Ankyrin and ,-spectrin accumulate independently of a-spectrin in Dro8ophila

We report the identification and Initial characterization of Drosophila nelanogaster ankyrin. Oligonucleotide primers based on the spctrin-binding domain of human brain ankyrin were used to amplify Drosophila genomic DNA. A cloned 184-bp PCR product was used to Isolate Drosophila ankyrin cDNAs. Ankyrin cDNA probes detected a 5.5-kb transcrpt from embryonic poly(A)+ RNA and a single polytene chromosome locus (1O1F-102A). The cDNA sequence encodes a 170-kDa protein that is 53% identical to human brain ankyrin (Ank2). Antibodies directed against a recombinant fragment of Drosophila ankyrin reacted with a 170-kDa polypeptide from Drosophila embryos, larvae, S2 cells, and adult flies. The ankyrin antibody immunopripitated aand ,-spectrin with ankyrin in detergent extracts of Drosophila embryo membranes. Antibodies against Drosophila ankyrin, a-spectrin and «spectri were used to detect these proteins in wild-type and a-sin mutant larvae. a-Spectrin levels were greatly diminished in mutant larvae, but levels of ankyrin and -spectrin were indis hable from wild type. The persistence of ankyrin and P-spectrin may explain the relatively mild phenotype of a-spectrin mutants during early Drosophila development. The protein ankyrin provides a link between integral membrane proteins and the spectrin-based membrane skeleton (1). That link appears to be important to the mechanical properties of the plasma membrane in mammalian erythrocytes. Defects in ankyrin result in membrane fragility, a decrease in the number of circulating mature erythrocytes, and consequent anemia (2, 3). The membrane defect results from the failure of spectrin and other proteins to assemble properly without ankyrin as an attachment site. Defects in 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 transmembrane protein in erythrocytes (5), ankyrin interacts with several different proteins in nucleated cells. Coimmunoprecipitations, cosedimentation, and in vitro binding assays have been used to demonstrate interactions between ankyrin and the sodium pump (6), sodium channels (7, 8), a nonerythroid anion-exchange protein (AE3; ref. 9), a cell adhesion molecule from brain (ABGP1; ref. 10), and others (reviewed in ref. 11). Each of these interactions may simply attach the spectrin-based membrane skeleton to the plasma membrane. Alternatively, it has been proposed that ankyrin can act in the opposite direction: to capture and stabilize membrane proteins in specific membrane domains defined by the membrane skeleton (12, 13). But it has not been possible to directly test the role of ankyrin in membrane domain segregation. Genetic studies have been important in unraveling the roles of membrane skeleton proteins in the erythrocyte, and they are now contributing to our understanding of the membrane skeleton in nucleated cells. Studies in Drosophila have so far shown that three membrane skeleton proteins originally identified in the mammalian erythrocyte are conserved in evolution and have essential roles in development. The hts protein is a homolog of adducin that is likely to be associated with actin in ring canals of the developing oocyte (14). Defects in hts block egg development. D4.1 is a homolog of protein 4.1 (15) that is encoded by the essential coracle gene. Defects in D4.1 block dorsal closure during embryonic development and suppress the dominant eye defect in epidermal growth factor receptor mutants. Drosophila spectrin is also conserved in its structure, composition, and actin-binding activity (16). Mutations ofthe a subunit are lethal during larval development (17). Surprisingly, some aspects of development do not appear to be grossly affected by diminished levels of a-spectrin. Mutant larvae, which retain only a small quantity of maternally derived a-spectrin, are initially able to crawl, feed, and respond to tactile stimuli. They appear normal in overall form and development of major larval structures, but they become lethargic and die before the first larval molt. Close inspection of the larval gut reveals a significant effect of a-spectrin mutations on the shape and adhesion ofone class of epithelial cells (17). We have initiated studies of ankyrin in Drosophila to further dissect the cellular roles of the membrane skeleton. An ankyrin gene was identified by amplification of genomic DNA with oligonucleotide primers based on a conserved region of mammalian brain ankyrin.t We used a collection of antibodies against this ankyrin homolog and the spectrin subunits of Drosophila to examine the fate of the membrane skeleton in the absence of a-spectrin synthesis. MATERIALS AND METHODS Amplification of Ankyrin DNA. Genomic DNA from adult Drosophila melanogaster was used in PCRs with synthetic primers based on the human brain ankyrin 2 sequence (18): AnkAl, CCA CTT TAT CAT CAG TCA TGC; and AnkS1, AAC GTG TCT GCC AGG TTC TGG. Reactions were carried out in a MiniCycler (MJ Research, Cambridge, MA) for 40 cycles (940C for 1 min, 450C for 2 min, 720C for 1 min) with Taq DNA polymerase (Perkin-Elmer/Cetus). Isolation of Ankyrin cDNA. A 0to 4-hr Drosophila melanogaster embryocDNA library (19) was screened with the fly ankyrin PCR product by standard methods (20). The probe was amplified from cloned plasmid DNA and hybridized at high stringency under conditions described by O'Neill and Belote (21). Additional cDNAs were recovered from a sizefractionatedlarvallibrary(generouslyprovidedbyCarlThummel, University of Utah), using the A fragment of clone A411 as probe (Fig. 1). *To whom reprint requests should be addressed. tThe sequence reported in this paper has been deposited in the GenBank data base (accession no. L35601). 10285 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 10286 Cell Biology: Dubreuil and Yu All A411 A631 Flank A631 B E X SB E E pNB-40 [ I 1 : II vectorr I I