Vo1.173, No. 3,1990

December 31, 1990

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 872-877

MOLECULAR CLONING AND CHROMOSOMAL LOCALIZATION OF A SARC0/ENDOPLASMIC RETICULUM-TYPE Ca2+-ATPASE

OF DROSOPHILA MELANOGASTER

Attila Magyar and Andr&s V~radi

Institute of Enzymology, Biological Research Center, Hungarian Academy of Sciences, 1518 Budapest, P.O.Box 7, Hungary

Received November i, 1990

We report here the characterization of a Drosophila melanogaster cDNA that encodes a sarco/endoplasmic reticulum-type Ca2+-ATPase. Previously we amplified the phos- phorylation site - FITC-binding site fragment of this cDNA by Polymerase Chain Re- action utilizing homology primers (V~radi,A., Gilmore-Hebert, M. and Benz,E.J.Jr. (1989) FEBS Letters 25__8.8 203-207) and in the present work we used this fragment as probe for isolation of a cDNA clone with the intire coding region. The isolated 3.3 kb clone has been sequenced and the primary structure of the encoded protein has been deduced. It consists of 1002 amino acids with all the characteristic features of the P-type ATPases. It shows 67-71% amino acid identity and an apparentlyidentical hydropathy profile with the mammalian sarco/endoplasmic reticulum-type CaZ+-ATPases. On ba- sis of sequence similarity we suggest that the first transmembrane segment of sarco/ endoplasmic reticulum type Ca2+-ATPases may be responsible for targeting of these transport proteins into the organellar membrane. The gene of this sarco/endoplasmic reticulum-type Ca2+-ATPase has been mapped on the right arm of chromosome 2, in section 60A. ~ 1990 Academic Pr . . . . I n c .

Intracellular organelles store Ca 2+ and the cytoplasmic concentration of Ca 2+ is partly controlled by its accumulation or release from the organelles. ATP-driven transport proteins, the organellar Ca2+-ATPases, pump Ca 2+ back into the storage organelles after release. They belong to the family of P-type ion-motive ATPases together with the Na+,K+-ATPase, the gastric H+,K+-ATPase, H+-ATPase and the plasma membrane Ca2+-ATPase. These enzyme share common molecular architecture, basic mechan- istic features of the ion transport (including the formation of a phosphorylated inter- mediate) and show amino acid sequence homology. In mammals the organellar (sarco/ endoplasmic reticulum-type) Ca2+-ATPases are encoded by closely related genes; so far three different classes of this enzyme have been characterized (SERCAI*, SERCA2 and SERCA3) (1,2,3). Both the fast twitch skeletal muscle cell form (SERCA1) and the slow twitch skeletal muscle/cardiac form (SERCA2) appear in two, alternatively spliced

*.Abbreviations used are • FITC, fluorescein isothiocyanate; FSBA, 5'fluorosulfonyl- benzoyladenozine; SR, sarcoplasmic reticulum: ER, endoplasmic reticulum. The no- menclature for the sarco/endoplasmic reticulum-type Ca2+-ATPases proposed by Burk et al. (3)is followed.

0006-291X/90 $1.50 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved. 872

Vol. 173, No. 3, 1990 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

variants. (4,5,6). The SERCA1 variants are found in the sarcoplasmic reticulum mem- brane of the muscle cells and their expression is developmentally regulated. One of the SERCA2 splice-product is also muscle cell specific, while the other one is expressed in many tissues including brain, kidney and stomach. The third form (SERCA3) is ex- pressed in a broad variety of muscle and non-muscle tissues. A chicken sarco/ endoplasmic reticulum-type to SERCA1 has also been sequenced (7). Recently we generated the phosphorylation site - FITC-binding site cDNA segment of the Na+,K+-ATPase and that of the Ca2+-ATPase of Drosophila melanogaster by uti- lizing Polymerase Chain Reaction and oligonucleotide primers designed on the basis of conserved amino acid sequence motifs around the two sites (8). Now we report the cDNA sequence and the chromosomal location of the Drosophila SR/ER-type Ca2+-ATPase.

MATERIALS and METHODS

Isolation and sequencing of cDNA : The Drosophila adult head cDNA-library con- structed in ;~gtl0 was a gift of Tom Kornberg, University of California at San Francisco. The DrosSRCaPF probe was generated by Polymerase Chain Reaction as described (8) and used for screening the cDNA-library under stringent conditions (65°C) following a standard procedure (9).. Several large cDNA clones were isolated by screening ap- prox. 5 x 105 library plaques and a 3.3kb clone, DrCa-33, containing the entire coding re- gion of a potential ATPase was subcloned into a Blueskript SK+ vector (Stratagene) mapped by restriction enzymes and sequenced by the chain termination method of Sanger et al. (10). Hydropathy analysis was carried out by the method of Kyte and Doolittle (11). Chromosomal mapping : The polytene chromosomes from salivary glands of late third instar larvae were prepared for in situ hybridization according to a standard protocol (12). A biotin - avidin labelling and detection kit of Bethesda Research Laboratories was used to label DrosSRCaPF probe and to visualize the hybridizing bands following the manufacturer's suggestion.

RESULTS and DISCUSSION

cDNA cloning and sequence analysis : A 550 bp cDNA fragment corresponding to the phosphorylation site - FITC-binding site of a Drosophila Ca2+-ATPase (DrosSRCaPF) had been generated by Polymerase Chain Reaction using homology primers as it is de- scribed in our previous work (8). This cDNA fragment was used to screen an adult head cDNA library and a 3.3 kb clone (DrCa-33) containing the entire coding region has been isolated and sequenced. The complete nucleotide sequence and the de- duced amino acid sequence are shown in Fig.1. Clone DrCa-33 comprised 3,236 bp and contains an open reading frame of 1002 codons encoding a protein with a M r of 109,540. At the beginning of this open reading frame there is an ATG codon in a sequence location (CAAGATG) harmonizing with the con- sensus initiation sequence observed in Drosophila genes (13). The encoded orotein is a SR/ER-type Ca2+-ATPase : The protein encoded by DrCa-33 is the member of the P-type ion transport ATPase family. It possesses all the funtionally important sequence motifs characteristic to each member of this family, namely the

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CGGCCACTGAAC GAAGAGGATAACGAATCAAG ATGGAAGACGGTCACTCGAAAACCGTC GAACAGTC CCTCAACTTCTTCGGAAC GGACC CCGAGCGCGGCTTAACCCTCGACCAGATCAAGGCTAAC CAGAAGAAATACGGACC CAATGAG 120 M E D G H S K T V E Q S L N F F G T D P E R G L T L D Q I K A N Q K K Y G P N E

20 TTGCC GACTGAGGAAGGAAAGAGCATC TGGCAGCTGGTCTTGGAGCAGTTCGACGATC TGCTGGTGAAGATTCTGC TGTTGGCAGCCATCATCTCATTTGTTC TCGCC CTGTTTGAGGAA 240 L P T E E G K S I W Q L V L E Q F D D L L V K I L L L A A I I S F V L A L F E E

60 CAC GAGGAAACGTTCACTGCATTTGTAGAG CCCCTAGTTATTTTACTTATCCTGATAGCCAACGCC GTGGTGGGAGTATGGCAGGAGCGTAACGCCGAGTCGGCCATTGAGGCCC TCAAG 360 H E E T F T A F V E P L V I L L I L I A N A V V G V W Q E R N A E S A I E A L K

100 GAGTACGAGCCTGAGATGGGCAAGGTGGTGcGCCAGGACAAGTCCGGCATCCAGAAGGTGCGCGCGAAAGAGATCGTGCCCGGTGACcTGGTTGAGGTGTcTGTC GGTGACAAGATC CC T 480 E Y E P E M G K V V R Q D K S G I Q K V R A K E I V P G D L V E V S V G D K I P

140 GCC GATATCC GTATCACCCACATCTAC TCGACCAC C CTTAGGATCGATCAGTCCATCCTCACCGGTGAGTC GGTCTC CGTCATCAAGCACACCGATGCCATCC CCGATCCCCGCGCCGTC 600 A D I R I T H I Y S T T L R I D Q S I L T G E S V S V I K H T D A I P D P R A V

180 AACCAGGACAAGAAGAACATCCTGTTCTCC GGCACCAACGTCGCAGCCGGCAAGGC CCGTGGCGTC GTCATCGG CAC TGGCCTGAGCACCGCCATCGGCAAGATCCGTACTGAGATGTCC 720 N Q D K K N I L F S G T N V A A G K A R G V V I G T G L S T A I G K I R T E M S

220 GAGACCGAGGAGATCAAGACCCCCC TGCAGCAGAAACTGGACGAGTTCGGTGAGCAGCTGTCCAAGGTCATTTC CGTCATTTGCGTTGCCGTGTGGGCCATCAACATCGGCCACTTCAAC 840 E T E E I K T P L Q Q K L D E F G E Q L S K V I S V I C V A V W A I N I G H F N

260 GACCCCGCTCACGGAGGCTCCTGGATCAAGGGTGCCATCTACTACTTCAAGATCGCCGTCGCTGTGGCTGTGGC TGCCATCCCCGAGGGTCTGCCCGC CGTCATCACCACCTGTCTGGCT 960 D P A H G G S W I N G A I Y Y F K I A V A V A V A A I P E G L P A V I T T C L A

3OO CTGGGCACCCGCCGCATGGCCAAGAAGAATGCCATC GTCCGCTCCCTGC CCTCCGTGGAGACCCTGGGC TGCACATCTGTCATCTGCTCTGATAAGACCGGCACACTCACCACCAACCAA 1080 L G T R R M A K K N A ! V R S L P S V E T L G C T S V I C S D K T G T L T T N Q

340 P ATGTCCGTTTCCCGCATGTTCATCTTCGACAAGGTTGAAGGCAACGACAGCAGC TTCCTCGAGTTC GAGATGACCGGCTCCAC C TACGAGC CCATCGGC GAGGTC TTCCTCAACGGTCAG 1200 M S V S R M F I F D K V E G N D S S F L E F E M T G S T Y E P I G E V F L N G Q

380 CGCATCAAGGCTGCTGACTAC GATACCCTGCAGGAACTGTCCAC CATCTGCATCATGTGCAACGACTCCGCCATTGATTACAATGAGT CAAGCAGGCC TTCGAGAAGGTTGGCGAAGCC 1320 R I K A A D Y D T L Q E L S T I C I M C N D S A I D Y N E F K Q A F E K V G E A

420 AcTGAGA••G•CcTGATTGTGCTGGCTGAGAAACTGAACAGCTTCAGcGTGAACAAGT•TGGCCTGGACCGCCGCTCGGCTGCCATCGCcTGC•GCGGTGAGATCGAAACCAAGTGGAAG 1440 T E T A L I V L A E K L N S F S V N K S G L D R R S A A I A C R G E I E T K W K

460 AAGGAATTCACC C TGGAGTTCTCTCGCGATCGTAAATCCATGTCCTCGTACTGCACCC CC CTGAAGGCTTCCCGC CTGGGCAC TGGCC CCAAGTTGTTC GTGAAGGGCGC CCC CGAGGGT 1560 K E F T L E F S R D R K S M S S Y C T P L K A S R L G T G P K L F V K G A P E G

500 FTTC GTTC TAGAGC GTTGCACACACGCCCGGGTTGG CACCAC CAAGGTTCCTCTGACCTCGGCC C TGAAGGCCAAGATCCTCGCCCTGAC CGGCCAGTAC GGTACTGGACGCGACACCCTTCGC 1680 V L E R C T H A R V G T T K V P L T S A L K A K ! L A L T G Q Y G T G R D T L R

540 TGCCTGGCTC TGGCCGTTGCC GATAGCCCCATGAAGCC CGACGAGATGGATCTGGGCGACTCCACCAAGTTCTACCAGTATGAGGTTAACC TGACC TTCGTCGGTGTTGTGGGCATGTTG 1800 C L A L A V A D S P M K P D E M D L G D S T K F Y Q Y E V N L T F V G V V G M L

58O GATCC CC CCC GTAAGGAAGTTTTC GATTCCATTGT CCGCTGCCGTGCC GCCGGTATTC GTGTTATTGTGAT CAC CGGCGACAACAAGGC CACTGCCGAGGCTATCTGC CGCAGAATCGGT 1920 D P P R K E V F D S I V R C R A A G I R V I V I T G D N K A T A E A I C R R I G

620 GTATTCGC CGAGGATGAGGACACCACTGGCAAGTC CTACTC GGGTCGCGAATTCGACGACC TTTCCCCCAC CGAACAAAAGGC TGCCGTCGC CCGCTCCCGCC TC TTCTCCCGC GTGGAG 2040 V F A E D E D T T G K S Y S G R E F D D L S P T E Q K A A V A R S R L F S R V E

660 C CCCAGCACAAGTCCAAGATTGTTGAGTTCC TGCAGAGCAT GAACGAAATC TCCGCTATGAC TGGTGATGGTGT GAACGACGCCC CC GCCCTGAAGAAGGCTGAGATCGGTAT TGCCATG 2160 P Q H K S K I V E F L Q S M N E I S A M T G D G V N D A P A L K K A E I G I A M

700 FSBA GGCTCTGGTACCGC CGTCGCCAAGTCTGCCGCCGAAATGGTGCTGGCTGACGACAACTTCTCCTCCATC GTGTCTGC CGTTGAAGAAGGTCGCGCTATC TACAACAACATGAAACAGTTC 2280 G S G T A V A K S A A E M V L A D D N F S S I V S A V E E G R A I Y N N M K Q F

740 ATCCGCTA•CTCATCTCTTCGAACATTGGTGAGGTCGT•TCCATCTTCCTTACTGCTGCCCTTGGCCTG•CCGAGGCTTTGATT•CcGT•cAGCTGCTCTGGGTCAACTTGGTTAcTGAT 2400 I R Y L I S S N I G E V V S I F L T A A L G L P E A L I P V O L L M V N L V T D

780 GGTC TCCCAGCCACCGCTCTGGGCTTCAACCC CCCTGATCTGGATATCATGGAGAAGCCCCC CAGGAAGGCCGATGAGGG TCTCATTTCCGGATGG TTGTTCTTC CGTTACATGGCTATT 2520 G L P A T A L G F N P P D L D I M E K P P R K A D E G L I S G W L F F R Y M A I

820 GGATTCTATGTCGGCGCTGCCAC C GTCGGTGCCC-CCGCCTGGTGGTTCGTGTTCTCTGATGAGGGACCCAAACTGTCCTACTGGCAGC TGACCCAC CAT CTG TCCTGCTTGGGCGGTGGC 2640 G F Y V G A A T V G A A A W W F V F S D E G P K L S Y W Q L T H H L S C L G G G

860 GACGAGTTCAAGGGCGTTGAC TC-CAAGATC TT CAGCGACCCCCATGCGATGACCATGGCTCTGTC CGTGCTGGTGACAATCGAAATGTTGAACG~T~CA~ ~G TCC~C~G 2760 D E F K G V D C K I F S D P H A M T M A L S V L V T I E M L N A M N S L S E N Q

900 TCGCTGATTACCATGCCCCCATGGTGCAACCTGTGGCTGATTGGATCAATGGCACTCTCCTTCACTCTTCACTTTGTTATTCT TTACGTCGATGTCC TC TCCACC GTC TTCCAAGTGACA 2880 S L I T M P P W C N 7. W L I G S M A L S F T L H F V I L Y V D V L S T V F Q V T

940 CCGTT GTCTGCAGAAGAATGGATAACTGTGATGAAATTCTCAATTCCTGTAGTTTTATTAGATGAGACATTGAAGTTTGTTGC TAGAAAAATCGCAGATGTTCCTGATGTCGTC GTCGAC 3000 P L S A E E W I T V M K F S I P V V L L D E T L K F V A R K I A D V P D V V V D

980 AGGATGTAGAGGTTTGGAC CAC CAAAGCCGATACGAATGTGAAACTGAATGTGAAGGAGGACGAGTTTT CCTGCCTTTATCATTC GTGTATTAATC GCTAAGCCAGC C CTGTTACTTGCA 3120

R M *

3217 AC TGCAACATTAAATCCAACATAAATCAATTTATTTGCCTTACAAACTAAAATAT TAATGGAAC TGTGGGTTAGTAACAACGGATTG

Figure1. Nucleotide sequence of clone DrCa-33 and deduced amino acid sequence of DRSERCA. Nucleotides are numbered beginning at the first base of the initiator me- tionine. The phosphorylation site (P), the FITC-binding site and the FSBA-site are un- derlined and labeled. The hypothetical hydrophobic membrane spanning structures are also underlined.

conserved residues around the phosphorylated side-chain (Asp351), the FITC-binding site harbouring Lys 515 and a peptide segment that binds ATP-analog, FSBA (around residue 700). It is closely related to the sarco/endoplasmatic reticulum-type Ca2+-ATPases : it exhibits 70,9 % amino acid sequence identity with SERCAlb, 71,3 % identity wTth SERCA2a and 67,7 % identity with SERCA3. The aligment of the DrCa-33 amino acid sequence with SERCAlb, SERCA2a and SERCA3 is shown in Fig.2.

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DRSERCA

SERCAIb

SERCA2 a

SERCA3

DRSERCA

SERCAlb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

DRSERCA

SERCAlb

SERCA2a

SERCA3

DRSERCA

SERCAlb

SERCA2a

SERCA3

DRSERCA

SERCAlb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2 a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2 a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

DRSERCA

SERCAlb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

DRSERCA

SERCAlb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

DRSERCA

SERCAIb

SERCA2a

SERCA3

MEDGH SKTVEQSLNFFGTDPERGLTLDQIKANQKKYGPNELPTEEGKS IWQLVLEQFDDL 60

--AA---ST-EC-AY--VSETT---P--V-RHLE---H .... A ..... L-EL-I----E--

--NA-T .... EV-GH--VNEST- -S -E -V-KLKERW-S .... A .... TLLE--I---E--

- -EA-LLSAADV-RR- SVTA-G .... E-VTDARER ............. L-E--V---E--

LVKILLLAAI I SFVLALFEEHEE TFTAFVEPLVILLILIANAWGVWQERNAE SAIEALK 120

--R ...... C ...... W----G---I ...... F .......... I .......... N ......

- -R ...... C ...... W----G---I ...... F ...... V---I .......... N ......

--R ...... LV ..... W----G- --T ......... M---V- --I .................

EYEPEMGKVVRQDKSGI QKVRAKE IVPGDLVEVSVGDK IPAD IRITHIYSTTLRI DQS IL 180

......... Y-A-RKSV-RIK-RD ..... I---A .... V ...... LS-K ..... V .....

............. RKSV-RIK--D ..... I--DA .... V ..... L-S-K ..... V .....

......... I-S-RK-V-RI--RD ..... I---A .... V---L-LIE-K ..... V .....

TGE SVSVIKHTDAIPDpRAVNQDKKN ILFSGTNVAAGKARGW IGTGLSTAI GKIRTEMS 240

........... EPV ............ M ...... I ..... L-I-AT--V--E ..... DQ-A

............ PV ............ M ...... I ..... M---VA--VN-E ..... D--V

....... T .................. M ...... I-S---L--AVA---H-EL .... SQ-A

ETEE I KTPLQQKLDEFGEQLSKVI SV ICVAVWAIN IGHFNDPAHGG SWINGAI YYFKIAV 300

A--QD--- .................. L ...... L ........ V . . . . R .........

A--QER ...................... I---I ....... V . . . . R .........

AV-PER .... R ...... R---HA ......... V ...... A ....... LR--V .......

AVAVAAIPEGLPAVITTCLALGTRRMAKKNAIVRSLPSVETLGCTSVICS DKTGTLTTNQ 360

-L ..........................................................

-L ..........................................................

-L ......................... R ................................

MSVSRMF IFDKVEGND S S FLEFEMTGSTYEP IGEVFLNGQRIKAADYDTLQEL STI C IMC 420

---,,---,---,-,,,-,,--,, ..... A-E ---LK-DKP-RSGQF-G -V--A- - -AL-

---C .... L---D-DTC-LN--TI ..... A ..... HKDDKPV-CHQ- -G-V--A-- -AL-

-- -C- --VVAEA-A-ACRLH--TI S-T- -T-E ---RQGE -LVRCGQF-G-V- -A- - -AL-

NDSAI DYNEFKQAFEKVGEATETAL IVL~KLNSFSVNKSGLDRRSAAIACRGE I E TKWK 480

---SL-F--T-GVY ........... TT-V--M-V-NTEVRN-S KVER-N--NSV-RQLM-

.... L .... A-GVY ........... TC-V--M-V-DTELK--SKIER-N--NSV-KQLMK

.... L .... A-GVY ........... TC-V- -M-V- DTDLK- -S -VER-G- -NSV-KQLMQ

KE FTLEF SRDRKSMS S YCTPLKASRLGTGPKLFVKGAPEGVLERCTHARVGTTKVPLTSA 540

............... V--S-A-S--AAV-N-M ......... ID--NYV ..... R--M-GP

............... V .... N-P--TSMS--M ......... ID .... I---S .... M-AG

............... V .... TRADPKAQ-S-M ....... S- I---SSV---SRT---SAT

LKAK ILALTGQYGTGRDTLRCLALAVADS PMKPDEMDLGDS TKFYQYEVNLTFVGWGML 600

V-E---SVIKEW ............. TR-T-P-RE--V-D--SR-ME--TD ..........

V-Q- -MSVIREW-S-S ......... TH-N-LRRE--H-K--AN-IK--T ...... C ....

SREH---KIRDW-S-S ......... TR-T-PRKED-Q-D-CSQ-V---TG ..... C ....

DPPRKEVFDS IVRCRAAGI RVIVI TGDNKATAEAICRRIGVFAEDEDTTGKSY SGREFDD 660

....... MG--QL--D ...... M ...... G--I ....... I-G-N-EVADHAYT ......

.... E--AS-VKL--Q ...... M ...... G--V ....... I-GQE--V-A-AFT ..... E

.... P--AAC-T--SR ..... VM ...... G--V ..... L-I-GDT--VL--AYT ......

LSPTEQKAAVARSRLFSRVEPQHKSKIVE FLQSMNE I SAMTGDGVNDAPALKKAE IG IAM 720

-FLA--RE-CR-ACC-A .... S ....... Y---YD--T ......................

-N-SA-RD-CLNA-C-A .... S ........... FD--T ......................

-- -EQ-RQ-CRTA-C-A .... A---R---N---F---T ......................

GS GTAVAKSAAEMVI~DDNFS S IVSAVEEGRAIYNNMKQF IRYL I S SNI GEVVS I FLTAA 780

........ T-S .......... T--A ....................... V .... C ......

........ T-S .......... T--A ....................... V .... C ......

............... S .... A---A ....................... V .... C ..... I

LGLPEAL I PVQLLWVNLVTDGLPATALGFNPPDLD IMEKPPRKADEGLI SGWLFFRYMAI 840

.................................... DR---SPK-P .............

................................... N .... NPK-P .......... L--

...................................... L--NPR-A .......... L--

GFYVGAATVGAAAWWFVFS DEGPKLSYWQLTHHLSCLGGGDEFKGVDCKI FS DPHAMTMA 900

................ MYAED--GVT-H .... FMQ-TEDHPH- E -L- -E--EA-EP ....

-C .............. IAA-G--RV-FY--S-F-Q-KEDNPD-E .... A--ESPYP ....

-V---L---A--T---LYDA---QVTFH--RNF-K-SEDNPL-A-I--EV-ESRFPT---

L SVLVT I EI~N/~NSLS ENQsLI TMPPWCI~NLIGSN/~SF~HFVI LYVDVL STVFQVT 9611 ......... C--L ......... MR .... V-I--L--IC--MS- --L ..... P-FMI -KLK

......... C--L ......... LR .... E-I--V--IC--MS---L .... EP-PLI --I-

......... C--L--V ...... LR .... L-P---L-AVVM-MA- - -L- -L-PP-pL I ....

PLSAEEWITVMKFS I PVVLLDE TLKFVARK IADVP DVVVDRM 1002

A-DLTQ-LM-L- I-L-- IG--- I -- - I--NYLED-EDERRK

--NVTQ-LM-L-I -L--I-M ......... NYLEPAILE

---GRQ-GV-LQM-L--I .... A--YSL-HHV-EKKDLK

Figure 2. Comparison of the amino acid sequences of Drosophila and mammalian sar- 2+ co/endoplasmic reticulum type-Ca -ATPases. Residues in the mammalian enzymes

that are identical with the corresponding residue in the Drosophila Ca2+-ATPase are replaced by dashes. The mammalian sequences are taken from Refs. 1, 2 and 3.

The residues essential in forming a Ca2+-binding site in the transmembrane domain of Ca2+-ATPases, i.e. Glu 309, Glu 771, Asn 796, Thr 799, Asp 800 and Glu 908 (14) are all preserved in the Drosophila enzyme further underlining its identity with a Ca2+-pump. A very similar molecular architecture of the encoded protein and the other SR/ER-type Ca2+-ATPases could be concluded by comparing the hydropaty profile of DrCa-33 pro-

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tein and that of SERCAlb, SERCA2a and SERCA3 (not shown). On the basis of the high percent values of sequence similarity and the other features which are common in the SR/ER-type Ca2+-ATPases and in the DrCa-33 encoded ATPase we assume that DrCa-33 encodes a sarco/endoplasmatic reticulum-type Ca2+-ATPase of Drosophila melanogaster, which we designate DRSERCA. On the basis of amino acid sequence comparison DRSERCA is equally closely related to all three SR/ER-type enzymes. In mammals both "muscle-type" Ca2+-ATPase genes (SERCA1 and SERCA2) encode two alternatevely spliced proteins (4,5,6). In our previous work we did not detect mRNA species of different size by Northern analysis and concluded that alternative splicing may not play role in processing of Drosophila sarco/endoplasmic reticulum-type Ca2+-ATPases (8). On the other hand we have demonstrated that the abundancy of the DRSERCA mRNA is developmentally regulated. A putative internal signal sequence which may target into the sarco/endoplasmatic reticulum membrane :The SR/ER-type Ca2+-ATPases, unlike other cation ATPases which are inserted into the plasma membrane, are integral components of the sarco- plasmic reticulum or the endoplasmic retieulum membrane. This type of Ca2+-ATPases requires the signal recognition particle for integration into the mem- brane of the organelles and the existence of uneleaved signal sequence(s)in the pol- ypeptide chain was suggested (15). This (hypothesized) sequence must be present in each SR/ER-type Ca2+-ATPase but absent from the plasma membrane-type members of the family. Recently Bonifacino et al reported that a 23-amino acid sequence contains information that determines targeting for retention (and then degradation) of the a chain of T cell antigen receptor (TCR ~) within the endoplasmic reticulum (16). This peptide sequence comprises the transmembrane domain of TCR ~ and when it was fused to the ex- tracellular domain of an otherwise cell surface protein (Tac antigen), the chimeric polypeptide retained in the ER.

The first transmembrane helix of the SR/ER-type Ca 2+ -ATPases is similar to the trans- membrane domain of TCR c~ which conferes retention in the ER :

MGLR I L L L K V A G F N L L M T L

L L V R I L L L A A C I S F V L A W F

L L V R I L L L A A L V S F V L A W F

L L V K I L L L A A I I S F V L A L F

TCR ~ aa225-243

SERCAI and SERCA2 aa60-78

SERCA3 aa60-78

DRSERCA aa60-78

A pentapeptide motif (R/KILLL) is conserved within the transmembrane segments in both type of proteins. The sequence of this transmembrane region is well preserved in each known SR/ER- type Ca2+-ATPase but it is entirely different in the plasma membrane ATPases. The conservative replacment of a charged residue in a transmembrane petide segment (R/K) is noteworthy and comparable with a functional role of this motif. We suggest that the first transmembrane helix of the SR/ER-type Ca-ATPases may be responsible for the ER/SR targeting of this enzyme i.e. may serve as an internal signal sequence. Chromosomal location of DRSERCA qene : Previously we have shown by Southern analysis that there is one DRSERCA gene in the Drosophila genom (8). This finding is further supported by the chromosomal mapping of the gene. Using DrosSRCaPF as

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probe the DRSERCA gene has been detected as one locus, at the right arm of chromo-

some 2, i n section 60A.

ACKNOWLEDGMENTS

The help in chromosomal mapping of Tibor TGrSk (Institute of Genetics, Biological Re-

search Center) is gratefully acknowledged. This work was supported by an OMFB

grant (OMFB 13433/88) and by an AKA grant from the Hungarian Academy of Sciences.

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