Proc. Natl. Acad. Sci. USAVol. 92, pp. 1485-1489, February 1995Cell Biology

The Drosophila yolkless gene encodes a vitellogenin receptorbelonging to the low density lipoprotein receptor superfamilyCHRISTOPHER P. SCHONBAUM, SCOTr LEE*, AND ANTHONY P. MAHOWALDDepartment of Molecular Genetics and Cell Biology, The University of Chicago, 920 East 58th Street, Chicago, IL 60637

Contributed by Anthony P. Mahowald, November 28, 1994

ABSTRACT Sequence comparisons of vitellogenins froma wide range oforganisms have identified regions of similaritynot only to each other but also to vertebrate apolipoproteins(e.g. apoB-100 and apoE). Furthermore, the chicken vitelloge-nin receptor, which also binds apolipoproteins, has beenfound to belong to the low density lipoprotein receptor(LDLR) superfamily [Bujo, H., Hermann, M., Kaderli, M. O.,Jacobsen, L., Sugawara, S., Nimpf, J., Yamamoto, T. &Schneider, W. J. (1994) EMBO J. 13, 5165-5175]. The yolkproteins of higher dipterans are exceptional, however, andinstead show similarity to lipoprotein lipases. The molecularcharacterization of the putative Drosophila melknogaster vitel-logenin receptor gene, yolkless (yl), described in this reportreveals that the protein it encodes (Yl), is also a member of theLDLR superfamily. The ovary-specific 6.5-kbylRNA codes fora protein of -210 kDa which contains all three motifs commonto the LDLR class of proteins. Within this superfamily, Yl maybe related more to the LDLR-related proteins (LRPs), whichbind both apolipoproteins and lipoprotein lipases. The simi-larity of Y1 to the other LDLR proteins is restricted to theputative extracellular domain. Most noticeably, the cytoplas-mic domain of Yl lacks the typical NPXY sequence which isinvolved in receptor internalization.

One of the best-studied mechanisms for the selective uptake ofproteins into cells is receptor-mediated endocytosis. Two keyadvances in the study of endocytosis have been the develop-ment of cell-free assays and the identification of mutationswhich disrupt specific steps of endocytosis (1, 2). In this regard,we have been interested in identifying mutations which affectendocytosis in Drosophila. We have focused on the uptake ofvitellogenins (yolk proteins) into the developing egg for sev-eral reasons. First, oocytes incorporate large amounts ofvitellogenins (3, 4). Second, in oocytes, a specialized mecha-nism has evolved so that the yolk proteins are stored in yolkgranules instead of being fated for degradation in lysosomes.Third, in many insects, juvenile hormone is required for theoocyte to initiate vitellogenin uptake (4, 5).

Obviously, one of the key components in the uptake ofvitellogenins is the receptor, and although vitellogenins froma variety of organisms have been characterized, much less isknown about their receptors. In vertebrates, one of the best-studied systems is the chicken oocyte 95-kDa receptor, whichbinds two components of chicken yolk-vitellogenin and verylow density lipoproteins (6). Mutants lacking this receptor failto accumulate yolk proteins in the oocyte, and plasma vitel-logenin levels are elevated (6, 7). Recently, Bujo et al. (8) haveshown that the 95-kDa receptor is closely related to themammalian very low density lipoprotein receptor (VLDLR).Frog and fish vitellogenin receptors appear to be related to thechicken receptor (9, 10).By using ligand-binding assays, vitellogenin receptors of

similar molecular mass have also been detected in several

insect species [e.g., mosquito, 205 kDa (11); locust 156-186kDa (12, 13); and cricket 200 kDa (14)], yet their relationshipto vertebrate receptors is not known. In the fruit fly Drosophilamelanogaster, the female-sterile mutation yolkless (yl) was agood candidate for the vitellogenin receptor gene, since fe-males mutant for yl exhibited a germ-line-dependent pheno-type in which yolk proteins failed to accumulate in the oocyte(15, 16); ultrastructural examination revealed >90% reductionin the number of endocytic structures (i.e., vesicles and tu-bules) in the cortex of the oocyte (17). In this report, wedescribe the molecular characterization of yl.t We concludethat the yolkless protein (Yl) is the yolk protein receptor andthat, like vertebrate vitellogenin receptors, Yl is a member ofthe low density lipoprotein receptor (LDLR) superfamily. Thisis, to our knowledge, the first reported sequence of an inver-tebrate vitellogenin receptor.

MATERIALS AND METHODSFly Culture. Stocks were maintained at 24°C on standard

cornmeal/agar/molasses medium. The yl stocks and the defi-ciencies Df(l)gl and Df(1)KA9 have been described (15, 17,18). All other mutations used in this paper are described byLindsley and Zimm (18).DNA Techniques. Standard techniques were used for

screening DNA libraries, isolating DNA, and subcloning (19).The iso-1 cosmid and A libraries (20) were screened to isolate200 kb of genomic DNA from the 12E/F cytological region.The chromosome walk was initiated by using cosmids suppliedby the Drosophila Genome Project (Heraklion, Crete) andPCR products of a microdissected 12E/F region (gift of R.Saunders, Dundee, Scotland).RNA Techniques. Total RNA was extracted from flies and

analyzed by Northern blot hybridization as described previ-ously (21) except that an aqueous buffer [5X standard saline/phosphate/EDTA (SSPE), 5x Denhardt's solution/0.4%SDS/0.1 mg of sheared salmon sperm DNA per ml] was usedfor hybridizations at 65°C. The Northern blot was probed witha random oligo nucleotide-primed (Boehringer Mannheim),32P-labeled 4-kb Not I genomic DNA fragment (see Fig. IA).P-Element Transformation. A 9-kb genomic DNA region

flanking the 6.5-kb transcript was transferred from the iso-i-derived cosmids into the CaSpeR4 vector, which contains awhite+ (w+) marker gene (22). pWY173 was introduced intow1118 hosts by P-element-mediated germ-line transformation(23, 24). pWY173, at 0.5 mg/ml, was coinjected with the phsir"helper transposase" plasmid (25) into w1118 embryos. Adultswere backcrossed to wl'18, and two w+ lines were retrieved inthe F1 generation; both lines contained an autosomal insertion.Both lines were crossed into y w yl backgrounds (y w y1262b and

Abbreviations: LDLR, low density lipoprotein receptor; LRP, lowdensity lipoprotein receptor-related protein; VLDLR, very low den-sity lipoprotein receptor.*Present address: Department of Molecular and Cellular Biology,Harvard University, 16 Divinity Avenue, Cambridge, MA 02138.tThe sequence reported in this paper has been deposited in theGenBank data base (accession no. U13637).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

1486 Cell Biology: Schonbaum et al.

y w ylICR756 f) and tested for rescuing activity in y w ylhomozygotes.DNA Sequencing. yl cDNAs, isolated from an ovary cDNA

A library (26), were subcloned in the pBluescript SK(+) orKS(+) vector (Stratagene). Unidirectional nested deletions(Erase-A-Base system; Promega) of the cDNA clones weresequenced by using double-stranded DNA templates (MagicMiniprep; Promega) and the chain-termination method (27).Sequencing was performed as described (21). dGTP and dITPreactions were run for most templates to resolve compressionartifacts. The BLAST algorithm (28) was used to compare theyl sequence to the GenPept, Brookhaven, SwissProt, andProtein Identification Resource (PIR) data bases availablethrough the National Center for Biotechnology InformationBLAST e-mail server. The cDNA sequence has been submittedto GenBank and assigned accession number U13637.

Anti-YI Antibody Production. Anti-155 antibodies wereraised against a bacterially expressed TrpE-Yl fusion protein.pAY155 contains a 666-bp yl cDNA fragment (nt 2398-3063)cloned in the pATH1 vector (29). This region encodes a 222-aapolypeptide with three of the C repeats (aa 752-973 in Fig. 3).Inclusion bodies were isolated (30) and fractionated by SDS/PAGE. After staining with 0.25 M KCl (31), the fusion proteinband was excised and allowed to diffuse out of the crushed gel.The eluted protein was filtered, concentrated, emulsified withFreund's complete adjuvant (Sigma), and injected into maleSprague-Dawley rats (50 ,tg of protein per rat). After twoboosts with 25 ,ug of protein each, the rats were exsanguinated.

Protein Extraction and Western Blotting. Ovaries weredissected in cold phosphate-buffered saline (PBS; 10 mMsodium phosphate, pH 7.2/130 mM NaCl) plus 1 mM phe-nylmethylsulfonyl fluoride (PMSF) and then immediatelytransferred to a 1.5-ml microcentrifuge tube on ice containingPBS plus 1 mM PMSF. After all ovaries had been dissected, thePBS was replaced with cold buffer A (25 mM Tris HCl, pH7.5/1 mM EDTA/1 mM EGTA/1 mM dithiothreitol/1 mMPMSF/5 ,g of pepstatin per ml/5 gg of leupeptin per ml) andhomogenized at 4°C. The homogenate was centrifuged at 800x g for 5 min and then the supernatant was centrifuged at100,000 x g for 1 hr at 4°C. The pellet was resuspended in 300,ul of buffer B (50 mM Tris HCl, pH 7.5/2 mM CaCl2/1 mMPMSF) and sonicated briefly on ice. Finally, 100 ,ul of 4%Triton X-100 in buffer B was added. For samples treated with2-mercaptoethanol and heat, an equal volume of 2x sampleloading dye (32) was added and then the mixture was boiled for10 min. For nondenatured samples, only 2x loading dyewithout 2-mercaptoethanol was added. Proteins were sepa-

_ telomere i1 kb

A

Df(1) KA9

Xb Xb G Xb

rated by SDS/PAGE (6% polyacrylamide) and transferred tonitrocellulose (33). Blots were incubated for -1 hr at roomtemperature with 5% nonfat dry milk (NFDM) in Tris-buffered saline plus Tween-20 (TBT: 20 mM Tris*HCl, pH7.5/0.9% NaCl/0.1% Tween-20). Blots were incubated for1.5-2 hr in TBT plus 1% NFDM with the primary antibody(anti-155) diluted 1:2000. After several washes with TBT, blotswere incubated for 1-2 hr with peroxidase-conjugated anti-bodies to rat IgG (Amersham) diluted 1:3000 in TBT plus 1%NFDM. Finally, the blots were washed and signals weredetected with the ECL Western detection system (Amersham).

RESULTS AND DISCUSSIONyl had been mapped by complementation to the intervaluncovered by the overlapping deficiencies Df(J)gl (liF10/12A-12E8) and Df(l)KA9 (12E3-13A5). In females transhet-erozygous for these two deficiencies, the only apparent defectis the yolkless phenotype (15). Using genomic Southern anal-ysis in a chromosome walk of the 12E/F region, we identifiedthe two deficiency breakpoints within --30 kb of each other(Fig. IA). Most of the walk ("150-180 kb) lies in the dense12E8 band, proximal to the Df(1)gl breakpoint. By Northernblot analysis, an abundant female- and ovary-specific 6.5-kbtranscript was identified (Figs. 1B and 2A) at the Df(1)glbreakpoint. In addition, 1.2-kb and 4-kb transcripts wereidentified proximal to the breakpoint. The 6.5-kb transcript isabsent from Df(1)g1/Df(1)KA9 females (unpublished data). A9-kb genomic DNA region (Fig. 1C) encompassing the 6.5-kbtranscript, introduced into flies by P-element transformation,restored yolk uptake and fertility to yl mutants.

Overlapping cDNAs covering 6.3 kb were isolated from anovarian cDNA library (26), and a 5844-nt open reading framewas identified which encoded a potential translation productof 220 kDa (Fig. 3). Antibodies to a bacterially expressedfusion protein, encoded by a vector containing a portion of thecDNA, detected a 210-kDa protein which was absent fromfemales lacking the yl gene (Fig. 2B). The 210-kDa protein isalso absent from ovaries from severalyl mutants (unpublisheddata). Comparison of the predicted amino acid sequence tovarious protein data bases detected highly significant similaritywith several members of the LDLR superfamily, includingLDLRs, VLDLRs, and the LDLR-related proteins (LRPs).The yl open reading frame contains all three motifs commonto this class of proteins (34, 35). Two of the types of motifs arebased on alignment with conserved cysteines: the A motif is atandemly repeated six-cysteine motif which contains a con-

centromere -

Df(1)gI

G N N

11 kb 7 kb

N

7.8 kb

PROBE

4 kb TRANSCRIPTS

EpWY173 RESCUE FRAGMENT

FIG. 1. Molecular characterization of theyl region. (A) Physical map of the genomic DNA encompassing yl. The approximate positions of thebreakpoints (filled rectangles) for Df(1)gl and Df(l)KA9 are indicated above the map. The hatched rectangle below the map indicates the DNAfragment used to probe the Northern blot shown in Fig. 2. (B) Approximate positions of the 6.5-kb yl transcript and proximal 1.2-kb and 4-kbtranscripts. (C) The 9-kb genomic DNA fragment in the pWY173 construct which rescues the mutant phenotype. E, EcoRI; G, Bgl II; N, Not I;(N), cosmid vector Not I site; Xb, Xba I.

B

C

(N)

6.5 kb (yI)

Xb

1.25 kb

G N

s m - - e 2 a m a a-M-ob

Proc. Natl. Acad ScL USA 92 (1995)

I I s

izgmMMORRORRM

Proc. NatL Acad Sc. USA 92 (1995) 1487

Ao L-a

E _- w o

.. .__ _

6.5 W> > ;.-:!'. sFs :.

': .:ZF

1.2_

B

BME+Heat

190-

125 -

88-

65-

L Yi

O C

4'

0

I* Yi

A12345678910111213-Prel

FIG. 2. Adultyl RNA and YI protein expression. (A)A female- andovary-specific 6.5-kb transcript was identified by Northern blot anal-ysis of total RNA (10 Zg per lane) from adult males, females, ovaries,and germ-line-deprived females (daughters of tud/tud females). RNAloading was checked by ethidium bromide staining. (B) Western blotanalysis using anti-Yl antibodies identified a 210-kDa protein in thehigh-speed pellet of ovary protein extracts from OreR (wild type) butnot Df(1)g1/Df(1)KA9 females. Like the LDLR, the cysteine-rich Y1protein migrates faster in the absence of 2-mercaptoethanol (BME)and boiling. Equal protein loading is shown by a preimmune reactingprotein (Prel).

"Al" MOTIFSC-DAGQ-FQCRDGG-CILOAKM-CDGRGDCKDSSDELD-CCRPPHW-FPCAQPHGACLAAELM-CNGIDNCPGGEDELN-CC-SKYE-FMCQQDRT-CIPIDFM-CDGRPDCTDKSDEVAGCC-PGEG-HLCANGR-CLRRKQWVCDGVDDCGDGSDERG-CCEPQKGKFLCRNRET-CLTLSEV-CDGHSDCSDGSDETDLCC--E-FRCHSGE-CLTMNHR-CNGRRDCVDNSDEMN-CC-SPSQ-FACHSGEQ-CVDKERR-CDNRKDCHDHSDEQH-CC-HVHQ-HGCDNGK-CVDSSLV-CDGTNDCGDNSDELL-CC-EPGM-FQCGSGS-CIAGSWE-CDGRIDCSDGSDEHDKCC-PPDM-HRCLSGQ-CLDRSLV-CDGHNDCGDKSDELN-CC-AEDQ--YQCTSNLKICLPSAVR-CNGTTECPRGEDEAD-CC-SIYE-FKCRSGRE-CIRREFR-CDGQKDCGDGSDELS-CC-RPHL-FDCQDGE-CVDLSRV-CNNFPDCTNGHDEGPKC

124166220252304

10621109115211931232127913181375

YL C. FC. .G...C#......CDG ..DC.DGSDE.. .CLDLR C.F...C..CG . .. CI ..... CD. ..DC.DGSDE, .C# = non-polar amino acid (e.g.L,V,I)

"B" MOTnESCDAKK-CALGAAKC-HMNPASGAECFCPKGFRLAKFEDKCCKEQDDL-CSQGC---ENTSGGYRCVCDAGYLIDKDNRTC

3 CENAT--CSHLCLLAEPEIGGHSCACPDGMRLAPDHRRCCQQQNGG-CSHIC-VGEGPYHSICLCPAGFYRDAGNRTCCRSASGRQVCQHKC--RATPAGAVCSCFDGYRLDADQKSC

; CQEQQP--CAQLC---ENTLGGYQCQCHADFMLRQDRVSCCAHAH---CHGLC--LOADYGYECMCGNRLVAEGER--C

346387700

1025141614561769

YL c. C...C.....G..C.C..Ge.L .CLDLR C ..C.LC-.. GY.C.C.DGF.L-...Ce = aromatic amino acid (F,Y)

served acidic (SDE) region required for ligand binding (36),while the B motif is a different six-cysteine motif foundoriginally in the epidermal growth factor precursor (Fig. 4A).The third type of motif is an -280-aa region with up to fivecopies of a module (C) which contains the sequence YWXD;this motif is also found in the epidermal growth factorprecursor.

MCQAEHQVHP SEQRIRVESP KMTASRRGFN LTSQTRAHPS SGGSTSSRYGNCQRTHLIIN GRHVAISLLL LVGLCGGTAA GTPGSADTRC DAGQFQCRDGGCIL9AKMCD GRGDCKDSSD ELDCDYRLCR PPHWFPCAQP HGACLAAELMCNGIDNCPGG EDELNCPVRP GFRFGDTAHR MRSCSKYEFM CQQDRTCIPIDFMCDGRPDC TDKSDEVAGC KQAEITCPGE GHLCANGRCL RRKQWVCDGVDDCGDGSDER GCLNLCEPQK GKFLCRNRET CLTLSEVCDG HSDCSDGSDETDLCHSKPDC DAKKCALGAK CHMMPASGAE CFCPKGFRLA KFEDKCEDVDECKEQDDLCS QGCENTSGGY RCVCDAGYLL DKDNRTCRAV VYGSKEQQPLLLYTTOMTIM GMHLREDNVR NHVYQVAGNL SKVIGVAYDG SHIYWTNIQNEAESIVKANG DGSNAEILLT SGLDAPEDLA VDWLTQNIYF SDNIMRHIAVCSNDGLNCAV LVTQDVHQPR SLAVWPQKGL MFWTDWGEKP MIGRASMDGSRSRPIVSDNI EWPNGIALDM HQQRIYWVDA KLGSVQTVRP DGTGRRTVLDGMLKHPYGLA IFEDQLYWSD WATKSVHACH KFSGKDHRIL AKDRTIYAVHIYHPAKQPNS PHGCENATCS HLCLLAEPEI GGHSCACPDG MRLAPDHRRCMLMEKRQRLF IGLGQVLLEI EHTAFGAHQV SKSYTLPCLI NEMVYNRINGSLIIADNDQR LILEFQPESH ESNVLVRSNL GNVSALAFDH LSRNLYWADTERAVIEVLSL QTRHRALIRF FPGQEVPIGL TVMPAEGYLY WLKAKRHSHIDKIPLSGKG EQVHVFEDDL GDDDIKLVTD YETQTIFWSD SDLGRISYSNYRVPHSQIFR GKLRRPYSLA MWHHDLFWNE LGTPRIYWTH KSNMGPRKVIDIMEKDDPAA IMPYVPVATP NGIPLAASSP VGQESHPCQQ QNGGCSHICVGEGPYHSICL CPAGFVYRDA GNRTCVEALD CEFRCHSGEC LTMNHRCNGRRDCVDNSDEM NCDEEHRHKP KVLCSPSQFA CHSGEQCVDK ERRCDNRKDCHDHSDEQHCE KFDKSKKCHV HQHGCDNGKC VDSSLVCDGT NDCGDNSDELLCEATLRCEP GMFQCGSGSC IAGSWECDGR IDCSDGSDEH DKCVHRSCPPDMHRCLSGQC LDRSLVCDGH NDCGDKSDEL NCGTDSSTMN ISCAEDQYQCTSNLKICLPS AVRCNGTTEC PRGEDEADCG DVCSIYEFKC RSGRECIRREFRCDGQKDCG DGSDELSCEL EKGHHNQSQI QPWSTSSRSC RPHLFDCQDGECVDLSRVCN NFPDCTNGHD EGPKCATACR SASGRQVCQH KCRATPAGAVCSCFDGYRLD ADQKSCLDID ECQEQQPCAQ LCENTLGGYQ CQCHADFMLRQDRVSCKSLQ SGATLLFSSF NEVRNLSEQP VMLNVAWSAN DSRITGFDLAMHRQMGYFSA EDEGIVYQVD LQTKVIVRAL GLPAPTKLSV DWVTGNVYVLSGAQEIQACS FVGRMCGRIV HVKSPRHVKH LAVDGYHARI FYIVIRTEGYGQTSSEIHMA RLDGSRRDML LQRSESFMTA LTTDPHQQLL YFVDQHMRTLERISYRLKTG PMRRPEIMLQ KSNALMHPSG LSVYENNAFI VNLGSMEAVQCALYGSRICH KISINVLNAQ DIWAGRSRQ PQKASHPCAH AHCHGLCLQADYGYECMCGN RLVAEGERCP HGSGNEVAVL GAVNSLELEH EHEQNGHFHWIMAFVLAAG SLTAGLGYMY YQYRQRGHTD LNIUWQNP LATLGGTKAFLEHERAEAGV GFTTETGTVS SRGSUD!FTTTSASSSYAAQ9rSVPNALQRLLRPRQSASG DPMAQELLLE SPSRESKLHA LDGGGAGGDG DGGCGVGRQVPDILVADMDD DAAKSAGQFG GNYAGNDANA RFVS

50100150200250300350400450500550600650700750800850900950

10001050110011501200125013001350140014501500155016001650170017501800185019001950

FIG. 3. YI sequence: 1984-aa sequence derived by conceptualtranslation of the ovarian cDNA 5844-nt open reading frame. YWXDor potentially related sequences present in C repeats of LDLRsuperfamily proteins are double underlined. The putative transmem-brane region is underlined. The potential internalization signals(NMHF, NDTF, and FAAQQF) are in boldface type.

BLRPYLVLDLRLDLR

-4500 aa-2000 aa.-860 a.a.-840 aa.

,A" motifs 'B" motifs31 2213 78 37 3

C clusters9311

L '- - -N-_ _. -- .R a 1 1 1-1-1 1 1--II I -P *mmil lIIU-C

N-EO33ImB U UYL was[ IIII I Erm El=-c

VLDLR N-10I I I I I -S -C

LDLR N-_______I_-C

3=A E =B "-- =C O=TM

FIG. 4. YI is a member of the LDLR superfamily. (A) The repeatedYl class A and class B motifs are aligned by cysteines. A consensusderived from those residues present in at least half of the motifs iscompared with consensus sequences for the LDLR (35, 37). (B)Comparison of the number and distribution of the different motifs inY1 relative to the human LDLR (35), chicken VLDLR/vitellogeninreceptor (8), and human LRP (37). In the diagram, the LRP and Ylare fragmented to illustrate the similar modular arrangement of thesemotifs among the four proteins. TM, transmembrane domain.

YI falls between the LDLR and the LRP both in size andnumber of the different motifs (Fig. 4B), yet the organizationof the repeats is similar among the three proteins. For example,A motifs are present in tandem arrays which are followed bya pair ofB motifs; clusters ofC motifs are flanked by B repeats;and the transmembrane domain is preceded by a B motif.While the LRPs contain two subclasses (B.1 and B.2) of Bmotifs (37), the LDL receptor and Yl have only the B.2subclass. In addition, relative to the first two clusters of Crepeats, the final cluster ofC repeats in Yl shows less sequencesimilarity with other LDLR superfamily C repeats, but it is stillconserved in length (-280 aa).

In contrast to the extracellular domain, the putative cyto-plasmic domain of Y1 (Fig. 3) shows no similarity to the otherLDLR superfamily members. Most conspicuous is the absenceof the NPXY sequence common to other LDLR proteins; this

I --- -I

Cell Biology: Schonbaum et al.

1488 Cell Biology: Schonbaum et al.

peptide, which can form a P-turn structure, is required forinternalization of the receptor (38, 39). However, the Ylcytoplasmic tail does have two similar potential internalizationsignals (NMHF and NDTF) as well as a sequence witharomatic residues separated by four amino acids (FAAQQF)which may serve as the internalization sequence. When placedin the transferrin cytoplasmic tail, a six-residue LDL receptorsequence with two aromatic residues (FDNPVY) promotedmore efficient internalization than NPVY alone (40).The similarity of the 95-kDa chicken vitellogenin receptor to

the mammalian LDLR had previously been demonstrated bySchneider and his colleagues (3, 41), and the recent cloningdata has shown that it is closely related to mammalianVLDLRs (8). Considering that vertebrates and invertebratesdiverged hundreds of millions of years ago, the identificationof Yl as a LDLR superfamily member suggests a high degreeof conservation of vitellogenin receptors. Yet YI does notappear to be more closely related to the chicken vitellogeninreceptor than to other LDLR superfamily members. First, Ylhas two sets ofA motifs, with five and eight tandem copies ofthe motif, while the chicken vitellogenin receptor has only asingle set of eight tandemly repeated A motifs. Second, thereis not particularly high amino acid identity between the eightA repeat domains of these two proteins. For example, whilethere is 84% amino acid identity between the eight A repeatdomain of chicken vitellogenin receptor and rabbit VLDLR(8), the eight A domains (repeats 6-13 in Fig. 4A) of Yl showonly 38% amino acid identity (48% with conservative changes)with the chicken vitellogenin receptor. Similar amino acididentities are seen between the eight A domains of Yl andmegalin, an LRP class protein (42).Homology of vitellogenin receptors and LDLRs has also

been suggested by the similarity of their ligands, since vitel-logenins from a broad range of species (e.g., birds, amphibia,and nematodes) have regions of sequence similarity to apoli-poprotein B-100 (43). However, the yolk proteins of higherdipterans, such as Drosophila, Ceratitis, and Calliphora, sur-prisingly show similarity instead to lipoprotein lipases (44-46).Yet this too reveals ties to the LDLR superfamily. In mam-mals, LRPs have been shown to bind lipoprotein lipases as wellas a-macroglobulin and apolipoproteins (47, 48). It is not clearhow the structure of the LRP is related to the ligand bindingspecificity, but independent binding sites for the multipleligands have been suggested (49). One possibility may be thatan ancestral LRP-like molecule has diverged so that thedifferent binding activities of LRPs have been separated toform different types of vitellogenin receptors. The ancientorigin of LRPs was revealed by the discovery of a nematode(Caenorhabditis elegans) LRP (50). It is not known if the C.elegans LRP functions as a vitellogenin receptor. However, thesimilarity of C. elegans vitellogenins to vertebrate apoB-100(43) suggests that the C. elegans vitellogenin receptor will bea member of this superfamily. Interestingly, in chickens,vitellogenin is bound by an -380-kDa oocyte-specific LRP aswell as by the 95-kDa vitellogenin receptor (51). Comparisonof the vitellogenin binding domains in these two proteinsshould help in understanding the basis for this interaction.Understanding the evolution of this system will require a

more detailed comparison of the binding activities and se-quences of the receptors and their ligands. The study ofvitellogenin receptors within insects should be particularlyinteresting. Similarity of receptors among higher dipterans hasbeen suggested by the ability of Drosophila melanogasteroocytes to incorporate the yolk proteins of other dipterans (52)as well as by the conservation of dipteran yolk protein se-quences (45, 46). In other insects, however, our knowledge ofthe receptors has been limited mainly to descriptions ofapparent molecular masses and biochemical properties; therehave been no previous reports of an insect vitellogenin recep-tor sequence. The molecular mass of Yl (210 kDa) is similar to

that of the vitellogenin receptors from mosquito (205 kDa)and cockroaches (200 kDa), while the receptor from locustsappears smaller (156-186 kDa) (11-14). Since the vitel-logenins of other insects [e.g., mosquito (53), moth (54), andboll weevil (55)] appear to resemble vertebrate apolipopro-tein-like vitellogenins more closely, it will be interesting tocompare their vitellogenin receptor sequences with Yl to seewhich features are conserved. The next step is to test thebinding specificity of the Drosophila receptor and the require-ment of the different domains for ligand specificity. It ispossible that the insect receptors, like LRPs, will have broadligand specificity, binding both apolipoprotein-like and li-poprotein lipase-like vitellogenins. If specificity is observed,then chimeras may allow us to determine the domains respon-sible for discriminating between the different vitellogenins.

We thank the Drosophila Genome Project for providing cosmid andPCR products from the 12E/F region and John Tamkun for the iso-1A and cosmid libraries. This work was supported by National Institutesof Health Training Grant T32 HD07136 (C.P.S.), National Institutesof Health Grant HD17607 (A.P.M.), and National Cancer InstituteCancer Center Grant CA14599-20.

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