to contribute to the ATP binding containing the FITC-reactive lysine, and (iii) a region of unknown function corresponding to residues 143-156 of the rabbit SR Ca 2+ATPase. The remaining oligonucleotide codes for a portion of the putative calcium binding region near the N-terminus which should be unique to the SR Ca 2+ATPase. These oligonucleotides were used to screen a cat cardiac cDNA library construction Agtl0. An initial screening of approx. 250000 independent plaques yielded five positive clones. One of these, H1, is 1.4 kilobases long and hybridizes to probes from the putative calcium binding domain, phosphorylation site, and the putative ATP binding site. HI was then used to rescreen the cat cardiac cDNA library. The screening of 150000 independent plaques in the cat library yielded 73 positive clones. These clones were rescreened with the RC oligonucleotide (calcium binding domain). One of these clones, pll.4, contained a 3.8 kb insert. Nucleotide sequencing of both strands by dideoxy chain termination method [10] revealed a single ORF for the full-length SR Ca2+-ATPase (Fig. 1). The cDNA clone contained a 158 bp 5' UTR and the single termination codon was followed a 807 bp 3' UTR with a poly(A) tail of 15 bases. There are two canonical polyadenylation signals and one out of the five independent clones we sequenced utilized the first signal with the remaining four using the second. The
204 quence amount out the of the
amino acid sequence of the feline cardiac Ca 2 +-ATPase exhibited a striking 98% homology with that of the rabbit [7,8], human [11], rat [12] and pig [13] cardiac/slow twitch SR Ca2+-ATPase sequences. Se-
GCGCCCGGCGAGGCGGAACGGGCCGCAGGAGGAGGAGGAGGGGGGAGAGGCCAGTCAGCGCCTGGC
-156 90
analysis reveals that there is a remarkable of homology at the nucleic acid level throughO R F and in the 5' and 3' UTRs between each cardiac/slow twitch SR Ca:+-ATPase clones
GCG•AGGGTGG•ACGAG•C•GCGGC••GAGTGCGAGG•GGAGGCGAGGAGGC•G•GGGGACTGGAGG•GAGGCCG••GGG•CC•GCAGCC
1 ATGGAGAACG•G•A•A•AAAGA•GGTGGAGGAGGTG•TGGGCTACTT•GGCGT•AA•GAGAGcA•AGGG•TGAGCCTGGAGCAGGTcAAG
I 91 31
181 61
271 91 361 121 451 151 541 181
631 211
721 241
811 271
901 301
991 331
1081 361
1171 391 1261
421 1351 451 1441 481 1531 511
1621 541
M
E
N
A
H
T
K
T
V
E
E
V
L
G
Y
F
G
V
N
E
$
T
G
L
S
L
E
Q
V
K
AAGCT•AAGGAGAGATGGGG•TCCAACGAG••ACCGGCTGAAGAAGGAAAAACCTTGTTGGAACTTGTGATTGAGcAGTTTGAAGACTTA K
l
K
E
R
W
G
S
N
E
L
P
A
E
E
G
K
T
L
L
E
l
V
I
E
Q
F
E
0
l
F
V
E
TTAGTTAGAATTTTATTG•TGGCAGCTTG•ATATCTTTTGTTTTGG•TTGG•TTGAAGAAGGTGAAGAAA•AAT•AcAGCCTT•G•AGAA L
V
R
I
L
L
L
A
A
C
I
S
F
V
L
A
W
F
E
E
G
E
E
T
l
T
A
C••TTTGTAA•TTTA•TTATA•TAGTAG•CAATGCTATTGTGGGTGTATGG•AGGAAAGAAATG•TGAGAATG••ATTGAAGCCCTTAAG
P
F
V
|
L
L
I
L
V
A
N
A
I
V
G
V
W
Q
E
R
N
A
E
N
A
I
E
A
l
K
G
O
I
GAATATGAG•CTGAAATGGGCAAAGTGTAT•GACAGGA•AGAAAGAGTGTA•AGcGGATTAAAGCTAAGGA•ATAGT•c•TGGcGATATT E
Y
E
P
E
M
G
K
V
Y
R
Q
D
R
K
S
V
Q
R
I
K
A
K
D
I
V
P
GTAGAAATTGCTGTTGGTGA•AAAGTTC•TG•TGATATAAGATTAACTTCCATCAAA••TACTACTCTGAGAGTTGA•CAGT••ATTCTc V
E
I
A
V
G
D
K
V
P
A
D
[
R
L
T
S
I
K
S
T
T
L
R
V
O
Q
$
I
L
N
M
L
S
F
A•AGGTGAGTCTGTGTCTGTCATCAAGCACACTGACCCTGTCCCAGACCCACGGGCCGTCAAC•AAGATAAGAAGAAcA•G•TGTTT••T T
G
E
S
V
S
V
1
K
H
T
D
P
V
P
D
P
R
A
V
N
Q
D
K
K
GGAACAAACATTGCTGCTGG•AAAG•CATGGGAGTGGTGGTGGcAACTGGAGTTAACA•AGAAAT•GGCAAGATCcGGGATGAGA•GGTG G
T
N
I
A
A
G
K
A
M
G
V
V
V
A
T
G
V
N
T
E
[
G
K
l
R
0
E
N
V
GCAA•AGAACAGGAGAGAACACCCCTC•A•CAGAAACTAGACGAGTT•GGGGAGCAGcTTTCCAAAGTCATAT•CCTTATTTGCATTGCA A
T
E
Q
E
R
T
P
L
Q
e
K
L
D
E
F
G
E
Q
L
S
K
V
I
S
L
I
C
I
A
F
K
I
A
V
M
A
K
K
W
L
T
T
N
Q
$
T
Y
A
C
A
L
C
G•••GGATCATAAA•ATTGGGCA•TTCAATGACC•AGTTCACGGAGGCTCCTGGATCAGGGGTGCTATTTACTA•TTTAAAATTG•GGTG V
W
I
I
N
I
G
H
F
N
D
P
V
H
l
l
S
W
I
R
l
A
I
Y
Y
GCCCTGG•TGTGG•GG••ATTCCTGAAGGT•TGCCTG•TGT•AT•A••AC•TG•CTGGCT•TTGGAA•T•GcAGAATGGcAAAGAAAAAT A
L
A
V
A
A
I
P
E
G
L
P
A
V
]
P
T
C
L
A
L
G
T
R
R
GCCATTGTTCGAAGTCTC•CTT•TGTGGAAACCCTTGGTTGTACTTCTGTTA•CTGCTCAGA•AAGA•TGGTA•GC•TACGA•GAA•CAG A
I
V
R
S
L
P
S
V
E
T
L
G
C
T
S
V
I
C
S
D
K
T
G
T
ATGT•AGTGTGCAGGATGTTcATT•TGGACAAGGTTGAGGGGGATA•TTGTTcC•TTAATGAGTTTA••ATAA•TGGGTCAACATATGCA M
S
V
C
R
M
F
%
L
D
K
V
E
G
O
T
C
S
l
N
E
F
T
I
T
G
c••ATTGGAGAAGTGCATAAAGATG•TAAAC•AG•GAA•TGT•A•cAATATGATGGTCTTGTGGAAT•GG•AACAATTTGTGC••TTTGT P
l
G
E
V
H
K
D
D
K
P
V
K
C
H
Q
Y
0
G
L
V
E
L
A
T
l
AATGATT•TG•A•TGGA•TA•AATGAGGCGAAGGGCGTGTATAAAAAGTTTGGGGAAGcTACAGAGAc•GCTcTCA••TG•cTAGTAGAG
N
0
S
A
L
D
Y
N
E
A
K
G
V
Y
K
K
F
G
E
A
T
E
T
A
L
T
C
L
V
E
L
M
K
AAGATGAATG•ATTTGATA•cGAATTGAAGGGT•TTTcTAAAATAGAAAGAG•AAATGcCTGcAACTCGGTcATAAAGcAGTTAATGAAA K
M
N
V
F
D
T
E
L
K
G
L
S
K
AAAGAAT T TACT CTCGAGT T T TCACGCGATAGAAAATCAAT K
E
F
ATGTTTGT M
F
T
L
E
F
S
R
D
R
K
S
I
E
R
A
N
K
G
A
P
E
G
V
I
D
R
C
N
S
V
[
K
Q
GT CAGT T T A T T G T A C A C C A A A C A A A C C A A G C C G G A C A T C G A T G A G C A A A M
S
V
Y
C
T
1
R
V
GAAGGGCGCTCCCGAAGGTGTCATTGACAGGTGCACCCACATTCGGGT V
A
C
T
H
P
N
K
P
S
R
T
S
M
S
K
l
V
TGGAAGTACCAAAGTCCCTATGACCCCTGGAGTC l
S
T
K
g
P
M
T
P
AAACAGAAGGT CAT GT CT GTCAT CAGGGAAT GGGGTAGT GGCAGCGACACACTGCGGTGCCTGGCTCTGGCCACTCATGACAACCEACTG K
Q
K
V
N
S
V
I
R
E
W
G
S
G
S
D
T
L
R
C
L
A
L
A
T
H
D
N
P
L
205 1711 571 1801 601
1891 631
1981 661 2071 691 2161 721
2251 751 2341 781 2431 811 2521 841 2611 871
2701 901
2791 931 2881 961
2971 991
AGAAGAGAAGAAATGAACCTTGAGGACTCTGCCAACTTTATCAAATATGAGA•TAATCTGACTTTCGTTGGCTGTGTGGG•ATG•TGGAC R
R
E
E
M
N
L
E
D
D
A
N
F
l
K
Y
E
T
N
L
T
F
V
G
C
V
G
M
L
O
G
T
E
L
E
F
l
A
H
G
G
R
A
L
N
P
l
G
L
S
M
A
L
E
N
I
l
T
P
R
N
Y
••TCCAAGAATTGAAGTGG•CTCTT•TGTGAAGCTTTGCCGG•AAG•AGG•ATCCGGGTGATCATGATCACAGGGGACAACAAAGGCA•T P
P
R
I
E
V
A
S
S
V
K
l
C
R
Q
A
G
l
R
V
I
M
I
T
G
O
N
K
GCTGTGGCCATCTGTCGCCGCATTGG•ATCTTTGGGCAGGACGAGGATGTGA•GTCAAAGGCTTTTACGGGTCGGGAGTTTGATGAGCTC A
V
A
1
C
R
R
I
G
I
F
G
Q
O
E
D
V
T
S
K
A
F
T
G
R
E
F
O
AGT•CTTCAG•CCAGAGAGATG•CTGCTTGAATG•CCGCTGTTTTGCTCGAGTTGAGCCTTCCCA•AAGT•AAAAATTGTAGAGTTTCTT S
g
S
A
O
R
D
A
C
L
N
A
R
C
F
A
R
V
E
A
F
H
K
S
K
l
V
•AGT•TTTTGATGAGATTA•AG•TATGACCGGGGATGGCGTGAATGATGCT•CCGCCCTGAAGAAATCTGAGATTGGCATTGCCATGGG• 0
S
F
D
E
l
T
A
M
T
G
D
G
V
N
O
A
P
A
L
K
K
S
E
l
G
[
TCTGGCACGG•AGTGGCTAAAA•CG•CTCTGAGATGGTCCTGGCC•ACGACAACTTCTCCACCATTGT•GCTGCTGTTGAGGAGGGGCGG S
G
T
k
V
A
K
T
A
S
E
M
V
L
A
D
D
N
F
S
T
I
V
A
A
V
E
E
GCGATTTACAACAATATGAAGCAGTTTATCCGCTACCTCATCTCTTCGAATGTGGGGGAAGTTGTCTGTATTTTC•TGACTGCAGCCCTT A
]
Y
N
N
M
K
O
F
I
R
Y
L
I
S
S
N
V
G
E
V
V
C
l
F
L
T
A
GGATTTC•TGAGGCTTTAATTCCTGTC•AG•TGCTCTGGGTCAATCTGGTGACAGACGGCCTGCCTG•CACCGCACTGGGGTTTAAC•CA G
F
P
E
A
L
l
P
V
O
L
l
W
V
N
L
V
T
D
G
L
P
A
T
A
L
G
F
•CTGATCTGGA•AT•ATGAACAAACCACCCCGGAAC•CGAAGGAACCACTGATCAGCGGATGGCTCTTTTTCCGTTACCTAGCTATTGGC P
O
L
D
I
H
N
K
P
P
R
N
P
K
E
P
L
l
S
G
W
L
F
F
R
Y
L
A
TGTTACGTTGGCGCTGCTACTGTGGGTGCTGCCG•GTGGTGG•TTTATTGCTGCCGACGGGGGTCCAAGGGTGTCCTTCTACCAACTGAGC C
Y
V
G
A
A
T
V
G
A
A
A
W
W
F
[
A
A
D
G
G
P
R
V
S
F
Y
O
CA•TTCCTACAGTGTAAAGATGACAACCCGGACTTTGAAGGAGTGGATTGTGCAATCTTTGAGTCCCCGTACC•GATGACAATGGCGCTG H
F
L
O
C
K
0
D
N
P
D
F
E
G
V
O
C
A
l
F
E
S
P
Y
P
M
T
TCTGTTCTAGTAACCATAGAGATGTGTAACGCC•TCAACAGCTTGTCTGAAAACCAGTCTCTGCTGAGGATGCC•••TTGGGAGAA•ATc S
V
L
V
T
I
E
N
C
N
A
k
N
S
L
S
E
N
O
S
L
L
R
M
P
P
W
TGG•T•GTGGGCTCCATCTGCCTGTCCATGTCGCTCCATTTCCTCATCCTCTACGTGGAACCTTTGCcACTTATTTTCCAGATCACACCG W
L
V
G
S
I
C
L
S
M
S
L
H
F
L
[
L
Y
V
E
P
L
P
L
I
F
Q
•TGAACTTGACCCAGTGGCTGATGGTGCTGAAAATCT•CTTGCCTGTGATTC•AATGGATGAGACACTCAAGTTTGTGGCC•GCAA•TAC L
N
L
T
O
W
L
N
V
L
K
l
S
L
P
V
I
L
H
0
E
T
L
K
F
V
A
CTGGAACCTGCAATACTGGAGTAACCGCTTCCTAAACCATTTTGCAGAAATATAAGGGTGTTCGGTTGCGTGCATGTGCGTTTTTAGCAA L
E
P
A
I
L
E
--
3061
CACATCTACCAGCCCTCTGCATGATTGATGTTGGGGGAAAAAGAAAAGTAGAAAAAAGTCCCCA
ACTCATTGTGTGTTATGTGGAGGAAA
3151
CGTGTATTACCAACGGGTTTAGCTTTTAAATCAAAATAATAATTACAAATGTACAATTTAGCTT
AACGAATCAGAAAGCCTCTCCAGCGA
3241
ACTTTGGTTTCTTTGCTGCAAGAAGACTGAGGTTTTGAAACCTTATCTAAGAACTTAGAAGCCA
TCAGCCAAGTCTCCATATTTCTCTGT
3331
GCAAAATGTCATAGCTTATATAAATGTACAATATTCAATTGTAATGCATGCCTTCAGTTGTAAG
TAGCTAGATTTCTCTATGGTGTCATT
3421
TAAACATGCTACTTTTTAATTGGCTCTGTACAGTTTGCTTATTTATAAATTTATTGAAAACACT
GCAGGTGTTGAAAGGTTAAAACGTAG AATGTAACTTATTTAATGAAATCAGA
3511
GCCTCCAGTTCATAACTTCAGTTATTTTTTTTGAGTGTGCAAACGGCTATTTTGCACTGTATTA
3601
AGCAGTAGACAGATGTTGGTGCAATACGGATATTGTGATGCATTTATCTTAATAAAATGCTAAA
3691
CTTTAGACTGTAAATAGAGATCAGTTGTTTTATTTCTGTGCTGGTATAGCAACAAGCATTGCAC
3781
ATCTACGATAAAAAAAAAAAAAAA
CGTCAATTTTCACAACGCATGTTTGA AGACATGGTTTCAGGTAAATAAATCT
Fig. 1. Nucleotide sequence and deduced amino acid sequence of the full-length feline cardiac SR Ca2+-ATPase cDNA. Nucleotides are numbered with nucleotide '1' being the A of the A T G start codon. The two polyadenylation sequences in the 3' U T R are underlined. The sequence has been submitted to the E M B L / G e n b a n k Data libraries under the accession n u m b e r Z11500.
despite the species differences. Kyte and Doolittle hydropathy profile [14] reveals 10 putative transmembrane helices and two large cytoplasmic domains containing the phosphorylation site (Asp-351) and the nucleotide binding fold as described for the rabbit SR Ca2 +-ATPase [8]. This work was supported in part by grant NHLBI RO1 44202 from the National Institutes of Health
(DRM) and a grant from the American Heart Association, SC Affiliate, 1988-90 (DRM). References 1 Carafoli, E. (1987) Annu. Rev. Biochem. 56, 395-433. 2 Shull, G.E., Schwartz, A. and Lingrel, J.B. (1985) Nature 316, 691-695.
206 3 Shull, G.E. and Lingrel, J.B. (1986) J. Biol. Chem. 261, 1678816791. 4 Hager, K.M., Mandala, S.M., Davenport, J.W., Speicher, D.W., Benz, E.J. Jr. and Slayman, C.W. (1986) Proc. Natl. Acad. Sci. USA 83, 7693-7697. 5 Serrano, R., Kielland-Brandt, M.C. and Fink, G.R. (1986) Nature 319, 689-693. 6 Solioz, M., Mathews, S. and Furst, P. (1987) J. Biol. Chem. 262, 7358-7362. 7 MacLennan, D.H., Brandl, C.J., Korczak, B. and Green, N.M. (1985) Nature 316, 696-700. 8 Brandi, C.J., Green, N.M, Korczak, B. and MacLennan, D.H. (1986) Cell 44, 597-607.
9 Green, N.M., Taylor, W.R. and MacLennan, D.H. (1988) in The Ion Pumps: Structure, Function and Regulation (Stein, W.D., ed.), pp. 15-24, Alan R. Liss, New York. 10 Sanger, F., Nicklen, S. and Coulson, A.R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 11 Lytton, J. and MacLennan, D.H. (1988) J. Biol. Chem. 263, 15024-15031. 12 Komuro, I., Kurabayashi, M., Shibazaki, Y., Takaku, F. and Yazaki, Y. (1989) J. Clin. Invest. 83, 1102-I 108. 13 Eggermont, J.A., Wuytack, F., De Jaegere, S., Nelles, L. and Casteels, R. (1989) Biochem. J. 260, 757-761. 14 Kyte, J. and Doolittle, R.F. (1982) J. Mol. Biol. 157, 105-132.
