Molecular characterization and expressionof the ... - Europe PMC

The EMBO Journal vol. 8 no. 1 pp. 1 93 - 202, 1989

Molecular characterization and expression of the (Na+ + K+)-ATPase alpha-subunit in Drosophila melanogaster Richard M.Lebovitz, Kunio Takeyasu1 and Douglas M.Fambrough Department of Biology, The Johns Hopkins University, 34th and Charles Street, Baltimore, MD 21218, USA 'Present address: Department of Physiology, University of Virginia School of Medicine, Charlottesville, VA 22908, USA Communicated by R.Miledi

The (Na+ + K+)-ATPase (sodium pump) is an ouabain-sensitive, electrogenic ion pump responsible for maintaining the balance of sodium and potassium ions in almost all animal cells. Robust, ouabain-sensitive rubidium uptake, indicative of the sodium pump, was found in tissue-cultured Drosophila cells, and both larvae and adults die when fed a diet containing ouabain. A monoclonal antibody to the avian sodium pump a-subunit was found to cross-react with the Drosophila sodium pump a-subunit. Immunofluorescence microscopy was used to obtain a semi-quantitative view of the expression of the sodium pump in Drosophila tissues: high levels of the sodium pump were detected in malpighian tubules, indirect flight muscles and tubular muscles, and throughout the nervous system. The cDNA encoding this sodium pump a-subunit in Drosophila melanogaster was cloned, sequenced and expressed in mouse L cells. At the amino acid level, its deduced sequence of 1038 residues (the first such sequence for an invertebrate) is -80% similar to c-subunit sequences reported for vertebrates. Only one gene was found in Drosophila, located on the third chromosome at position 93B. A restriction site polymorphism has been found, and several mutations exist that may involve the a-subunit gene. Key words: Drosophilalion transport/(Na+ + K+)-ATPase/ ouabain/sodium pump

Introduction The concept of the sodium pump was first proposed by Dean (1941) on the basis of the observation that cells accumulate potassium but pump sodium out. Levi and Ussing (1948) in muscle fibers and Hodgkin and Keynes (1955) in axons later demonstrated active transport of these ions, providing clear evidence for the sodium pump and emphasizing its role in the physiology of excitable cells. It is now recognized that virtually all animal cells have a sodium pump, or (Na+ + K+)-ATPase, which is pivotal to a variety of physiological processes, including osmoregulation, cell volume regulation, transport of certain amino acids and sugars, and maintenance of membrane excitability. The sodium pump is a membrane-bound ATPase, inhibited by ouabain and related cardiac glycosides, that couples the release of energy from ATP cleavage to the translocation of ions across the plasma membrane. It comprises an ©IRL Press

100-kd catalytic a-subunit and a smaller glycoprotein f-subunit. The sodium pump is a member of a class of ion transport ATPases that undergo transitions between two very different conformational states (El and E2) during ion transport, that are phosphorylated on an aspartyl carboxyl group during the transport cycle, and that are sensitive to vanadate. Other members of this class include some (Ca2+)-, (K+ + H+)- and (H+)-ATPases (Pedersen and Carafoli, 1987). The catalytic subunit of the sodium pump has a highly conserved amino acid sequence, sequence similarity near 90% in birds (Takeyasu et al., 1988b), fish (Kawakami et al., 1985) and mammals (Shull et al., 1985; Ovchinnikov et al., 1986). Here we report the nucleotide and deduced amino acid sequence of the a-subunit for Drosophila melanogaster, the first complete sequence of an invertebrate a-subunit. It has 80% amino acid sequence similarity to the corresponding a-subunits of (Na+ + K+)-ATPase molecules of vertebrates, suggesting that the (Na+ + K+)-ATPase arose very early in the evolution of the animal kingdom and that its structure and function are so central to survival that only a modest degree of sequence divergence has occurred. Although the (Na+ + K+)-ATPase is a well-studied enzyme, it has not been examined extensively in invertebrates. We have chosen Drosophila for such a study for a variety of the typical reasons, including amenability to genetic analysis and the opportunity to study the behavior of mutants. It has been suggested that a deficit in (Na+ + K+)-ATPase activity might underlie the Drosophila behavioral mutant, bang senseless (Jan and Jan, 1978; Ganetzky and Wu, 1982) or some other member(s) of the bang-sensitive group of behavioral phenotypes. In our study the chromosomal location of the a-subunit gene is identified. It does not correspond to any of the known bang-sensitive loci. An invertebrate may also provide some special insights into the evolution of the (Na+ + K+)-ATPase. The Drosophila genome appears to contain only one gene encoding the a-subunit of the (Na+ + K+)-ATPase, whereas those vertebrate genomes examined to date have at least three unlinked a-subunit genes (Kent et al., 1987; Shull and Lingrel, 1987; Sverdlov et al., 1987; Takeyasu et al., 1988a). What this difference means physiologically and evolutionarily is not known, but it appears that for Drosophila a single molecular form of the catalytic subunit can account for sodium/potassium ATPase activity in all tissues. -

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Results and discussion Ouabain-sensitive ion transport in Drosophila A unique feature of the sodium pump is its sensitivity to ouabain. In high concentrations ouabain is lethal to most animal species. We found this to be true for Drosophila as well. Adults fed a sucrose solution supplemented with 1 mM 193

R.M.Lebovitz, K.Takeyasu and D.M.Fambrough Table I. Survival of Drosophila hatched and reared on media containing the (Na+ + K+)-ATPase inhibitor, ouabain Ouabain concentration (mM)

No. of flies that eclosed

Per cent control (58)

0 0.017 0.17 1.7 3.4

58 43 53 23 5

100 74 91 40 9

j8o t 70-

8 50-

'E4030

Eggs collected from Canton-S females on molasses-agar Petri dishes were evenly suspended in a small volume of water. Equal aliquots were placed on Drosophila Instant Medium (Carolina Biological Supply Co., Burlington, NC) hydrated with water containing various concentrations of ouabain. After 2 weeks at 20°C, the number of adults that had emerged was counted.

ouabain became sluggish and died after -2 days. When eggs were hatched and larvae raised on medium containing various concentrations of ouabain, they were killed in a dose-dependent manner (Table I). Although adult flies tolerate rather high doses of ouabain, Drosophila tissue culture cells (line TS:5697-7, Simcox et al., 1985) are quite sensitive. Sodium pump activity was monitored in a cell line as 86Rb+ uptake from a lowpotassium Ringer's solution (see Materials and methods). Ouabain inhibited 86Rb+ uptake with an IC50 of 250 nM (Figure 1). 86Rb+ uptake in Drosophila cells is about eight times more sensitive to ouabain than uptake by chick fibroblasts (IC50 = 2 x 10-6 M) and several orders of magnitude more sensitive than uptake by mouse L cells (IC50 > 10-4 M) under these same conditions (Takeyasu et al., 1988b). By making a few approximations, one can estimate the number of sodium pump molecules per Drosophila cell from the uptake data, as follows. The ouabain-inhibitable 86Rb+ uptake was 425 d.p.m. per culture well with 106 cells. The medium contained 2.5 ACi/ml 86Rb+ and 1.6 mM potassium. Thus, this uptake calculates to 7.2 x 107 K+ ions taken up per cell per minute (assuming that rubidium and potassium are transported identically). If one assumes a turnover rate of 1000/min for the Drosophila sodium pump at 22°C (true turnover rate not known), with two K+ ions transported per cycle, then each cell should have -4 x 104 pump molecules and have a pump site density of 350 sodium pumps/jLm2 of surface area. The disparity between the sensitivity of cells to ouabain and the ability of whole organisms to tolerate relatively high doses might be explained by several factors. First, Drosophila may have an effective detoxification mechanism. Second, Drosophila tissues most accessible to orally administered ouabain may make little use of the sodium pump. In this regard, lepidopterans appear to lack a midgut sodium pump and possess, instead, a potassium pump that is not coupled to sodium exchange, is not a member of the same ion-transport enzymes to which the (Na+ + K+)ATPase belongs, and is not inhibited by ouabain (Harvey et al., 1983). It is possible that Drosophila has a midgut physiology similar to that of lepidopterans. Consistent with this possibility, binding to anti-a-subunit monoclonal antibody a5-IgG to the Drosophila midgut was not detected by immunofluorescence microscopy (see below). Third, insect hemolymph is reportedly quite high in potassium ion concentration (Dethier, 1977). High [K+]o antagonizes

o2010 0-

0 0

6

7

5

4

- log [ouabainJ

Fig. 1. Ouabain inhibition of ion transport by the (Na+ + K+)ATPase in tissue cultured Drosophila cells. The relative rate of 86Rb+ uptake by Drosophila TS:5697-7 cells was measured as a function of ouabain concentration (see Materials and methods). Ouabain-inhibitable 86Rb+ uptake was 80% of total uptake (540 c.p.m. per well total uptake) under the conditions of the measurements. The IC50 for ouabain inhibition was determined to be 250 nM, indicated by the slash on the curve.

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Fig. 2. Monoclonal antibody a5-IgG detects a 100-kd ca-subunit polypeptide in Drosophila. Detergent extracts of whole heads (two for each sample) or thoraces (10 per sample) of adult Canton-S flies were subjected to SDS-PAGE followed by electrophoretic transfer to nitrocellulose paper. The paper was incubated with a5-IgG for 4 h at room temperature, rinsed, and incubated with 125I-labeled goat anti-mouse IgG for 1 h. After removal of unbound antibody, the blot was exposed to X-ray film overnight. The mol. wt markers are indicated to the left.

ouabain binding to the (Na+ + K+)-ATPase. Fourth, the peripheral and central nervous systems of insects are ensheathed by a layer of cells and connective tissue that acts as a diffusion barrier between the neurons and the circulating hemolymph (Treherne and Pichon, 1972). This active isolation of the nerves could impede the diffusion of ouabain into the tissues, delaying the time it takes to accumulate toxic concentrations.

Distribution of the (Na+ + K +)-ATPase a-subunit in Drosophila tissues Our laboratory has generated a number of monoclonal antibodies to the avian sodium pump oa-subunit (Takeyasu

194 -.1

Drosophila (Na+

+

K+)-ATPase a-subunit gene

Fig. 3. Indirect immunofluorescent localization of the (Na+ + K+)-ATPase a-subunit in the whole adult fly. Cryosections (8 of whole adult male flies were double labeled with monoclonal antibody a5-IgG and TRITC-conjugated second antibody. The top of the montage is dorsal, left is anterior. Dim, dorsal lateral flight muscle; b, brain; e, eye; p, proboscis and mouthparts; le, leg muscle; a, abdomen; m, malpighian tubules.

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et al., 1988a). These were tested for binding to Drosophila tissues and for binding to blots of Drosophila proteins separated by SDS-PAGE. One of these monoclonal antibodies, ct5-IgG, was positive in both of these tests. Figure 2 illustrates the binding of ct5-IgG specifically to an -100-kd polypeptide after separation of Drosophila proteins by SDS -PAGE. Sephadex beads which covalently bound ca5-IgG also retain specifically the 100-kd polypeptide (data not shown). The apparent mol. wt of this polypeptide is correct for a sodium pump ct-subunit. As shown below, the monoclonal antibody binds to the 100-kd polypeptide encoded by the cloned Drosophila cDNA, and this cDNA encodes a polypeptide with long stretches of amino acid sequence identity with mammalian ca-subunits, confirming the identification of this polypeptide as the Drosophila sodium pump ct-subunit. Monoclonal antibody ct5 was used in immunofluorescence microscopy to obtain a semi-quantitative view of the tissue distribution of the sodium pump oa-subunit in Drosophila. As shown in Figure 3, which is an immunofluorescence micrograph of a section through a whole adult fly labeled with ca5-IgG and TRITC-second antibody, the sodium pump is expressed very highly in three tissue types in D. melanogaster, muscle, the nervous system and malpighian tubules. This distribution is consistent with that previously proposed on the basis of physiological criteria (Jan and Jan, 1978; Wessing and Eichelberg, 1978). The nervous system contains copious amounts of the (Na+ + K+)-ATPase a-subunit; strong immunofluorescent labeling with a5-IgG is observed in the brain, optic lobes, retina (photoreceptor cells) and ventral thoracic neuromere. Indirect flight muscle and tubular leg muscle show a pattern of (Na+ + K+)ATPase distribution that resembles the plasma membrane labeling seen in chicken skeletal muscle fibers (Fambrough and Bayne, 1983). The labeling pattern in malpighian

tubules, the dipteran excretory organ, appears to be basolateral, as is typical for vertebrate polarized epithelia. Although fluorescent antibody labeling was not detected in the midgut and most of the internal abdominal tissues, a small amount of antigen was detected at the posterior end of the fly where the reproductive organs and rectum are located.

With respect to this observation, it is worth noting that ouabain-inhibitable ATPase activity has been reported in the rectum for another arthropod, the locust (Peacock, 1981). cDNA cloning

The a-subunit of the (Na+ + K+)-ATPase is highly conserved at both nucleotide and amino acid sequence levels across a variety of vertebrate classes (Kawakami et al., 1985; Shull et al., 1985; Ovchinnikov et al., 1986; Takeyasu et al., 1988b). This high degree of homology suggested that it would be very straightforward to isolate its cDNA from Drosophila using heterologous probes from the chicken (Takeyasu et al., 1988b) and sheep (Shull et al., 1985). To screen a Drosophila cDNA library, we chose three different probes, spanning different portions of the coding sequence for the ct-subunit. A 1 100-bp carboxy and a 600-bp amino terminus encoding sequence were selected from chicken cDNAs. The third probe was a 400-bp sheep cDNA, encoding the middle of the a-subunit sequence, including the FITC-binding sequence and the phosphorylation site. Initially, two different clones were selected from a late pupal cDNA library. Da-14 is 3 kb and hybridizes to all three heterologous sequences. Da-16 contains sequence homologous only to the sheep probe, encoding the middle portion of the ct-subunit. The two clones cross-hybridize. Using the longer Da-14 as a probe, we subsequently recovered three different full-length clones from a 6-h Drosophila embryo library. All three cDNAs are polyadenylated and share identical restriction sites (Figure 4). 195

R.M.Lebovitz, K.Takeyasu and D.M.Fambrough GENE 3' 13.4 kb R

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R K I

K

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6kb R

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I

I

I

1.0

kb

cDNA 47.1 K Xb

ATG

S

I

I

S

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A A A A A A A

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Fig. 4. Diagrammatic structure of Drosophila (Na+ + K+)-ATPase ai-subunit gene and cDNA. The DNA encoding the a-subunit of the Drosophila (Na+ + K+)-ATPase is contained in three contiguous genomic EcoRI restriction fragments of 6, 4 and 3.4 kb. The adjacent 5' SacIIEcoRI fragment that may contain 5' regulatory sequences is also diagrammed. A diagram of the a-subunit cDNA clone Da-47, drawn to twice the scale used for its gene, is shown underneath the schematic drawing of the gene. The arrows relate positions of the same restriction sites in the genomic DNA and cDNA. Restriction sites are: S, Sacl; R, Eco RI; K, KpnI; Xb, XbaI; Sp, SphI; Xm, XmaI. Positions of codons for start (ATG) and stop (TAA) translation are shown. The positions at which the phosphorylation site and FITC-binding site of the a-subunit are encoded in the cDNA are indicated.

From restriction mapping and sequencing (see below), we determined that the three cDNAs contained different lengths of 3' untranslated sequence. Da-75 has 385 untranslated base pairs; Da-47 contains an additional 17 bp preceding its poly(A) tail. Another clone, Da-72, has 700 bases of 3' untranslated sequence.

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200-

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Expression of Drosophila a-subunit in mouse L cells To show that the protein encoded by Da-47 corresponds to the same protein detected by ci5-IgG in adult flies, we expressed the cDNA in a mouse cell line and looked for the appearance of a5-IgG immunoreactivity in transfected cells. Mouse Ltk- cells, a cell line deficient in the gene for thymidine kinase (tk), were chosen as the recipients for the Drosophila cDNA because they do not react with a5-IgG, and the affinity of their endogenous sodium pump for ouabain is several orders of magnitude less than it is in Drosophila, providing the possibility of a ouabain-binding assay for expression of the Drosophila a-subunit. In addition, the absence of the tk gene (Kit et al., 1963) makes possible selection for transfectants by culture in HAT medium after the Ltk- cells have been cotransfected with the foreign DNA of interest and a functional tk gene. Drosophila c-subunit cDNA Da-47 was cloned into mammalian expression vector pSVDF (Takeyasu et al., 1987) between an SV40 promoter and polyadenylation cassette. Mouse L cells were cotransfected by calcium phosphate precipitation with the herpes tk gene and the expression vector containing the Drosophila cDNA, and transfected clones were selected in HAT medium. Forty colonies selected in this way were examined for expression of the Drosophila cDNA by assaying for high-affinity [3H]ouabain-binding sites. Assays typically were performed in 24-well culture dishes (Costar) containing confluent layers of cells (-- 2 x 105 cells/well). The binding solution contained 200 nM [3H]ouabain 4 10 mM unlabeled ouabain (to determine nonspecific binding, see Materials and methods). We selected lines expressing .200 c.p.m./ well bound ouabain. Specific binding to control L cells, attributable to low-affinity binding to the endogenous mouse pump, was half this amount. By this criterion, six Ltk+ cell lines were chosen for further characterization. 196

1169568-

43-

Fig. 5. Immunoprecipitation of Drosophila a-subunit encoded by

Da-47 from transfected mouse L cell lines. [35S]Methionine-labeled were extracted from mouse cell lines with buffer containing non-ionic detergent Triton X-100 and incubated overnight with ca5-IgG immunobeads. Protein affinity purified in this manner was eluted by high pH from the beads, subjected to SDS-PAGE in 10% acrylamide gel, and the gel subjected to fluorography after impregnation with PPO. Two cell lines, Ltk-Dal and Ltk-Da2, transfected with pSVDa-47, expressed a 100-kd protein. This protein was absent from the parental line, L, and from another HAT-resistant transfectant (Ltk) that evidenced no high-affinity ouabain binding. A band at the 43-kd position, common to all four cell lines, probably represents actin, a frequent contaminant of immuno-purifications. Mol. wt marker positions are indicated in kilodaltons at left.

proteins

Two stably transfected lines, Ltk-Dal and Ltk-Da2, selected by their growth in HAT medium and an increased number of ouabain-binding sites compared to the parental L cell line, were analyzed for reactivity with a5-IgG by immunoprecipitation and by immunofluorescent labeling. In one experiment cells were treated for 30 h with 10 mM butyrate, a compound which increases the expression of transfected genes, then grown in media in which the only source of methionine was [35S]methionine. Detergent extracts of the 35S-labeled cells were incubated with a5-IgG covalently coupled to Sepharose beads. High pH eluates from the immunobeads were subjected to SDS -PAGE and

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Fig. 6. Immunofluorescence localization of Drosophila a-subunit expressed in mouse L cells. Ltk-Da2 cells, expressing the a-subunit of the Drosophila sodium pump, were plated on glass coverslips and grown in medium containing 10 mM butyrate to enhance expression of the SV40 early promoter/enhancer (Gorman and Howard, 1983). Cells were fixed lightly with formaldehyde, made permeable with saponin, and labeled with a5-IgG followed by TRITC-conjugated second antibody. The field illustrated by this photomicrograph was typical. Some cells fluoresced brightly after immuno-labeling while others were more lightly immuno-labeled. The distribution of fluorescence is quite similar to that reported previously for mouse L cells expressing the avian a-subunit (Takeyasu et al., 1988b). Panel A, phase contrast; panel B, rhodamine immunofluorescence. The bar represents 25 zm.

fluorography. The results (Figure 5) demonstrated that both Ltk-Dal and Ltk-Da2 but not parental L cells expressed a 100-kd polypeptide immunopurified with the a-subunitspecific immunobeads. This mol. wt is identical to that determined in our protein immunoblot analysis of Drosophila proteins (see Figure 2), indicating that the cDNA indeed encodes the protein recognized by a5-IgG. Analysis of Ltk-Dal and Da2 cell lines with [3H]ouabain did not yield a simple binding isotherm characteristic of high-affinity ouabain sites (data not shown), indicating that normal expression of the Drosophila a-subunit in mouse cells may require additional factors, such as the Drosophila 3-subunit. It has been suggested that the f-subunit plays an essential role in the assembly and transport of the a-subunit to the cell surface (Noguchi et al., 1987; Fambrough, 1988). In this system, the endogenous mouse (3-subunit may interact only weakly with the Drosophila a-subunit, accounting for the low level of [3H]ouabain-binding sites on the surface of transfected cells ( -2000 sites/cell). In fact, immunofluorescent labeling of the cells with a5-IgG followed by TRITC-conjugated second antibody (Figure 6) revealed that most of the Drosophila a-subunit was internal. Messenger RNA We determined the size of the a-subunit message in different tissues and developmental stages of Oregon-R flies. In all

Fig. 7. Northern blot analysis of a-subunit transcripts. Total RNA isolated from various Drosophila tissues and stages was separated by agarose gel electrophoresis and blotted onto nitrocellulose paper. The blot was hybridized with 32P-labeled Da-14 [containing most of the coding sequence for the Drosophila (Na+ + K+)-ATPase a-subunit]. Arrowheads at right indicate the position of chicken ribosomal RNA markers: top arrow at 4.0 kb; bottom arrow at 1.8 kb. For this analysis, organs were dissected from late-third-instar larvae and homogenized directly in RNA extraction buffer. For the individual RNA samples, the following number of organs and body parts were used: larval salivary glands, 15; larval malpighian tubules, 21; larval brains, 20; embryos, 40 eggs collected over 3 h; whole pupae, 10; adult abdomens and thoraces, 10; adult heads, 10. Judging from the ribosomal bands, the lanes with RNA from pupae and adult bodies contain the most RNA; the lane with RNA from malpighian tubules contained the least RNA. RNA loading in the other lanes was about equal. Longer autoradiographic exposures of this same blot revealed labeled bands in the lanes of RNA from salivary glands and malpighian tubules.

the preparations surveyed, Da-14 hybridized to RNAs of three different lengths: 4.8, 3.8 and 3.6 kb (Figure 7). The two smaller messages may correspond to our cloned cDNAs Da-47 and Da-75 (3.6 kb) and Da-72 (3.8 kb). Each of the three transcripts is present in all tissues, at all developmental stages examined, but in varying amounts and proportions. The adult head and larval brain express more of the 4.8-kb transcript than the smaller two. In contrast, the adult abdomen contains more of the smaller transcripts. Generally, this latter pattern of mRNA expression was observed; embryo, larval salivary glands and malpighian tubules all exhibit this pattern. The thorax of the adult fly, however, expresses all three transcripts in about equal abundance (data not shown). Nucleotide and deduced amino acid sequence Da47 contains an open reading frame encoding 1038 amino acids (Figure 8), slightly longer than reported for the vertebrate sodium pump a-subunit isoforms (see below). Twelve untranslated nucleotides precede the first potential start codon, which is in a sequence ATAACATGG, similar to the consensus sequence C/AAAA/CATG for initiation of translation in Drosophila (Cavener, 1987). There is a second possible start codon in the sequence GCAAGATGC in frame with the first (which would yield an a-subunit 40 amino acids 197

R.M.Lebovitz, K.Takeyasu and D.M.Fambrough GG CMG T CGC ACA ATCC MT TAA MT MCAIG CCC [TA C TCC CAT T MA T CCC AM CC UC TCr MAT (M G= GCC ACG CGTr ATA CCC ACC CAT GAC GAC MAT ARG EET AIA LEU AMG SER ASP [YR GLU HIS CLY AMC AIA ASP SIR TYR ARG VAL ALA UHR VAL ItS ALA IHR ASP ASP ASP ASN THR ALA A.SH GLY GIN [YR 114 220 MC MA MA CCC AM MG CC CCC MA CIT MT MA M C AAT ITM GAT GAT CrC MAA CG GAG TIC CAT ATC CAT TIT CAT MAA MCTCT CCr CGCAA A¶EM TAT CACCC YR CIN ARG LYS SER AEC AG LYS MET PR) ALA LYS 1/A ASN LYS LYS GIl ASN LEU ASP ASP LEU LYS CIN CU) IEU ASP ILE ASP PHE HIS LYS ILE CAR PR GLU Gl) ET CMA [TCGGGMAM TTC TCT TIT CMG ACA CAT CCC CAA MAT CGT CTA ACT CAC GCC AC CCC AAG CAA AMC TIC GAG CGC GAT (MT CCC AAT CTC ACC CCA CCC MCACGMGCG CCC PHE CEN [HR HIS PM) CLU ASN CLY LEU CAR HIS ALA ARC ALA LYS Gil) ASN LEU GiL) ARG ASP CLY PR) ASN LSU THR PR) PRO LYS GIN [HR PR CU)l TIe V/AL LYS PEE CYS 342 GAA GAC CTC TTC GGr~ GTG CCC ATG TIG CTC TMC ATC Ca[ GCr ATT CTC ITC TIT GTC GCC kTAT1CT ATC 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THR LYS ASH LEU ALA PEE PHESECR AMA [G[ CCC GAT CMC MC GIC MTG (MC CGG ATr GCC CCC TIC CCC ICC GGT CIACAMCACCAGG AIG CCC MT GCCC MCC MAC CCC GTC CMG CCG CTG CCC AMG GGT GIG GCMC ThR ASN AIA V/AL CU) ALA LEU PR) LYS CLY VrAL 1/AL ILE SIR CYS CLY ASP HIS ThR VAL MET CLY ARC ILE ALA ALA IEU ALA CAR GLY LEU ASP TiR GLy THR pR) I1.5 ALA 912 AMG GAG MTC CMC CAT TIC MTC CMC CIT MTT MC GGC GIG CCC GIG TIC CMGIGCC GMC MCTC=TIC GIG AT[ GCCC Tic MTC GTGGGC TARC CACTGIcG GCM GCCI GTC MTC TITT 1026 TMGLY 1/AL [HR PEE pEE 1/AL IIE AILA pEE IIS LEU CLy [YR HIS flI IMU ASH ALA 1/AL IIE PHEE LYS CU) IIE HIS HIS PEE ITSE HIS LEU IIE TIR GLY V/AL ALA 1/AL PELEU AM AIGWrAG ACC CCC CCC TCA AMG MAC G GIG GIG AMG MAT CGI 1140 CIT CGr GITCGCTGCC ACC GIR ACr GIGM TGIMC TIC MIC GGr MTC MTC GTC CCC MAC GTG CCC GA MTGG IEU) ILE CLY HIE IIE 1/AL ALA ASN VAL PR) CLU GLY IEU IM) ALA THR 1/AL THR V/LC M H M E L Y CPE L A Y S fST)1A Y GAG GCCC GIGC ACC CIT CCC TCC ACA ICC MCC MTC ICC TCC CAT AMG MC CCC MCC CIC MCC CMG MAC CCA MGC ACC GTC CCC CRC MTG TGG TIC CAT MAT CM MTC MTC GAG U) HR M) LY IR HR CR lE IS CS SE AH LS lE CY THR IEU ThR GIN ASH MIC Mr [HR 1/AL MLA HIS PET flPP PEE ASH ASH CIN IIS ITS CLU)15 CU) A 1AL CCC GCM ACA Mr GAG GAT CMG TCC T CGTr CAA TMC CAT MAG MC MGC CCT GO TIC AMACCGOGIC TCr CCC MTT CCC MCT CIC MCT MC CGT CCGM TIC AMAA GMGC CAA 1368 lEE GLCU) ASH GIN CAR GLY 1/AL GIN [YR ASH MCGlUR CAR PRO CLY PEE LYS MLA IM) SER MRC ITS MIA ThR IEU CYS ASH MC MAC)PELSCYCYGN MIA ASPHR CCIGI CTC AMG IGC MTG G CMC GIG CTG GIGCC CAT GIG MG MAC MTT CCC AMG CG~ T AMSAA CAT (MC GIC CC ATMC CTC AMG MAA CM GIC ACMr(MACT CCC TCC GAG GCCT U M ATl LYAH1A E S t C LYS C AHLYS 1482 ASH CLY 1/AL PR) ILS LEU LYS LYS CU) 1/AL CAR CLY ASH MIA SER CU) MLA MLA ISU [EU LY CfAE AMG MTT CCC CMG GIG CCC TI1C MAC TCC MCC MAC MAA TMC CM GIG TCC MTC CMC GRA MCc GAGCAT MC MPC CAT CCC COC TMc TCG CI GnkAMG AMCAG CCC CCCCMCGmG 1596 IYS HE MLA CU) 1/A PR) PEE AMSH CR TIR ASH LYS TYR GIN 1/AL SE IIS HIS CU) THR CU) ASH 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shorter). Following the termination codon, TAA, there are 402 bases of 3' untranslated sequence before the poly(A) tail. The deduced amino acid sequence of the Drosophila a-subunit is 80% homologous to the rat oal a-subunit isoform (Figure 9) when compared from amino acid 53 in the rat cIll sequence (a + of Shull et al., 1986) and 71 in Drosophila. The Drosophila at-subunit sequence shows about 198

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