Proc. Nati. Acad. Sci. USA Vol. 91, pp. 10285-10289, October 1994 Cell Biology Ankyrin and ,- accumulate independently of a-spectrin in Dro8ophila RONALD R. DUBREUIL* AND JIANQING YU Department of Pharmacological and Physiological Sciences and The Committee on Cell Physiology, University of Chicago, 947 East 58th Street, Mail Code 0926, Chicago, IL 60637 Communicated by Hewson Swit, June 30, 1994

ABSTRACT We report the identification and Initial char- skeleton in nucleated cells. Studies in Drosophila have so far acterization ofDrosophila nelanogaster ankyrin. Oligonucleo- shown that three membrane skeleton originally tide primers based on the spctrin-binding domain of human identified in the mammalian erythrocyte are conserved in brain ankyrin were used to amplify Drosophila genomic DNA. evolution and have essential roles in development. The hts A cloned 184-bp PCR product was used to Isolate Drosophila is a homolog of adducin that is likely to be associated ankyrin cDNAs. Ankyrin cDNA probes detected a 5.5-kb with in ring canals of the developing oocyte (14). transcrptfrom embryonic poly(A)+ RNA and a single polytene Defects in hts block egg development. D4.1 is a homolog of chromosome locus (1O1F-102A). The cDNA sequence encodes protein 4.1 (15) that is encoded by the essential coracle gene. a 170-kDa protein that is 53% identical to human brain Defects in D4.1 block dorsal closure during embryonic de- ankyrin (Ank2). Antibodies directed against a recombinant velopment and suppress the dominant eye defect in epidermal fragment of Drosophila ankyrin reacted with a 170-kDa poly- growth factor receptor mutants. peptide from Drosophila embryos, larvae, S2 cells, and adult Drosophila spectrin is also conserved in its structure, flies. The ankyrin antibody immunopripitated a- and composition, and actin-binding activity (16). Mutations ofthe ,-spectrin with ankyrin in detergent extracts of Drosophila a subunit are lethal during larval development (17). Surpris- embryo membranes. Antibodies against Drosophila ankyrin, ingly, some aspects of development do not appear to be a-spectrin and «spectri were used to detect these proteins in grossly affected by diminished levels of a-spectrin. Mutant wild-type and a-sin mutant larvae. a-Spectrin levels larvae, which retain only a small quantity of maternally were greatly diminished in mutant larvae, but levels ofankyrin derived a-spectrin, are initially able to crawl, feed, and and -spectrin were indis hable from wild type. The respond to tactile stimuli. They appear normal in overall form persistence of ankyrin and P-spectrin may explain the rela- and development ofmajor larval structures, but they become tively mild phenotype of a-spectrin mutants during early lethargic and die before the first larval molt. Close inspection Drosophila development. of the larval gut reveals a significant effect of a-spectrin mutations on the shape and adhesion ofone class ofepithelial The protein ankyrin provides a link between integral mem- cells (17). brane proteins and the spectrin-based membrane skeleton We have initiated studies of ankyrin in Drosophila to (1). That link appears to be important to the mechanical further dissect the cellular roles of the membrane skeleton. properties of the plasma membrane in mammalian erythro- An ankyrin gene was identified by amplification of genomic cytes. Defects in ankyrin result in membrane fragility, a DNA with oligonucleotide primers based on a conserved decrease in the number of circulating mature erythrocytes, region ofmammalian brain ankyrin.t We used a collection of and consequent anemia (2, 3). The membrane defect results antibodies against this ankyrin homolog and the spectrin from the failure of spectrin and other proteins to assemble subunits of Drosophila to examine the fate of the membrane properly without ankyrin as an attachment site. Defects in skeleton in the absence of a-spectrin synthesis. spectrin, which produce a similar phenotype, do not affect the accumulation of membrane-bound ankyrin (4). In contrast to its interaction with a single major transmem- MATERIALS AND METHODS brane protein in erythrocytes (5), ankyrin interacts with Amplification of Ankyrin DNA. Genomic DNA from adult several different proteins in nucleated cells. Coimmunopre- Drosophila melanogaster was used in PCRs with synthetic cipitations, cosedimentation, and in vitro binding assays have primers based on the human brain ankyrin 2 sequence (18): been used to demonstrate interactions between ankyrin and AnkAl, CCA CTT TAT CAT CAG TCA TGC; and AnkS1, the sodium pump (6), sodium channels (7, 8), a nonerythroid AAC GTG TCT GCC AGG TTC TGG. Reactions were anion-exchange protein (AE3; ref. 9), a cell adhesion mole- carried out in a MiniCycler (MJ Research, Cambridge, MA) cule from brain (ABGP1; ref. 10), and others (reviewed in ref. for 40 cycles (940C for 1 min, 450C for 2 min, 720C for 1 min) 11). Each of these interactions may simply attach the spec- with Taq DNA polymerase (Perkin-Elmer/Cetus). trin-based membrane skeleton to the plasma membrane. Isolation of Ankyrin cDNA. A 0- to 4-hr Drosophila mela- Alternatively, it has been proposed that ankyrin can act in the nogaster embryo cDNA library (19) was screened with the fly opposite direction: to capture and stabilize membrane pro- ankyrin PCR product by standard methods (20). The probe teins in specific membrane domains defined by the membrane was amplified from cloned plasmid DNA and hybridized at skeleton (12, 13). But it has not been possible to directly test high stringency under conditions described by O'Neill and the role of ankyrin in membrane domain segregation. Belote (21). Additional cDNAs were recovered from a size- Genetic studies have been important in unraveling the roles fractionatedlarvallibrary(generouslyprovidedbyCarlThum- of membrane skeleton proteins in the erythrocyte, and they mel, University ofUtah), using the A fragment ofclone A411 are now contributing to our understanding of the membrane as probe (Fig. 1).

The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. L35601). 10285 Downloaded by guest on September 24, 2021 10286 Cell Biology: Dubreuil and Yu Proc. Natl. Acad. Sci. USA 91 (1994) All RESULTS A411 A631 Synthetic oligonucleotide primers based on the conserved Flank spectrin-binding domain ofthe human ANK2 gene were used A631 B in the PCR with Drosophila genomic DNA. A 184-bp product E X SB E E pNB-40 [ 1I : II (Flank, Fig. 1) was amplified, cloned in pBluescript, and vectorr I I sequenced. The predicted sequence of the fly 1 kL ankyrin fragment was -40%o identical to the sequences of mammalian ankyrins (18, 30). FIG. 1. Drosophila ankyrin cDNA clones. Primers based on the Isolation and Characterization of Drosophila Ankyrin sequence ofhuman brain ankyrin 2 were incubated with fly genomic cDNA. The cloned fly ankyrin sequence was used as a probe DNA to produce the fly ankyrin (Flank) PCR product. Ankyrin cDNAs were isolated from embryonic (A411 and A631) and imaginal to screen libraries of Drosophila embryonic cDNA. The disc libraries (All). A composite restriction map of the cDNAs is probe failed to hybridize with fly DNA under standard shown at the bottom (E, EcoRl; S, Stu I; B, BamHI; X, Xba I). Dots high-temperature conditions, but several cDNA clones were mark the boundaries of clone A411 probes. isolated from a 0- to 4-hr embryonic cDNA library after hybridization in 50%o formamide at 42TC. Additional clones Southern and Northern Blots and Polytene Hybridizations. from the 5' region of the coding sequence were recovered Blots of fly genomic DNA and RNA were prepared by from a size-selected larval cDNA library by using the A standard methods (20). Biotin-labeled probes (Polar-Plex fragment of clone A411 as probe. Overlapping regions of labeling kit, New England Biolabs) were used in Southern these cDNAs produced identical digestion patterns with blots (1 fly per lane). A 32P-labeled C fragment of cDNA 411 several restriction enzymes, suggesting that they are all (Fig. 1) was used in Northern blots [8 pg of poly(A)+ RNA partial cDNA products derived from a single class ofankyrin per lane]. Polytene chromosomes from Oregon-R Drosophila transcript (Fig. 1). larvae were prepared by standard methods (22). Southern blots of Drosophila genomic DNA were probed DNA Sequencing. The cDNA sequence was determined on with the EcoRI A fragment of cDNA A411 (Fig. 2A). The both strands by using a Sequenase kit (United States Bio- probe spans a BamHI restriction site in the cDNA sequence chemical) and synthetic oligonucleotide primers. A compos- and therefore detects two bands in BamHI-digested genomic ite sequence was assembled by using cDNA clones All and DNA (lane B). An Xba I-Stu I fragment (A411 C fragment in A631 (Fig. 1). Sequence assembly and dot-plot comparisons Fig. 1) from upstream of the BamHI site detects only the were generated by using the Genetics Computer Group lower 5.8-kb band, and an EcoRV fragment probe from (Madison, WI) programs (23), as previously described (24). downstream ofthe BamHI site detects only the upper >10 kb Production of Antibodies. Recombinant fragments of fly band (not shown). The A411 A probe does not span an EcoRP ankyrin and 3-spectrin were produced as fusions with glu- site in the cDNA sequence, but it still detects two bands (2.8 tathione S-transferase and used to generate rabbit antibodies. kb and >10 kb) in EcoRI-digested genomic DNA (lane E). The B fiagment of ankyrin cDNA clone A631 (Fig. 1) was There may be an intron in the ankyrin gene that contains an expressed in the pGSTag vector (25). A 1.1-kb EcoRV EcoRI site, but the possibility of a related cross-hybridizing fragment from the C-terminal coding region of 3-spectrin gene cannot be ruled out. cDNA (26) was expressed in pGEX3X. Proteins were puri- fied from bacteria by glutathione affinity (27) and preparative A B electrophoresis (28). Both antibodies were affinity purified before use. Immunoprecipitation and Antibody Reactions with Larval 4-. 5.5 kb Proteins. Dechorionated embryos [500 pl + 1 ml of Tris- 0 12 buffered saline (TBS) with protease inhibitors (19)] were W. disrupted by several strokes in a Dounce homogenizer fol- lowed by brief sonication (three 5-sec sonications with a Branson sonifier and microtip at 20 W). Membranes were collected by centrifugation at 20,000 rpm in a Beckman JA21 0 I'. rotor for 30 min and resuspended in buffer plus 1% Triton X-100. The suspension was again sonicated and after 20 min on ice, a clarified supernatant was obtained by centrifugation at 70,000 rpm for 30 min in a Beckman TLA 100.1 rotor. B E Control experiments showed that the plasma neuroglian (29) was efficiently recovered in the mem- C brane pellet and in the detergent-soluble extract. The embryo extract was mixed with 60 pg of affinity-purified ankyrin -s.¢K#AA2 antibody and excess Pansorbin (Calbiochem) immunoadsor- bent for 4 hr at 40C with end-over-end rotation. Pansorbin cells were rinsed three times in buffer, then dissolved in SDS sample buffer for gel electrophoresis. Proteins were trans- ferred to nitrocellulose and allowed to react with antibody as previously described (16). FIG. 2. Southern, Northern, and polytene hybridizations with First-instar larvae were collected from a y/y; ankyrin cDNA probes. (A) BamHI (lane B) and EcoRI (lane E) Drosophila digests ofDrosophila genomic DNA probed with the A411 cDNA A RG41/TM3[y+] stock. Wild-type and mutant larvae were fragment. (B) Northern blot of poly(A)+ RNA from ovaries (lane 0), distinguished by the y+ marker on the balancer chromosome 0-to 4-hr embryos (lane 1), 4- to 8-hr embryos (lane 2), and 8- to 12-hr 3 (17). Thirty to 40 larvae were triturated in SDS sample embryos (lane 3) probed with the A411 C fragment. (C) Probe C was buffer by several strokes with a microhomogenizer and also used to probe Drosophila polytene chromosomes. A single loaded onto polyacrylamide gels with the equivalent of 4 labeled band was detected, at position 1O1F-102A on mutant or wild-type larvae per lane. (arrows). Downloaded by guest on September 24, 2021 Cell Biology: Dubreuil and Yu Proc. Natl. Acad. Sci. USA 91 (1994) 10287 A 5.5-kb transcript ofDrosophila ankyrin was detected in but the repetitive signal is lost (Fig. 4B). The diagonal Northern blots of poly(A)+ RNA from Drosophila ovaries continues through the spectrin-binding domain of ankyrin, and embryos probed with the A411 C fragment (Fig. 2B) or although it is interrupted at the junction between domains the Flank probe (not shown). The A411 A and C fragments (arrows). The C-terminal domain of fly ankyrin is shorter were also used to probe Drosophila polytene chromosomes. than the corresponding region of human ankyrin and shows These probes both specifically labeled the right arm of only limited sequence similarity. Comparisons of the human chromosome 4 at position lOlF-102A (Fig. 2C). ankyrin isoforms (Ankl, ref. 30, and Ank2, ref. 18) at high Sequendng of Drosophila Ankyrin. A composite cDNA stringency (Fig. 4C) reveal that sites of divergence between sequence from clones All and A631 included a single long fly and human ankyrin are also sites of divergence between open reading frame encoding 1549 amino acids (Fig. 3) with human isoforms. The diagonal of identity through the repet- a predicted molecular weight of 170,096. A putative start itive domain in high-stringency plots (Fig. 4 B and C) indi- codon was identified by the presence ofan upstream in-frame cates that degenerate positions in the ankyrin repeats, as well stop codon and downstream sequence similarity to human as consensus positions, are conserved between fly and hu- ankyrins. However, the 4 residues immediately upstream of man ankyrins. the ATG codon (AATT) are unlike the consensus start sites Antibody Reactions with Drosophila Ankyrin and Spectrin. commonly used in Drosophila (31). A recombinant fragment ofDrosophila ankyrin (GST-A631B) Dot-plot comparisons ofDrosophila and human sequences was used to produce an ankyrin antibody in rabbits. Affinity- indicate that the domain structure of ankyrin is conserved purified antibody to fusion protein reacted with a 170-kDa (Fig. 4). Most of the 23 fly ankyrin repeats are at least 35% polypeptide in S2 cells (Fig. SA, lane 1), fly embryos (Fig. identical to the repeats ofhuman ankyrin (Fig. 4A, lower left). SB), larvae (Fig. 6), and adults (not shown). Staining oflower When the stringency of comparison is raised to 50%, the molecular weight polypeptides was also detected in some diagonal ofidentity between fly and human ankyrins remains, preparations, but it is not known if these are proteolytic OCAR7TCTTAOLTAA (;AGTTC-WRACAAMTACAPLACAAGRAG7GAAACCTGVOCTA7CAATCGAATGGCTrrAr-ACAACAAaAAqyw.Akq%-Kmjkhkrjkr-hhqv--&O--..- .. I - - - - 150 T L GD V N K I Q T R S E S C A I N C X A L D N K N GI N DA T 151 AT11CGTYCYACGTGCTGCTCGCAGCGGAGACATM ki TAAAAAGGTGATGGA kcCTGIQGAGAAATATCAGACATAAAlTAGCTOCAATGCAcG4TCTTA) hi kc k7 c 300 I S F L R A A R S C D I X XV tD D F L D C C E I S D I N S C N A N L kTGCACTTrCATrTAGCTGCAAA 6GGACGGATAI,TGTGGACATATGC G A L H L A A K D G Y V D I C 301 zrCOCATAAAGTCVGATAAki ri LTOCCACAAAAAAGGX;AAACACAGCxCrTrCACTTGCGTCTTTGCG GtrGCAGCATGATGTCATCAATCAGC¶ VrAATTTTATATAATGCTAATGTm W T 450 C 3 L L R R C I K I D N A T X X G GAACGTGCA( N T A L H I A S L A G Q H D V I N Q L I L Y N A N V N V Q IICVTCITrATGGJS L N G 451 NA TAS TCACCCCGCTTThTATrcrGGAGACAAGAAAACCAkcCGACAATTGTIGTCCwAACCCTATT(riaGXCAATGGGGCAAAGTCCG7CATT C) F T P L Y Nt A A Q E N H D N C C R ,ATMCAGAAGATGGCTTCACACC ACTTGCGGTAGCAATGCAGCAkc,GGGACACGAkcCAAAATTGTAGC$T 600 T L L A N G A N P S L S T E D G F T P L A V A M Q Q G H D X I V A 601 QrCrrAClSTGAAAATGA kcC TGGTGGGCAGGT¶VG XCCTACCAGCTCTTCAki x V L L Z N D V R G TAT7GCTVC IAAAAAAATGATGTAAATOCAGCITAAGTTATACTTCAACATGATCCcc'CAATGCTGACATCGTTTCCAAG uATCAGGATTLIC c 750 K V R L P A L H I A A K X N D V N A CACCCCTTGCAC ICAAGGA A XK L L L Q H D P N A D I V S K S G F S P L H 751 ATAGCAGCTCAITATGG zr.AACGTCGACATTCCCAC12ITTTGCTTTGAACM kc W I A A R Y G N V ,TGTAAACTATlGTAGCCAAACATAACCATAKCTCCCTTACATGIC;IATtGm;TAAATGGGAAAGCTATCACrM rh A 900 D I A T L L L N N V N Y V A K H N TYGCACITT 9TrACTGTCCG8 I T P L H V A C K W C K L S L L L C R 901 IAACCJYG C :G rs 11 CTGTLT TACACCACTICACTC )cCTCCGGTCATGTAGAAGTTATTAAGICAI'tIGCAGCAAAACGOCCCIIC, ki IT G 1050 C A X I D A A T R D G L T .GATACTrACAAAAACGAAGAAI P L H C S GH V E V I X H L L Q Q N A P I L T K T X N G L S TICGCTICACATGA L H Nf 1051 kcCGACGAAGCAGCTCATrDM kc KATTTGlCAACAA mkGATCAAGPACTQGTGATTACTfI TCTACOCCGCTGC7CAkc 11 M G 1200 A A Q G 2 H D Z a A H L L L D N K A P V D 2 V T V D Y L CIG1AGTGTTAAAGTAGC IAGCTflM hCTTGATTACAAG AGVTCCG T A L V A A H C C H V K V A K L L L D Y X 1201 OCAAATCCAAASGCCC2 )cCOCGC1VAACGGT=TCAClITTICC¶TCAThTOCCX'OTCAAGAAAAAATCGTATTAAGATGGTG&ACTA rl A N P N A R A L N G F T P L !ACETATMAGCACGCAGCTAACATITGCGCAACCACGGAATCTGGIXCTTAACACCIITSTTACATGTAGCTr 1350 H I A C X X N R I X 2{ V E L L I X H C A N I G A T T E S G L S P L H V A 1351 AGTTAKATIDAATGTh77M7AATATrCTAATATAhIDACTACAACATGAGWC 11TAGTGCGGA CTTTACCAACAATACGCGGKGAAACA 11 S F }( C C I N I V I Y L L H 'AC'CT~ACTCAC1CITAAICAGOCGGATATTAS1tC AAATMACTI IrTCGCAGCGCAAAAk 1500 Q E A S A D L P T I R G T P L H L A A R A N Q A D I I R I L L R 8 A K 1501 GTGIATGGCTATTGTAT ;7TGAAGCCCACCA ITTCATGTAGCTTCTG;AUATTAGGAAAILT V D A I V R rATrAATATAATTATGTTATrAC S GCAGCATGGAGCAGAGATAMTGC 175TCAATCAATGACAAATATTCc.GGGCGCTOCAILT.TATTGCTOCAAAAk 1650 E C Q T P L H V A S R L C N I N I I S L L L Q A E I N A Q S N D X Y S HG A L H I A A X 1651 GAAGGACAAGACGAATAim74GTCCAAGTOTTACTCGW aAJATGCrGGGAAAA ki,TAATGCT T Z G Z N I V rACCAAA GGT0ACTtCAC00 CMATTIOCTSGCAAATACOGAAAkcC.CAGRAT7XTGTrCAAATATT'AIACTACAAAAILT r 1800 Q Q V L L B N G A N N A V T X K G F T P L H L A C K Y TOGAGClTOTMTT G Q N V V Q I L L Q N G A S I 1801 GAKTITCAGOGAAAhA k?,WTGTMACCCCATrACACcCGTCGCCACGCACTAkc,CAATAATCCI ICCTCAACTAGAACTCCTS1TAAAA 17 D F Q C X N D V T P L H V A AAMTGGGIVATCGCCAAATTTATG GCC=GAAATGGTCAATGTGC mTATTCACATAPtOC 1950 T H Y N N P S I V E L L L K N C S S P N L C A R N G Q C A I H I A C?FGGhhAX X 1951 MSTTAT TSCAAMTT( tx1ATGCAICICI7GCAACACC,CGGTGCAGACGTTAAIk7TATTATTAGCr_ CAAGAGTGGCTCITCTccGTYACA7 .TGGCGGCtA GGG GAA C CT rxTGATGGTCCACTACTTCSJ!AAGAGMUMGw 2100 N Y L Z I A X Q L L N G A D V KGTTATrAGCGCC Q N I I S K S C F S P L H L A A Q C C N V D Xt V Q L L L E Y G V I S A 2101 AATAAGAAGAAACCTPAACACATTACATGGGC7TI k7 TOCCCAGGAGGGCCA ITGTCTIGSI WVTCACAGATTTTACTGGAGCACGGTTGCTAACATTTCAGAGCCGACTAO;G ,TTATGTATGCC.o 2250 A A X N C L T P L H V A A GAATGTTATACTCCCCITrCAl CCATSATGG'rCAC Q EGC V L V S Q I L L H G A N I S R T R N C Y T P L H M A A H Y G H 2251 CT£CAtCITGCIVAGTO IrTFrATACAGACGC7 17 CACATTGAGRTGT 7TCTAATAICCICGGTATACCCCGCTTCATCAAGCCCGCTCAGCAAGGTCACATTATGAT rr ,T 2400 L D L V X F F I 2 N D A D I E K S S N I TATrAATCTTTTATrACGCCAITAAAGCGAATICCAAATGCTCTA C Y T P L H Q A A Q Q C H I X I I N L L L R H K A N P N A L 2401 ACAAAGhCOGGAACACJ :AhAC7CTCChACATTGCAAG7IT, rAACCT!GGATACGTI7A'AACTGTAATC!GiGAATCCTCAAAATTGTCACTTCAAACATCCGMThrAAMTCAAATAT TGGTGCGATTGAAGAAAACT r- a T X D G N T A L H I A S N L G Y V N CAAGCTAATGSACTCCAGAGT 2550 T V S L K I V T S T S V I N S N I G A I E E X L X V N T P E L 2551 ATGCAAGAAACCTTACOY TTTI C0A1¶vCGATGACGIG A. IT TGTCATGATCTTrCGAVTCA7IT,rAACCATTACAAATACAIkTGrAACTTGTGA AAAAGCCAACTACG CCCAGGATCAGAAGAACIrCGA:'T QT:ACTCTGCGAT 2700 X Q E T L L S D S D D S C D D L TACTACAAAC L D H N H Y K Y N A T D D L X A N Y G Q D Q X N F D S SN T D H D 2701 CTTACQTFGIVAGCTII70RCTAAATAAGAAAGAATARI IG ID kCTACCCAA2QAAA"9 GACCTCTATr&GATTZACTGAAATCGOOCACAAAKCCCGATAACGTTGT-TArC'CGUkATCCCAAGTCATTAGGGOT ID q 2850 L T D V S V L N K X 8 I L P N B N C =TGGTC Sc 8 L T E I G H K P D N V V I A R S Q V H L G F L V S 2851 GCACGTGGCGGhTCCATC10CGCGGATATCGCCACAA1.74rGGITSCGCATrATI .10CGTACC¶c )U A kAAGGCCTGCGCTGKACCAACACGA ATAACATGTCGAThTrSMXGCC(.0 a M;"GGTI~AGC 3000 R CGS R G Y R H N G V R I I V GCAAAGGGTAGTCAACCCTCCGSCCSTSMAAT P P K A C A E P T R I T C R Y V XP Q R V V N P P P L IN 3001 CGCA¶WTGr1o Llq GG"AAI !GGAA2TITrAAGMITITCCAATAACT7rMAGCAAGTTCCACACTATGGCAtCCCCTAGAAAAAACG&GCGCGAAAS T In r L V S R I L N S P V TATTATACTCCGGTCCGACAAT PTC;GCGTGAOCAT 3150 D G ) F L S P I T L E V P H Y G T rGGAkGAAGT L R X N E R E I I I L R S D N G I S WI R E N 3151 MCTTAThTAAAGRCATAXkATAGGTGAAGATATAAACa:CAAACGGAGGAATF IT V TCATTCGGAIrCGCAhCTCGGGATCGTrAC7CAAkAT TGCAC7TTTCC IrTGCGlWCGAGGCA AXkGGCACGTrTj 3300 N L Y X D I I G E D I N T E E F rATrGGACCIrIAT QI H S D R I V R I V T Q N V P H F F A V V S R V R Q E VI H V I G P D 3301 GGTCGhACCGOTTTCTCTrACAGCAGTGCCCCAGGTCci:AAAGCTATATI'TCCC!OGCCTCATGCIrMAACTAAATAAATCGAGTCGGDI .GI G G CTTCAGGCTCAGTCAMTGACCT(SCSTGAA$'TASCSa G( 3450 T V F S T A V P Q V X A I F P P H DGACAGOG ;GTCGCTCTCA A L T K K I R V G L Q A Q S V D L V E C S K L L G Q G VI A V S 3451 CCCGTACTAACTGTGM0CCAC&CCGTCGAAAATFI TX WACAAAGCTATACJ KTTAAGCATrT(PCCAGCTCCAAAAGCATGTACAAAT.rAGTATGTTAATGCATGMAI c In Pk( P V V T V Z P R R R X F HI X A I T L S I P A P K A C T N CAA1GAAMTCAAGTTCOCCTACTCTlTCGAkcICTCTTCC 3600 S 1 V N A C Y G N C N S S S P S L R LI L C S 3601 ATAGTOG~GTCAAACCOCGAGCAACATGGGAGCAIr(rGTTACTGGATCAACJ KCCGTTOICAAlTIrTGrTAGAGACAGTGTAACATTI G G rACCACTAC7GTCTGCCCGGl= T InrATCATAGACcc I S Q T R A T W E D VI T G S T rlGCtTAATAGATGCCGTAAI :GCAGGACGAATG 3750 P L S FI V R D S V T F T T T V S A R F W L I D C R N I I D Ai G R 3751 GCCACAGAAC1TTTAGTT(rCAT TOSCCARAAGCCTIm?ITTATGTCAAG7TI IDrGTAATAITI r( A T QGCAAAACGAATITCCCAAACGGAA4GXCAATMATCATATMOATc Q Go (C 3900 Z L Y S HI L A K V P FI Y V X F VI i 3ACUTGATGATAhAAG&GATAAGACCCSIAGrCAACAAGAATAT A K R I S Q S E A K F S V F C K T D D X E D K TI L E Q Q Y 3901 R GCAACGTTCCA hiUGWCAGGATh7CGAAGTAki kTGCAAAATCAAAAT TIGTATAICTiT(MGATTCGCOMMMAACATrOTOMCCC AT0TTAAAGAAAGGCGAACAGCX I W TI zc F T Z V A K SI R D I E V LI Q N Q I VI FATACCAAOTOTCAACCTITTrQCGAAAAC 4050 Y L I F A G N I V P:IL X K C E Q L Y T K F Q P F Cc E NI:CGGCTTTCCT~TRi L S F 4051 AGTOCTCACATTAAAGATvrCAAGAATTTCCACATGorrl( rCGCATCTGClT'ATG0;ACCTACCCG 3);ATSGTAGCACCAGATGAAGTGCCXC CTGAGMCGCITTGTACTCTGhACci V IC 8 A H I X D Q E PNGH i :ATFCAGTOGGATTOAAACTrATAACAAAC 4200 4 C R I C F N T Y P I V G P D V P IL P A1TC7CG6AAGA X L C T L N I S V D F X T II S NIHf L E R 4201 GhTAACCTGCATAGCTTrGWAAMCATTGTATAAITrGt X CATGGAAAATTAAACC(_CACAATG.AAuLAATATGTATTrrGAGTAAAGGAA( Tj Pi D N L H S L NI D C I N A Hf G kCAACAGGTGAAGAAAAITrTn rACAAAGCT1GTATAAT7GCAAGTGACATCAAAACTAATTCAT 4350 X L N HI N E Np I V F G V X E V K K I Q Q D I T X A C I I 8N Si D I K3 L I H 4351 GKGGCTGhTGTAATATTAkckGACGACATATGTAGCCAC11 Gc TIfAGGAAGIOGACTG CCTTT&C¶TSrc,KGCAAATGTATA.GGIGTTTCCCAA A( cc 3A B A D V I L DI D I C S R LI G S D V I GhAA¶CTAAACGACAGCOGTAAAGCAG 4500 P L L A N V L G V S Q ) E F L L N D V LAGCAIr2GCAAISIC kGTGGGATATAACTTGTkAAAA7C.A S K Q S 1 A N 4501 TrACAGCTATGGCTAGAAkcCCAIrIACGCATATGACGx CGCAATOTACTAGCTT(mAAoCCCT .II'TACAAAASCTGGCCACt-AGTATAC A(GAGT7TGGCACTCA7CAACCGoc, LI L Q L W L E HI C C I L T Gc N V L A EI A L .TTCCGACTGCC 4650 TY K I G R S D I %V 8 K S F X N A E F G T H Q P E X VIL P T A 4651 GAAWG GAAlT ICCATrATGAACAGTATTI G I G X D G .ID Y Z Q ^ rACAAAG7CTACAO=CTTAOCAAGATATITrTAAAAACGCAGhTC-CTAAGTTrAGTATTGATRAAATATATCTACIGCAACOCAGGrCTChCTAAAAGr.AGTAAATTGATACT 4800 4801 AATr.CAATAMlsAACATrlf ArACG7TAC GAAAGhAGCAATTATAATAlsTTGr.AACCAGA ATGTCGATTTACCTGAGTrAATCGATGAGTAASCWCC ATGMiATAGTCCGATATCTTAAATAATT5ACCT 4950 4951 AAA=GhTCCAArAkCISSTTC7R~ACATTT7CTGAJC~kTCGACTkATTICAA5TGTACTTlrCAATTTATATAATAAACAATATTAACAGTGTTAAA1A 5069 FIG. 3. Nucleotide and deduced amino acid sequence of Drosophila ankyrin. The sequence originally amplified by PCR is underlined. Downloaded by guest on September 24, 2021 10288 Cell Biology: Dubreuil and Yu Proc. Natl. Acad. Sci. USA 91 (1994)

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I ...... I . . . . H. u...... repeots spect inbindg C-term Fly Ankyrin Human Ankyrin 1 Fly Ankydn

FIG. 4. Dot-plot comparisons of Drosophila and human ankyrins. (A) Human Ank2 vs. fly ankyrin; stringency = 35% identity. (B) Same as A, but with 50% identity. (C) Human Ankl vs. human Ank2, stringency = 50%/ identity. The comparison window is 100 residues. products, products of alternatively spliced ankyrin tran- ABGP1 (10), also failed to coprecipitate with ankyrin (lanes scripts, or crossreactive products of other genes. Antibody 9-11, upper arrow). Together, these results imply a specific against a Drosophila (3-spectrin fusion protein (GST-KCar) association of ankyrin with spectrin but not with a- or reacted primarily with the 265-kDa ,3-spectrin subunit in S2 neuroglian. cells and crossreacted weakly with the 430-kDa 1H-spectrin Accumulation of Ankyrin and (-Spectrin In a-Spectrin subunit (Fig. 5A, lane 2). Mutants. Wild-type and a-spectrin mutant larvae (RG41/ Coimmunoprecipitaton of Ankyrin and 1- and a-Spectrin. RG41) were isolated as previously described (17). Paired Detergent-soluble membrane proteins from 0- to 12-hr Dro- nitrocellulose blot lanes of mutant and wild-type larvae were sophila embryos were used as the starting material for allowed to react with antibodies against a-spectrin (3A9; ref. precipitation of ankyrin and associated proteins. The 170- 16), 13-spectrin, and ankyrin (as in Fig. 5). As shown previ- kDa ankyrin polypeptide was precipitated with affinity- ously (17), a-spectrin levels were greatly diminished in purified antibody to ankyrin (Fig. SB, lane 2) but not with a-spectrin mutants relative to wild type (Fig. 6), but levels of nonimmune serum (lane 1). (3-Spectrin (lane 7) and a-spectrin ankyrin and spectrin appeared to be unaffected by the (not shown) were also detected in the ankyrin precipitate but a-spectrin mutation. were not precipitated as efficiently as ankyrin. We monitored the behavior of a-actinin during immunoprecipitation as a control for the specificity ofthe ankyrin-spectrin interaction. DISCUSSION a-Actinin is not known to interact with spectrin or ankyrin Human ankyrins can be divided into three domains: (i) an and did not coprecipitate with ankyrin (lane 10, lower arrow), N-terminal domain consisting of 23 imperfect repeats of a although it was readily detectable in the input and unbound 33-amino acid motif; (ii) a central domain that binds to fractions (lanes 9 and 11, respectively). Neuroglian, a Dro- (-spectrin; and (iii) a C-terminal domain that is highly vari- sophila homolog of the vertebrate ankyrin-binding protein able between ankyrin isoforms (reviewed in ref. 1). The repetitive sequence motif is found in a number of proteins A B other than ankyrin, including the Drosophila proteins cactus P U P U P U (32) and Notch (30, 33). But the spectrin-binding domain is unique to the membrane skeleton protein ankyrin. According _~ ... --M to its overall sequence organization, the conserved sequence 205- from the spectrin binding domain, and spectrin binding _mf - < we i9 6- activity, the protein describe here is Drosophila ankyrin. - The fly ankyrin probes we have tested hybridize with a single transcript on Northern blots of embryonic poly(A)+ RNA and with a single polytene locus on chromosome 4. Igb'- . 45 -

1 2 1 2 3 4 5 6 7 8 9 10 11 Arifbodye

FIG. 5. Antibody reactions withDrosophila ankyrin. (A) Western

blot of Drosophila S2 cell proteins allowed to react with affinity- a-Sqectrin purified antibody to ankyrin (lane 1, 4 pg/ml) or to P-spectrin (lane 2, 1:1000). Ankyrin antibody consistently detected a 170-kDa band FIG. 6. Ankyrin and spectrin levels in (arrowhead) and in some preparations (*) detected additional smaller a-spectrin mutants. RG41/RG41 a-spec- polypeptides. (B) Western blots of ankyrin immunoprecipitates. trin mutant larvae (-) and RG41/TM6b Embryo membrane extract (I = input, 1% of total), immunoprecip- /.--Spectrii __ wild-type larvae (+; ref. 17) were tritu- itate (P, 17% of total), and unbound fractions (U, 1% of total) were rated in SDS sample buffer and loaded on analyzed. Lanes 1-5, stained with anti-ankyrin antibody (10 pg/ml). gels at 4 larvae per lane. Western blots of Similar amounts of control nonimmune (lane 1) and immune (lane 2) total larval proteins from mutants and rabbit IgG were detected by the secondary antibody (Ig). Lanes 6-8, wild type were probed with antibodies stained with anti-p-spectrin antibody (1:1000). Lanes 9-11, stained Ankyrin against a-spectrin (Top, 1:1000), P-spec- with neuroglian antibody (upper arrow = neuroglian) and a-actinin trin (Middle, 1:1000), and ankyrin (Bot- antibody (lower arrow = a-actinin). tom, 4 pg/ml). Downloaded by guest on September 24, 2021 Cell Biology: Dubreuil and Yu Proc. Natl. Acad. Sci. USA 91 (1994) 10289 Southern blot hybridization patterns are also consistent with data before publication. We also thank Tony Mahowald for critical the presence of a single ankyrin gene in Drosophila. In reading ofthe manuscript. This work was supported by a grant from addition, the original probe used to isolate cDNAs was the Otho S. A. Sprague Research Foundation. derived from a region of the spectrin-binding domain that is 1. Bennett, V. (1992) J. Biol. Chem. 267, 8703-8706. conserved among different ankyrins, yet it detected only a 2. Davies, K. A. & Lux, S. E. (1989) Hum. Genet. Dis. 5, single class of cDNA in cDNA library screens. These data 222-227. suggest that there is a single conserved ankyrin gene in 3. Palek, J. & Lambert, S. (1990) Semin. Hematol. 27, 290-332. Drosophila. 4. Bodine, D. M., Birkenmeier, C. S. & Barker, J. E. (1984) Cell Ankyrin links spectrin to the cytoplasmic domains ofmany 37, 721-729. different integral membrane proteins in mammals. It has been 5. Bennett, V. (1990) Physiol. Rev. 70, 1029-1065. proposed that the array of ankyrin repeats constitutes an 6. Nelson, W. J. & Veshnock, P. J. (1987) Nature (London) 328, array of binding sites for different membrane proteins (re- 533-536. repeat in 7. Srinivasan, Y., Elmer, L., Davis, J., Bennett, V. & Angelides, viewed in ref. 1). Conservation of this domain fly K. (1988) Nature (London) 333, 177-180. ankyrin suggests that it interacts with at least some of the 8. Smith, P. R., Saccomani, G., Joe, E.-H., Angelides, K. J. & same proteins in the fly. A cell adhesion molecule in mam- Benos, D. J. (1991) Proc. Natl. Acad. Sci. USA 88, 6971-6975. malian brain (ABGP1) is known to interact with ankyrin in 9. Morgans, C. W. & Kopito, R. R. (1993) J. Cell Sci. 105, vitro (12), although the binding site on ankyrin is not yet 1137-1142. known. The cytoplasmic domain of ABGP1 resembles the 10. Davis, J. Q., McLaughlin, T. & Bennett, V. (1993) J. Cell Biol. cytoplasmic domains of several other cell adhesion mole- 121, 121-133. cules, including Drosophila neuroglian (29). Unexpectedly, 11. Bennett, V. & Gilligan, D. M. (1993) Annu. Rev. Cell Biol. 9, an interaction between neuroglian and fly ankyrin was not 27-66. in the 12. Nelson, W. J. & Veshnock, P. J. (1986) J. Cell Biol. 103, detected immunoprecipitation experiments. Either 1751-1765. immunoprecipitation conditions were not favorable for their 13. McNeill, H., Ozawa, M., Kemler, R. & Nelson, W. J. (1990) interaction or the sequence similarity in the cytoplasmic Cell 62, 309-316. domains of these proteins is unrelated to ankyrin-binding 14. Yue, L. & Spradling, A. C. (1992) Genes Dev. 6, 2443-2454. activity. 15. Fehon, R. G., Dawson, I. A. & Artavanis-Tsakonas, S. (1993) The most conspicuous difference between fly and mam- Development 120, 545-557. malian ankyrins is in the size and sequence ofthe C-terminal 16. Dubreuil, R., Byers, T. J., Branton, D., Goldstein, L. S. B. & domain. The major products of the Anki and Ank2 genes in Kiehart, D. P. (1987) J. Cell Biol. 105, 2095-2102. mammals are 30-40 kDa larger than fly ankyrin. Mammalian 17. Lee, J., Coyne, R., Dubreuil, R. R., Branton, D. & Goldstein, that are similar to in size have been L. S. B. (1993) J. Cell Biol. 123, 1797-1809. isoforms fly ankyrin 18. Otto, E., Kunimoto, M., McLaughlin, T. & Bennett, V. (1991) reported (34), but the sequence differences that are respon- J. Cell Biol. 114, 241-253. sible for their smaller size are not yet known. 19. Brown, N. H. & Kafatos, F. C. (1988) J. Mol. Biol. 203, Genetic studies in Drosophila have demonstrated an es- 425-437. sential role for the membrane skeleton in nucleated cells (17). 20. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Null mutations of Drosophila a-spectrin are lethal. But Cloning: A Laboratory Manual (Cold Spring Harbor Lab. embryonic development and larval crawling, feeding, and Press, Plainview, NY), 2nd Ed., pp. 1.93-1.104, 9.31-9.55. tactile responses are surprisingly normal in these mutants 21. O'Neill, M. T. & Belote, J. M. (1992) Genetics 131, 113-128. until their death late in the first instar. The polarized distri- 22. Engels, W. R., Preston, C. R., Thompson, P. & Eggleston, the pump in gut W. B. (1986) Focus 8, 6-8. bution of sodium also persists epithelial cells, 23. Devereux, J., Haeberli, P. & Smithies, 0. (1984) Nucleic Acids despite defects in the shape and adhesion of the same cells Res. 12, 387-395. (17). Our results demonstrate that null mutations in a-spec- 24. Dubreuil, R. R., Byers, T. J., Sillman, A. L., Bar-Zvi, D., trin do not constitute a complete knockout of the membrane Goldstein, L. S. B. & Branton, D. (1989) J. Cell Biol. 109, skeleton. Both ankyrin and 3-spectrin continue to accumu- 2197-2206. late in the absence of a-spectrin synthesis. Unassembled 25. Ron, D. & Dressler, H. (1992) BioTechniques 13, 866-869. subunits in mammals turn over with a half-life of hours (e.g., 26. Byers, T. J., Brandin, E., Winograd, E., Lue, R. & Branton, D. see ref. 12), suggesting that these proteins accumulate in (1992) Proc. Natl. Acad. Sci. USA 89, 6187-6191. Drosophila because they are assembled. Partial function of 27. Smith, D. B. & Johnson, K. S. (1988) Gene 67, 31-40. and may account for the relatively mild 28. Byers, T. J., Dubreuil, R. R., Branton, D., Kiehart, D. P. & ankyrin f-spectrin Goldstein, L. S. B. (1987) J. Cell Biol. 105, 2103-2110. effects ofa-spectrin mutations in early development. Later in 29. Bieber, A. J., Snow, P. M., Hortsch, M., Patel, N. H., Jacobs, larval development there must be a critical requirement foran J. R., Traquina, Z. R., Schilling, J. & Goodman, C. S. (1989) intact membrane skeleton, resulting in the observed lethality Cell 59, 447-460. of a-spectrin mutations. 30. Lux, S. E., John, K. M. & Bennett, V. (1990) Nature (London) 344, 36-42. We thank Yuanping Liang and Chris Schonbaum from the Ma- 31. Cavener, D. R. (1987) Nucleic Acids Res. 15, 1353-1361. howald laboratory for help with Northern blots and polytene hy- 32. Kidd, S. (1992) Cell 71, 623-635. bridizations, Dr. Alan Bieber for neuroglian antibody, Dr. Belinda 33. Wharton, K. A., Johansen, K. M., Xu, T. & Artavanis-Tsa- Bullard for a-actinin antibody, Dr. Vann Bennett for suggesting the konas, S. (1985) Cell 43, 567-581. use of oligonucleotide primers from the spectrin-binding domain of 34. Peters, L. L., Turtzo, L. C., Birkenmeier, C. S. & Barker, brain ankyrin, and Dr. Anthony Otsuka for sharing ankyrin sequence J. E. (1993) Blood 81, 2144-2149. Downloaded by guest on September 24, 2021