The Drosophila Clathrin Heavy Chain Gene: Clathrin ... · Drosophila Clathrin Heavy Chain Gene 1121...

Copyright 0 1993 by the Genetics Society of America

The Drosophila Clathrin Heavy Chain Gene: Clathrin Function Is Essential in a Multicellular Organism

Christopher Bazinet,*9t" Alisa L. Katzen,* Margaret Morgan,O Anthony P. MahowaldO and Sandra K. Lemmont

Departments *Genetics and ?Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106, George Williams Hooper Foundation, University of Calijiornia, San Francisco, Calijiornia 94143, and $Department of

Molecular Genetics and Cell Biology, The University of Chicago, Chicago, Illinois 60637 Manuscript received October 22, 1992 Accepted for publication April 5, 1993

ABSTRACT The clathrin heavy chain (HC) is the major structural polypeptide of the cytoplasmic surface lattice

of clathrin-coated pits and vesicles. As a genetic approach to understanding the role of clathrin in cellular morphogenesis and developmental signal transduction, a clathrin heavy chain (Chc) gene of Drosophila melanogaster has been identified by a combination of molecular and classical genetic approaches. Using degenerate primers based on mammalian and yeast clathrin HC sequences, a small fragment of the HC gene was amplified from genomic Drosophila DNA by the polymerase chain reaction. Genomic and cDNA clones from phage libraries were isolated and analyzed using this fragment as a probe. The amino acid sequence of the Drosophila clathrin HC deduced from cDNA sequences is 80%, 57% and 49% identical, respectively, with the mammalian, Dictyostelium and yeast HCs. Hybridization in situ to larval polytene chromosomes revealed a single Chc locus at position 13F2 on the X chromosome. A 13-kb genomic Drosophila fragment including the Chc transcription unit was reintroduced into the Drosophila genome via P element-mediated germline transformation. This DNA complemented a group of EMS-induced lethal mutations mapping to the same region of the X chromosome, thus identifying the Chc complementation group. Mutant individuals homozygous or hemizygous for the Chc', ChZ or Chc" alleles developed to a late stage of embryogenesis, but failed to hatch to the first larval stage. A fourth allele, Chc4, exhibited polyphasic lethality, with a significant number of homozygous and hemizygous offspring surviving to adulthood. Germline clonal analysis of Chc mutant alleles indicated that the three tight lethal alleles were autonomous cell-lethal mutations in the female germline. In contrast, Chc' germline clones were viable at a rate comparable to wild type, giving rise to viable adult progeny. However, hemizygous Chc4 males were invariably sterile. The sterility was efficiently rescued by an autosomal copy of the wild-type Chc gene reintroduced on a P element. These findings suggest a specialized role for clathrin in spermatogenesis.

I N the 30 years since ROTH and PORTER (1964) observed the uptake of yolk protein by developing

mosquito oocytes via coated pits and vesicles, the molecular machinery mediating endocytosis and other intracellular transport processes of cells has come under intense scrutiny [for reviews, see GOLDSTEIN et al. (1 985), BRODSKY (1 988), and PEARSE and ROBIN- SON (1 990)]. T h e roles of clathrin in the internalization, sorting, processing and secretion of molecules critical for proper development, and the differentiation of the transport machinery itself in specialized cell types, constitutes a rich area of overlap between cell and developmental biology.

Clathrin is the major component of the polyhedral protein lattice found on the cytoplasmic surfaces of clathrin-coated pits and vesicles. T h e basic clathrin subunit is a trimer of three clathrin heavy chains (HCs) (M, -180,000) joined at or near their carboxy

sity, Jamaica, New York 11439.

Genetics 134 1 1 19-1 134 (August, 1993)

' Present address: Department of Biological Sciences, St. John's Univer-

termini. Bound to each HC is a clathrin light chain of M , -23,000-27,000. Other major polypeptide species, known as assembly proteins or adaptors, are associated with clathrin coats, where hey are believed to modulate the assembly behavior of clathrin and/or provide a link between the clathrin lattice and the cytoplasmic domains of membrane proteins targeted to coated pits and vesicles (KEEN 1990; PEARSE and ROBINSON 1990; SCHMID 1992).

Our knowledge of the specific roles of clathrin in animal cells is derived largely from physiological and morphological studies. Clathrin is found in coated pits and vesicles at the plasma membrane, where its role in endocytosis is well documented, although a pathway for non-clathrin-mediated endocytosis is known to exist (SANDVIG and VAN DEURS 199 1). Clathrin is also localized to the trans-Golgi network, where, although not involved in constitutive secretion, it is thought to participate in the formation of regulated secretory granules, such as in endocrine and exocrine cells, and

1120 C. Bazinet et al.

in the transport of newly synthesized lysosomal hydro- lases from the Golgi to the lysosome via mannose-6- phosphate receptors (TOOZE and TOOZE 1986; BUR- GESS and KELLY 1986; PFEFFER and ROTHMAN 1987; GRIFFITHS and SIMONS 1986). However, experiments designed to directly inhibit or remove clathrin function in animal cells for analysis of its roles in these processes have been limited. For example, introduction of anti-clathrin antibodies into mammalian tissue culture cells resulted in a partial, but not complete, reduction of transferrin, Semliki Forest virus and fluid-phase endocytosis, although as predicted, the antibodies had no effect on the constitutive transport of newly synthesized influenza hemaglutinin to the cell surface (DOXSEY et al. 1987; CHIN et al. 1989).

In recent years genetic approaches to the study of clathrin function have become available. Elimination of yeast HC function by gene disruption resulted in a -seral fold reduction in growth rate, or lethality, depending on the genetic background in which the gene disruption experiments were performed (PAYNE and SCHEKMAN 1985; LEMMON and JONES, 1987; MUNN et al. 199 1). Yeast cells surviving without clathrin HCs have abnormal morphology, aberrant nuclear division, reduced mating efficiencies, genetic instability, and are sporulation-defective (LEMMON and JONES 1987; PAYNE et al. 1987; PAYNE and SCHEKMAN 1989; LEMMON et al. 1990, 1991). Although the transport of constitutively secreted proteins appears normal in these cells (PAYNE and SCHEKMAN 1985), the Kex2 protease and dipeptidyl aminopeptidase A, normally found in the trans Golgi, are mislocalized to the plasma membrane (PAYNE and SCHEKMAN 1989; SEE- GER and PAYNE 1992a). In addition, a temperature sensitive clathrin HC mutant displays a severe, but transient, effect on sorting of soluble vacuolar proteins to the vacuole (SEEGER and PAYNE 199213). These results indicate that clathrin plays a role in sorting and localization of proteins in the trans Golgi.

In the slime mold Dictyostelium discoideum recent experiments utilizing antisense RNA technology have shown that cells containing no detectable clathrin HC are capable of vegetative growth at a rate half that of the control Chc+ strain (O’HALLORAN and ANDERSON 1992b). These cells lack coated pits, coated vesicles and contractile vacuoles. They exhibit a marked reduction in the endocytosis of fluid-phase markers, defects in osmoregulation, and are unable to progress through the starvation-induced developmental cycle. The surprising observations that “simple” unicellular eukaryotes are, at least under certain laboratory conditions, viable in the absence of the clathrin HC raises the question of how essential clathrin function is in more complex organisms.

Genetic analysis of clathrin structure and function in a metazoan should advance our knowledge of cel-

lular transport processes in at least two key areas. First, the molecular analysis of mutations affecting many developmental pathways has identified a rapidly growing family of membrane proteins involved in developmental signaling (for recent reviews see GREENWALD and RUBIN 1992; JESSELL and MELTON 1992). Examples include the lin-12 and let-23 gene products involved in Caenorhabditis elegans vulval development (GREENWALD 1985; AROIAN and STERN- BERG 1991); the Drosophila faint little ball, Notch and Delta gene products, members of the EGF receptor family, involved in oogenesis and neural-epidermal developmental decisions (WHARTON et al. 1985; PRICE, CLIFFORD and SHUPBACK 1989; KOPCZYNSKI et al. 1988); the FGF receptor in vertebrate mesoderm formation (AMAYA, MUSCI and KIRSCHNER 199 1); and the c-Kit proto-oncogene product of the mouse W locus (CHABOT et al. 1988; GEISSLER, RYAN and HOUSMAN 1988; NOCKA et al. 1989). By analogy to the cellular processing of the EGF receptor-ligand complex (e.g., see FELDER et al. 1990), we expect that many of these receptors and their ligands are internalized via clathrin-coated pits. This is highlighted by the observations that the sevenless-bride of sevenless complex resides in large multivesicular bodies after internalization in the cells of the developing Drosophila eye (KRAMER, CA- CAN and ZIPUSKY 1991; CAGAN et al. 1992). In addition, the induction of a neurogenic phenotype by carefully timed heat shocks of embryos mutant for the Drosophila dynamin gene, shibire (POODRY 1990), indicates that developmental pathways can be affected by perturbations of endocytosis. Regulation of the functions of clathrin and its various accessory proteins, including variations in the intracellular routing or turnover of receptors and receptor-ligand complexes, thus may provide a means for modulation of many developmental signal transduction pathways.

Second, the genetics of clathrin in a metazoan should also yield new insights into the differentiated specializations of the endocytic and secretory machi- neries themselves. For example, neuron-specific alternate splicing of the mRNA’s derived from the two vertebrate clathrin light chain (LC, and LCt,) genes results in the insertion of polypeptide sequences unique to neuronal light chains (JACKSON et al. 1987; KIRCHHAUSEN et al. 198713; WONG et al. 1990). Pre- sumably, these sequences modify light chain structure to accommodate some specialized transport function of neurons such as axonal transport, neurotransmitter secretion, the recycling of synaptic vesicle membranes, or as suggested recently, the remodeling of neuronal membranes associated with learning-related synaptic reorganization (BAILEY et al. 1992). Other notable examples of cell-type specific differentiation of clathrin-associated transport machinery include: changes in relative stoichiometries of the two clathrin light

Drosophila Clathrin Heavy Chain Gene 1121

chains in cells exhibiting regulated secretion (ACTON and BRODSKY 1990); differential expression of the 100-kD a-adaptin types (a and c) in certain tissues (ROBINSON 1989); the requirement of the yolkless gene product of Drosophila for the efficient endocytosis of yolk protein only in developing oocytes (DIMARIO and MAHOWALD 1987); and the rapid turnover of the Drosophila embryonic oolemma in multivesicular bodies (MAHOWALD, ALLIS and CAULTON 198 1).

As an approach to understanding the function of vesicular transport systems in development, we have initiated a systematic search for Drosophila genes encoding various components of the clathrin-mediated transport apparatus and for mutations in these genes. Here we report on the identification of a Drosophila clathrin heavy chain (Chc) gene and the initial characterization of lethal mutations in this gene.

MATERIALS AND METHODS

Cloning of Drosophila Che sequences: For polymerase chain reaction (PCR) amplification of Drosophila clathrin HC gene fragments, purified embryonic DNA (a gift from A. R. LOHE) was used as a template using the degenerate probes shown in Figure 1. Conditions were exactly as described in the homology-probing PCR method of GOULD, SUBRAMANI and SCHEFFLER (1 989). Reactions employed 300 ng of template DNA and 10 ng each of the degenerate primers per 100-rl reaction. After initial incubation for 5 min at 94", 2.5 units Taq polymerase (Perkin-Elmer Cetus) were added to each reaction tube, and mixtures were put through 30 cycles with the following parameters: 2 min at 94", 2 min annealing at 50" and 3 min at 72". Reaction products were separated on a 3% NuSieve/l% Seakem (FMC Bioproducts) agarose gel. Bands were cut out of low melting agarose gels, melted with 5 volumes of TE buffer (10 mM Tris-HC1, 1 mM EDTA, pH 8.0), phenol extracted and ethanol precipitated, digested with Sac1 and XhoI and cloned into SacI/XhoI-digested pBluescript KS+ (Strata- gene).

Chc cDNAs were obtained by screening a Drosophila head cDNA library (ITOH et al. 1986) using the 350-bp fragment cloned by PCR. For restriction analysis, cDNA inserts were amplified by PCR using oligonucleotide primers (24 mers, New England Biolabs) flanking the unique EcoRI cloning site of Xgtl 1 (HERRMAN et al. 1990). Using the same cloned 350-bp PCR fragment as a probe, genomic Drosophila clones carrying Chc sequences were isolated from a genomic library of Oregon-R DNA cloned into the XEMBL 4 vector (from V. PIRROTTA via P. HARTE).

DNA sequencing: DNA sequencing was by the method of SANGER, NICKLEN and COULSON (1 977), using the genetically modified version of T7 DNA polymerase (Sequenase 2.0, U.S. Biochemical Corp.). The cDNA was subcloned into smaller fragments from which nested deletion sets were generated using the ExoIII-S 1 nuclease method (HENIKOFF 1987). Synthetic primers were used to cross gaps remaining after sequencing of the nested deletion sets. Both strands of a single large (apparently full-length) cDNA (clone 6) were sequenced completely, making sure that restriction sites used for subcloning were bridged to generate sequence overlaps that extended across the entire clone. Sequences were compiled using the MacVector sequence analysis pro- gram (IBI) and aligned using the GAP and PRETTY pro-

SENSE PRIMERS:

PRIMER 1 DEGENERACY = 16,384

760

Y D P E R V K N F L 5 a . . . CCTCGAGTATGATCCTGAACGAGTTAAMATTITTT . .3

.~

(XhoI) C C C GA C C G C CC A T A G G G

PRIMER 2 DEGENERACY = 1 2 . 2 8 8

782

I V C D R F D F V H 5 ' . . . CCTCGAGATTGTTTGTGATCGATTCGATTTCGTACA..3'

iXho I1 A A C CA C T C T C c c G G

G T T

ANTISENSE PRIMERS:


8 5 4

3' . . CTCCAACTCTTCGCATTAGCRAAATTCGAAC . . . 5 ' E V E K R N R L K

T C T TT C GT CG C (Hind1111 G G G G T T T T


884

N A L A K I Y I 3' . . TTACGAAAACGATTCTAAATATAWWGAGC . . . 5

G CG C C T G G G ( S K I ) G G G T A T T T

FIGURE 1 .-PCR primers used in amplification of Drosophila HC gene sequences. A cluster of primary sequences identical in the yeast and rat clathrin HC polypeptides were chosen for primer design on the basis of their close proximity to each other, thereby minimizing the distance that would have to be traversed by the polymerase and the probability that an intron would lie between any two primer sites selected. Degeneracies of each primer pool are indicated. The locations of these sites within the complete Drosoph- ila HC sequence are indicated in Figure 4. Above the first amino acid residue of each sequence is the corresponding codon number from the yeast HC sequence (LEMMON et al. 1991).

grams from the University of Wisconsin Genetics Computer Group Sequence Analysis Software Package.

Construction of P[w+;Chc] transposons: For construction of P[w+;Chc] transposons, DNA fragments carrying clathrin HC gene sequences were subcloned from genomic X clones into the pCasPer4 vector of PIRROTTA (1988) by a two-step procedure. First, 7.0- and 2.0-kb EcoRI/XbaI fragments from bacteriophage X genomic clones XCHCl and XCHC2 (see Figure 6), respectively, were subcloned into XbaI/EcoRI-digested vector. The pCasPer4 clone carrying the 7-kb EcoRI-XbaI fragment is designated pCHC2 (Figure 6). In the second step, the 6.0-kb XbaI fragment from XCHC2 was introduced into each of the products of the first step cloning reopened at the XbaI site to reconstitute the 3' end of the gene and generate P element constructs, pCHC9 and pCHC3 (Figure 6). Plasmids were purified by cesium chloride equilibrium density centrifugation for use in germline transformation experiments.

All other nucleic acid biochemistry and cloning proce- dures were as described in AUSABEL et al. (1 988) and SAM- BROOK, FRITSCH and MANIATIS (1 989).

General Drosophila methodology: Drosophila melanogaster was propagated by standard techniques using standard cornmeal, agar and molasses medium. Ethyl methanesulfon-


ate (EMS) mutagenesis of Drosophila and the subsequent screen for lethal mutations in the region around the Chc gene have been described by CAMPBELL et al. (1 99 1). The wild-type (parental) X chromosome in this screen carried a mutation at the white locus; for brevity, we refer to it throughout as w(K), as in CAMPBELL et al. (1 99 1). Chc', Chc', Chc) and Chc' are EMS-induced mutant alleles derived from this screen and are named according to the nomenclature of LINDSLEY and ZIMM (1 992). They have also been referred to as EM13, EM15, EM41 and EM9, respectively, and belong to complementation group VI as will be described by A. L. KATZEN, T. KORNBERC and M. BISHOP (in preparation). Other Drosophila mutations are as described in LINDSLEY and ZIMM (1 992).

Homozygous germline clones were induced essentially as described by PERRIMON, ENCSTROM and MAHOWALD (1 984), using the dominant female-sterile mutation ovo"'. ChclFM7c females were mated to ovoDJ males and late first instar larvae were y-ray irradiated (1400 rad) to induce mitotic recombination. The Chc/ovoDJ offspring were mated to Oregon-R males and screened for Chc/Chc clones by their ability to lay eggs, and subsequently by examining ovaries, dissected from Chc/ovoDJ females in Ringers solution, for ovo+ ovarioles under a binocular dissecting microscope.

Generation of P[w+; Chc] transformants and complementation of Chc mutants: For transformation, DNA was injected into freshly laid eggs from a y w strain, using the helper plasmid phSr (STELLER and PIRROTTA 1986) as a transient source of transposase. Eggs were mechanically dechorionated by gently rolling them on a piece of double- stick tape (3M) and injected as described by SPRADLINC (1 986). Five independent autosomal inserts of the pCHC9 construct were isolated. A single X-linked insert of the pCHC3 construct was isolated. For complementation analysis, the X-linked P element was mobilized by introduction of the stable A2-3 transposase source (ROBERTSON et al. 1988) into males carrying the X-linked element and new autosomal insertions were detected by screening for transmission of the w+ marker from these dysgenic males to their male offspring.

For complementation of candidate clathrin HC mutations by autosomal P[w+;Chc] insertions, heterozygous X-lethal/ FM7c virgin females were mated to males of the genotype y w/Y;P[w+;Chc]/+. The X-lethal mutations examined be- longed to three uncharacterized lethal complementation groups (V, VI and VIII) mapping to the Chc region (A. L. KATZEN, T. KORNBERC and M. BISHOP, in preparation). The presence of males carrying the mutant chromosome among the progeny of this cross in numbers significantly greater than observed in control crosses, indicates complementation of the lethal X-linked mutation by the autosomal P element insertion.

Cytological analysis: Hybridization in situ to third instar larval polytene chromosomes was as described by PARDUE (1 986) using genomic clone XCHC 1 as a probe. Embryonic cuticles were prepared in Hoyer's mountant, as described by WIFSCHAUS and NUSSLEIN-VOLHARD (1 986), and examined by phase-contrast microscopy.

RESULTS

Identification of a Drosophila clathrin HC (Chc) gene: We inspected the alignment of the yeast and rat HC amino acid sequences (LEMMON et al. 199 1 ; KIRCHHAUSEN et al. 1987a) for blocks of primary sequence highly conserved across this wide phyloge- netic gap, reasoning that some of these identities

probe

5 ' H 3 '

8B 1 I , I

6 t I , I 4

9A I I I ,

8A - 1 8 - 15 I I

I I I

H 1 kb

FIGURE 2.--EcoRI maps of representative Drosophila HC cDNA clones isolated from the Drosophila head cDNA library of ITOH et al. (1986). The location of the 350-bp probe, as determined initially by hybridization of restriction digests of cDNA clones and subsequently by sequencing, is shown above the maps.

would probably be conserved in any Drosophila HC gene. To maximize our chances of cloning the Dro- sophila gene by PCR, we chose a cluster of four conserved protein sequence motifs near the middle of the rat and yeast HCs (indicated in Figure 4) and designed degenerate oligonucleotide primers as shown in Figure 1. Reactions with primer pools 2 and 3 or 2 and 4 yielded single products in the expected size range (not shown). T h e -350-bp fragment produced from using primer pools 2 and 4 was cut out of the gel, subcloned and sequenced. Except for a few differences noted in the degenerate primer portion of these molecules, the sequences of 10 independent clones analyzed were identical and translated into a polypeptide sequence that was more than 90% identical with the corresponding section of the rat clathrin HC. This indicates that all of the clones obtained by this strategy were amplification products of a single Drosophila Chc gene, and that there are no introns in this region of the gene. In addition, hybridization with a "P-labeled preparation of the gel-purified PCR fragment to RNA blots from adult flies revealed the presence of a single transcript approximately 5.5 kb in length (data not shown). This is comparable in size to the yeast and mammalian HC transcripts (PAYNE and SCHEKMAN 1985; LEMMON and JONES 1987; KIRCHHAUSEN et al. 1987a) and is sufficient to encode a polypeptide of the expected size, 190 kD.

Roughly two dozen plaque-purified cDNA clones were initially characterized by PCR and restriction analysis. Crude restriction maps of all of the cDNAs were congruent, indicating no major splicing variants and confirming the source of the transcripts as a single gene (Figure 2). T h e 3' ends of most of the cDNAs were found to lack authentic poly(A) tails due to


incorrect priming at an internal A-rich site a few hundred base pairs upstream of the true 3’ terminus of the transcript (see Figure 3). The two largest cDNAs, both approximately 5.5 kb in length, were subcloned into plasmids for more detailed restriction analysis and nucleotide sequencing. In addition to a difference at their 3’ ends attributable to the misprim- ing at the A-rich internal site, they differed from each other by the presence in cDNA clone 8B of a small (-38 bp) stretch of sequence in the 5”untranslated region that was not present in clone 6. The biological significance of this apparent splicing polymorphism remains to be determined.

Sequence of the Drosophila clathrin H C The nucleotide sequence of cDNA clone 6, with the corresponding translated polypeptide sequence, is shown in Figure 3. The cDNA encodes a protein of 1678 amino acid residues, with a predicted M , of 19 1,163. A comparison to the rat and yeast HCs is shown in Figure 4. As expected from the PCR fragment originally sequenced, the Drosophila HC is approximately 80% identical at the amino acid sequence level to the rat protein (KIRCHHAUSEN et al. 1987a). When con- servative substitutions are allowed, the similarity rises to over 90%. Drosophila and yeast sequences (LEM- MON et al. 1991) show 49% identity overall, roughly equivalent to the degree of similarity observed between the yeast and mammalian proteins. The Dic- tyostelium HC (O’HALLORAN and ANDERSON 1992a) is 57% identical to the fly HC (not shown).

The longest stretch of amino acid sequence identity shared by the Drosophila, yeast, and rat HCs lies between amino acid residues 1308-1327 in the Dro- sophila sequence (Figure 4). The next 50 amino acid residues are also highly conserved, even more so than the region between residues 1460-1587 (of the mammalian HC) recently implicated by NATHKE et al. (1 992) in light chain binding and trimerization. The greatest divergence between the HC sequences is seen at the carboxy-terminal ends. In the triskelion, the carboxy termini of the HCs are believed to lie at or close to the hub (NATHKE et al. 1992) and are therefore expected to be positioned near the vertices of the assembled clathrin lattice. Although it was originally proposed that the carboxy terminus is important for trimerization of HCs (KIRCHHAUSEN et al. 1987a), truncation of the carboxy terminus of the yeast HC does not block triskelion formation (LEMMON et al.

Mapping of the Chc gene: Genomic DNA clones were isolated from phage libraries using the 350-bp cloned PCR fragment. In order to begin a genetic analysis of the clathrin HC gene in Drosophila, the gene was mapped by hybridization in situ to polytene chromosomes from larval salivary glands. A single site of hybridization was consistently detected at position

199 1).

13F2 of the X chromosome according to the cytological nomenclature of BRIDGES (1 938) (Figure 5).

Comparison of the restriction map of genomic clones (Figure 6) with the restriction map of the region around the scalloped (sd) gene, known from classical and molecular genetics to map to the same location of the X chromosome (CAMPBELL et al. 199 l), identified an overlap in the maps. This was confirmed by cross-hybridization of clones obtained in the sd walk with genomic Chc clones (data not shown). This estab- lishes the order of known genes in the area as (distal)- sd-Chc-myb-shi-(proximal) in agreement with the data of VAN DER BLIEK and MEYEROWITZ (1 99 1).

The approximate limits of the Chc transcript within the genomic map, as inferred from cDNA sequences, were mapped by hybridization of synthetic oligonucleotides (indicated in Figure 3) to Southern blots of EcoRI, XbaI and EcoRI-XbaI doubly digested genomic clones. This placed the two presumptive ends of the Chc transcript within 10 kb of each other on the genomic map (Figure 6). The 3’ end of the cDNA was placed between the distal XbaI site on the map and the end of X clone CHCl, based on its hybridization to the 6-kb XbaI fragment from X clone CHCS, but not to any fragment from XCHCl under the same conditions. The 5’ end of the cDNA was placed between the more proximal XbaI site and the proximal end of XCHC2, based on its hybridization to the 9-kb EcoRI fragment from XCHCl and the 6-kb EcoRI fragment from XCHC2, but not to the 6-kb XbaI fragment from XCHC2.

Identification of clathrin HC mutations: In a screen for EMS-induced lethal mutations in the nearby myb gene (CAMPBELL et al. 1991; A. L. KATZEN, T. KORNBERG and M. BISHOP, in preparation), many of the lethally mutable complementation groups in the 13F-14A region of the X chromosome had already been identified. To test whether any of these groups corresponded to mutations in the Chc gene, we reintroduced the cloned wild-type clathrin HC gene into the genome via P element-mediated germline transformation (RUBIN and SPRADLING 1982). Two different constructs in pCasPer4 (PIR- ROTTA 1988), identical at their 3’ ends and differing only in the amount of 5”upstream genomic DNA (Figure 6), were injected into mechanically dechorionated Drosophila embryos from a yellow-white (y w ) stock, and transformants (w’ individuals) were identified among the progeny of the resulting flies back- crossed to the same y w stock.

To test for complementation of a given lethal mutation by the P element-borne clathrin HC gene, males carrying various autosomal P[w’;ChcJ inserts were mated to virgin females heterozygous for the X-linked lethal mutation and the male progeny of this cross were scored for the presence of X-linked markers.

1124 C . Bazinet et al.

CGTTAGAGCTTGATAAGAGCTCAGTTTGTCTTCAGTGTGTGTGCCCGTCATC~AGCAACAAACCGACGTGATTATTTC~ACTAAACGTTCCAATGGAAAC 100 V

CGM'I'CTTAAACCAGAAACCGACTAAACTCGAACACAATTAAACAACAAT~TCCTCGAAACAAATCAAATACAAACTGTTACCAAGTGTGCAATTCAA 200

GAAAACATCGTATTGGCATTGTTTTCGCAAAGATACATTTACGTTGTAAAGGTCTGTCAATAACTAGACCTCACTGTAGTAGTAAA~~ACGCAACCAC 300 M T Q P 4

TGCCCATCCGCTTTCAGGAGCATTTACAGCTCACAAATGTCGGGATCAACGCCAATTCATTTTCATTCAGCACGCTCACGATGGAATCGGATAAGTTTAT 400 L P I R F Q E H L Q L T N V G I N A N S F S F S T L T M E S D K F I 38

TTGCGTGCGGGAGAAGGTGATGATACCGCCCAAGTGGTCATCATTGACATGAACGATGCCACCAATCCCACGCGGCGTCCCATCTCAGCGGATTCGGCT 500 C V R E K V N D T A Q V V I I D M N D A T N P T R R P I S A D S A 71

ATCATGAATCCAGCTAGCAAGGTCATTGCGCTCAAAGCGCAAAAGACGTTGCAGATCTTCAACATTGAGATGAAGTCGAAGATGAAGGCGCATACCATGA 600 I M N P A S K V I A L K A Q K T L Q I F N I E M K S K M K A H T M 104

ACGAGGATGTGGTGTTCTGGAAGTGGATCTCCCTTAATACGCTAGCTCTGGTCACAGAAACGAGTGTGTTCCATTGGTCCATGGAGGGCGATTCGATGCC 700 N E D V V F W K W I S L N T L A L V T E T S V F H W S M E G D S M P 138

GCAAAAGATGTTTGACCGGCATTCATCGCTGAACGGTTGTCAAATCATCAACTACCGCTGCAATGCCTCCCAGCAGTGGCTACTCTTGGTCGGCATTTCG 800 Q K M F D R H S S L N G C Q I I N Y R C N A S Q Q W L L L V G I S 171

GCACTGCCAAGTCGCGTTGCCGGTGCCATGCAGTTGTATTCGGTGGAGCGTAAGGTGTCCCAGGCGATCGAGGGGCATGCGGCCAGTTTTGCCACGTTTA 900 A L P S R V A G A M Q L Y S V E R K V S Q A I E G H A A S F A T F 204

AAATCGATGCCAACAAGGAGCCGACCACGCTGTTCTGCTTTGCAGTTCGTACGGCCACTGGGGGCAAGCTTCATATTATCGAAGTT~;GTGCTCCGCCGAA 1000 K I D A N K E P T T L F C F A V R T A T G G K L H I I E V G A P P N 238

CGGCAATCAGCCGTTTGCCAAGAAGGCTGTCGATGTCTTCTTTCCGCCGGAAGCACAAAATGATTTCCCTGTGGCTATGCAAGTCTCTGCCAAGTACGAC 1100 G N Q P F A K K A V D V F F P P E A Q N D F P V A M Q V S A K Y D 271

ACCATCTACTTGATAACCAAGTATGGATACATACATCTGTATGACATGGAGACGGCCACGTGCATATACATGAATCGTATATCGGCCGATACGATCTTTG 1200 T I Y L I T K Y G Y I H L Y D M E T A T C I Y M N R I S A D T I F 304

TTACCGCACCGCATGAGGCAAGTGGCGGCATCATTGGCGTCAATCGCAAGGGACAGGTCCTCTCCGTGACCGTCGACGAGGAGCAGATCATTCCCTACAT 1300 V T A P H E A S G G I I G V N R K G Q V L S V T V D E E Q I I P Y I 338

CAACACCGTTCTGCAGAATCCCGATTTGGCCCTTCGCATGGCCGTGCGCAACAATTTGGCTGGTGCCGAAGATCTCTTTGTGCGAAAGTTCAACAAGCTC 1400 N T V L Q N P D L A L R M A V R N N L A G A E D L F V R K F N K L 371

TTTACAGCCGGCCAGTATGCTGAAGCGGCTAAAGTTGCTGCCCTGGCACCCAAGGCCATTCTGCGTACGCCACAGACGATCCAGCGTTTCCAACAGGTGC 1500 F T A G Q Y A E A A K V A A L A P K A I L R T P Q T I Q R F Q Q V 404

AGACACCAGCTGGCTCCACGACTCCGCCGCTGCTGCAATACTTTGGCATTCTCCTCGACCAGGGCAAGCTGAACAAGTTCGAGTCTCTCGAGCTGTGCCG 1600 Q T P A G S T T P P L L Q Y F G I L L D Q G K L N K F E S L E L C R 438

TCCCGTCTTGCTGCAGGGCAAGAAGCAGCTGTGCGAGAAGTGGCTGAAGGAGGAGAAGTTGGAATGCAGCGAGGAGTTGGGTGATCTGGTCAAGGCCTCC 1700 P V L L Q G K K Q L C E K W L K E E K L E C S E E L G D L V K A S 471

GATCTTACACTTGCCCTGTCCATCTATCTGCGCGCAAATGTGCCCAACAAGGTTATCCAATGCTTTGCTGAGACTGGGCAGTTCCAGAAGATTGTACTCT 1800 D L T L A L S I Y L R A N V P N K V I Q C F A E T G Q F Q K I V L 504

ACGCCAAGAAGGTCAACTATACGCCCGATTACGTGTTCCTGCTGCGCTCCGTGATGCGAAGCAACCCGGAGCAAGGAGCTGGTTTCGCCTCTATGTTGGT 1900 Y A K K V N Y T P D Y V F L L R S V M R S N P E Q G A G F A S M L V 538

GGCCGAGGAAGAGCCACTGGCGGACATCAATCAGATTGTGGACATCTTCATGGAGCACTCCATGGTGCAGCAGTGCACTGCATTCCTGCTGGACGCCCTC 2000 A E E E P L A D I N Q I V D I F M E H S M V Q Q C T A F L L D A L 571

AAGCATAACCGTCCCGCCGAGGGTGCCCTCCAGACGCGCCTGCTGGAAATGAATCTGATGTCTGCTCCGCAGGTGGCCGACGCCATCCTGGGCAATGCTA 2100 K H N R P A E G A L Q T R L L E M N L M S A P Q V A D A I L G N A 604

M F T H Y D R A H I A Q L C E K A G L L Q R A L E H Y T D L Y D I K 638 TGTTCACCCACTACGATCGGGCCCACATTGCCCAGCTGTGCGAGAAGGCTGGACTGCTCCAGCGCGCCCTCGAGCACTACACGGATCTGTATGACATTAA 2200

GCGGGCCGTTGTGCACACGCACATGCTGAATGCCGAATGGCTGGTCAGTTTCTTTGGCACGCTGTCGGTGGAGGACTCGCTGGAATGTCTGAAGGCAATG 2300 R A V V H T H M L N A E W L V S F F G T L S V E D S L E C L K A M 671

CTTACGGCGAATTTGCGCCAGAACTTGCAGATCTGTGTGCAGATTGCCACCAAGTACCACGAACAGCTGACCAACAAGGCACTGATTGACCTGTTCGAAG 2400 L T A N L R Q N L Q I C V Q I A T K Y H E Q L T N K A L I D L F E 704

GTTTCAAGAGCTACGACGGACTGTTCTACTTCCTGAGCAGCATTGTCAACTTCTCACAGGATCCCGAAGTGCACTTCAAATACATTCAGGCGGCATGCAA 2500 G F K S Y D G L F Y F L S S I V N F S Q D P E V H F K Y I Q A A C K 738

GACTAATCAGATTAAGGACGTGGAGCGAATTTGCCGTGAATCAAACTGCTACAATCCCGAACGGGTGAAGAACTTCTTGAAGGAGGCCAAGCTGACGGAT 2600 T N Q I K E V E R I C R E S N C Y N P E R V K N F L K E A K L T D 771

CAGCTACCATTAATTATTGTTTGTGATCGTTTTGATTTCGTGCACGACTTGGTGCTTTACCTGTATCGTAACAATCTGCAGAAGTACATTGAGATCTATG 2700 Q L P L I I V C D R F D F V H D L V L Y L Y R N N L Q K Y I E I Y 804

V Q K V N P S R L P V V V G G L L D V D C S E D I I K N L I L V V K 838 TGCAGAAAGTGAATCCATCCCGCTTGCCAGTGGTAGTGGGTGGTCTTCTTGATGTTGATTGCAGTGAGGATATAATTAATCTAATTCTCGTGGTCAA 2800

GGGACAATTCTCAACCGACGAACTGGTCGAGGAGGTCGAGAAGCGCAACCGTCTCAAGCTTCTCCTTCCCTGGCTGGAGTCCCGAGTTCACGAGGGCTGC 2900 G Q F S T D E L V E E V E K R N R L K L L L P W L E S R V H E G C 871

GTCGAGCCAGCCACCCACAACGCGTTGGCCAAGATCTACATTGACTCGAACAACAATCCCGAGAGATATCTTAAGGAGAATCAGTACTACGATAGCCGTG 3000 V E P A T H N A L A K I Y I D S N N N P E R Y L K E N Q Y Y D S R 904

V V G R Y C E K R D P H L A C V A Y E R G L C D R E L I A V C N E N 938 TGGTCGGTCGCTACTGCGAGAAGCGGGATCCCCATTTGGCGTGTGTCGCCTACGAGCGTGGATTGTGCGATCGCGAGCTGATCGCCGTTTGTAACGAGAA 3100

Drosophila Clathrin Heavy Chain Gene

TTCTCTGTTCAAGAGCGAAGCACGCTACTTGGTTGGTCGCCGCGACGCCGAACTCTGGGCCGAGGTCCTTTCGGAGAGCAATCCATACAACCGCCAGTTG S L F K S E A R Y L V G R R D A E L W A E V L S E S N P Y K R Q L

ATCGATCAGGTGGTACAGACCGCTTTtiTCCGAGACCCAGGATCCCGATGACATCTCGGTAACGGTCAAAGCATTCATGACCGCCGATTTGCCCAATGAGC 1 D Q V V Q T A L S E T Q D P D D I S V T V K A F M T A D . L P N E

TGATCGAACTTCTCGAGAAGATTATTCTGGACTCGTCCGTCTTTAGCGACCATCGC~TCTGCAAAACTTGCTCATTCTCACAGCCATCAAGGCTGATCG L I E L L E K I I L D S S V F S D H R N L Q N L L I L T A I K A D R

CACCCGAGTCATGGACTACATTACCGGCTGGAGAACTACGATGCACCGGACATCGCGAACATTGCGATCAGTAATCAGTTGTACGAAGAAGCCTTCGCC T R V M D Y I N R L E N Y D A P D I A N I A I S N Q L Y E E A F A

ATCTTCAAGRAGTTCGATGTGAACACATCGGCCATTCAGGTGCTCATCGATC~GTGAACAACCTGGAGCGGGCTAACGAGTTCGCCGAGCGGTGC~TG I F K K F D V N T S A I Q V L I D Q V N N L E R A N E F A E R C N

AGCCGGCCGTTTGGTCGCAGCTGGCCAAGGCCCAACTGCAGCAGGGTCTGGTCAAGGAGGCTATCGACTCGTACATCAAGGCTGATGATCCGAGCGCCTA E P A V W S Q L A K A Q L Q Q G L V K E A I D S Y I K A D D P S A Y

CGTCGATGTCGTCGATGTGGCCAGCAAGGTGGAGTCTTGGGATGACCTCGTTCGCTATCTGCAAATGGCACGCAAGAAGGCGCGCGAATCTTACATCGAG V D V V D V A S K V E S W D D L V R Y L Q M A R K K A R E S Y I E

AGCGAATTGATCTATGCCTATGCGCGCACTGGACGTCTGGCCGATCTGGAGGAGTTCATTTCGGGTCCCAACCATGCCGATATCCAG~GATTGGCAACC S E L I Y A Y A R T G R L A D L E E F I S G P N H A D I Q K I G N

GTTGCTTCAGCGACGGCATGTACGATGCAGCGAAGCTACTGTACAACAATGTGAGCAACTTTGCCCGTCTGGCCATCACTTTGGTCTACCTGAAGGAGTT R C F S D G M Y D A A K L L Y N N V S N F A R L A I T L V Y L K E F

CCAAGGGGCCGTGGACTCGGCGCGGAAAGCCAACTCAACGCGCACATGGAAGGAGGTGTGCTTCGCCTGCGTGGACGCCGAGGAGTTTAGGCTAGCTCAG Q G A V D S A R K A N S T R T W K E V C F A C V D A E E F R L A Q

ATGTGCGGCCTGCACATTGTGGTGCATGCCGATGAGCTAGAGGATCTGATTAACTACTATCAAAACCGTGGATACTTCGATGN\TTGATTGCGCTACTCG

AGTCGGCTTTGGGGCTGGAACGTGCGCACATGGGAATGTTCACCGAATTAGCTATACTTTATTCAAAATTCAAACCTTCCAAAATGCGCGAACACTTGGA

M C G L H I V V H A D E L E D L I N Y Y Q N R G Y F D E L I A L L

E S A L G L E R A H M G M F T E L A I L Y S K F K P S K M R E H L E

GCTGTTCTGGTCTCGCGTTACATTCCAAAAGTTCTGCGTGCCGCTGAATCGGCTCACTTGTGGTCGGAGCTGGTGTTCCTGTACGATAAGTACGAGGAG L F W S R V N I P K V L R A A E S A H L W S E L V F L Y D K Y E E

TACGATAACGCCGTCCTGGCCATGATGGCTCATCCCACGGAGGCGTGGCGCGAAGGGCACTTCAAGGACATTATCACCAAGGTAGCCAACATTGAGCTGT Y D N A V L A M M A H P T E A W R E G H F K D I I T K V A N I E L

ACTACAAGGCTATCGAATTCTATTTGG~CTTCAAGCCGCTCCTGTTGAACGACATGCTGCTCGTGCTGGCACCCAGGATGGATCACACTCGTGCTGTTAG Y Y K A I E F Y L D F K P L L L N D M L L V L A P R M D H T R A V S

TTACTTCTCCAAAACCGGCTATTTGCCXCTCGTCAAGCCTTATCTGCGTTCAGTCCAATCTCTCAATAACAAGGCAATCAACGAAGCCCTGAACGGACTC Y F S K T G Y L P L V K P Y L R S V Q S L N N K A I N E A L N G L

TTAATCGACGAGGAGGACTACCAGGGTC:GCGCAATTCGATCGATGGATTTCATAACTTTGACAACATTGCGTTGGCACAGAAACTC~AGCACGAAC L I D E E D Y Q G L R N S I D G F D N F D N I A L A Q K L E K H E

TTACCGAATTCCGTAGGATTGCCGrCTACTTGTACAAGGGAAATAATCGCTGGAAACAGAGCGTTGAGCTCTGCAAAMGGATAAACTCTACAAGGATGC L T E F R R I A A Y L Y K G N N R W K Q S V E L C K K D K L Y K D A

TATGGAGTACGCCGCCGAATCTTGCAAGCAAGATATTGCCGAAGAGTTGTTGGGTTGGTTCCTAGAACGTGACGCTTACGATTGTTTTGCAGCTTGTCTT M E Y A A E S C K Q D I A E E L L G W F L E R D A Y D C F A A C L

TATCAGTGTTACGACTTGCTGCGCCCTGATGTTATCTTGGAGTTGGCCTGGAAACACAAAATCGTTGACTTTGCCATGCCCTATTTGATTCAGGTTCTGC Y Q C Y D L L R P D V I L E L A W K H K I V D F A M P Y L I Q V L

GCGAATACACAACAAAGGTGGACAAACTGGAGTTGAACGAGGCTCAGCGCGAGAAGGAGGA~GATTCCACTGACCACAAAAACATTATTCAGATGGAGCC R E Y T T K V D K L E L N E A Q R E K E D D S T E H K N I I Q M E P

ACRACTGATGATCACCGCTGGCCCAGCAATGGGCATTCCTCCACAATATGCACAG~TTATCCACCTCGTGCAGCAACGGTAACGGCAGCAGGAGGACGC Q L M I T A G P A M G I P P Q Y A Q N Y P P G A A T V T A A G G R

AACATGGGCTATCCCTACTTGTAGGACTTGCGCCCGATAATGAGATCATCAAAACAACTTTAAAAACAAATGTATCAGCATTAAAGGAATACCAATAAAA N M G Y P Y L ’ - 8B

TAAGAAAAATAATCTATTAATTGCATGTGCGCCAAAAATTACCAAAACGAAACAACACAGT~CATAACTTTGAACAT‘:F,TTAAATTAAAGCAATGCAA

TTAACAATCCAATTTTTGGTTTGTTTGTAGACAGTTAAAAATTGATTATATGTTTTGATAATTACAGCACTCTTTATTCAAATATAACAGAATAT

-AAw 5630

3200 971

3300 1004

3400 1038

3500 1071

3 600 1104

3700 1138

3800 1171

3900 1204

4000 1238

4100 1271

4200 1304

4300 1338

4400 1371

4 500 1404

4 600 1438

4700 1471

4800 1504

4 900 1538

5000 1571

5100 1604

5200 1638

5300 1671

5400 1678

5500

5 600

1125

FIGURE 3.-Nucleotide sequence of Drosophila HC cDNA clone 6 . The sense strand is shown, with the deduced amino acid sequence displayed below in single letter code. The additional sequence near the 5’ end of cDNA clone 8B, which may represent an alternative splicing product, is the insertion of 38 bases (GTATGATAGTACTTAGCGTACATTTTTGTTAAATTGCT) between positions 80 and 81 of clone 6 (arrowhead). The locations of the 30 mers used for mapping the 5’ and 3’ ends of the transcript by hybridization to genomic restriction fragments are indicated by underlining. All cDNAs except clone 6 ended in the poly A-rich region between nucleotides 5400 and 5450. This was probably due to promiscuous oligo-dT priming during first strand synthesis of the cDNAs. Shown with an arrow over the sequence is the 3’ end of clone 8B, which ends at position 5420 of clone 6 . These sequence data are available from EMBL/GenBank/DDBJ under accession no. Z14 133.

Complementation by the cloned clathrin HC gene some), whereas the lack of complementation would be residing on an autosomal P element construct was indicated by the absence of this class among the prog- indicated in this instance by the presence among the eny of the cross. Representative lethal alleles from male progeny of w+ individuals carrying the lethal three previously unknown complementation groups mutation (on the mutant, non-balancer X chromo- mapping in the vicinity of scalloped and myb (A. L.


D . m . 1 MTQPLP1RFQEHLQLTNVG;NANSFSFSTiTMESDKFIC;REKVNDTAQWIIDMNDATNPTRRPISADSAIMNPASKV;ALKAQKT .Li IFNIEMUSKM R a t 1 -A-I-----------Q-L---PANIG-------------I----GEQ----------PS--I---------------------- S . C . 1 .MSD---E-T-LVD-MSL--SPQFLD-RST-F---H-VT---TKDG-NS-A-V-LAKGNEV--KNMGG-----H-SQM--S~-NG-IV----L-T---L

G--.---""""-

100 KAHTMNEDVkWKWISLNTiALVTETSVFiWSM.. .EGDSMPQKMFDRHSSLNGCQIIN;RCNASQQWLiLVGISALPS~VAG~QLYS~RKV~QAIE~ 1 0 0 "-" 100 - S F - L D - P - I - - R - L - E T - - G F - - A R - I L T S N V F D G N V N A - - - L Q E N G - I - - R I - - F - K Q - N I - - - - D -

T'--T"-""-- V----DNA-Y----...--E-Q-V---------A--------TD-K-K----T----QQN--V--------D-----p---

1 9 7 HAASFATFK;DAN.KEPTT~FCFAVRTAT: .GGKLHIIE;GAPPNGNQPFAKKAVDVFFPPEAQNDFPV~QVS~Y~T~YLITKYGYI~LYD~TATC~ 2 0 0 -V-I-TNILLEG-GST-VQV-VTGN-N--TGA-E-R---IDHDASLPSQYQ-ETT-I----D-T----I-V---E--GI---L----F- 1 9 7 ------Q--MEG-AE-S.--------GQA..----------T--T-----P---------------------I-E-H-WF------------L--G---

"-EL--G-NL

294 YMNRISADTIFVTAPHEASGGIIGVNRKGQVLSVTVDEEQIIPYINTVLQNPDLALRMAVRNNLAGAEDLFVRKFNKLF~KFNKLFTAGQYAE~V~LAPKAILR 294 __-___ 3 0 0 FV---T-ESV-TA--YNHEN--ACI-K-----A-EISTS--V---LNK-S-VA---IV-T-GG-P--D---QKQ-ES-LLQND-QN-----..-SSTS--

GE----------TA--------------C-E--N-----TN---------------------E--A----A--AQ-N-S-------N---G---

394 TPQTIQRFQQVQTPAGSTTPPLLQYFGILLDQGKLNKFESLELCRPVLLQGKKQLCEKWLKEEKLECSEELGDLVKASD~TLALSIYLRANVPNKVIQCF 394 --D--R---S-PAQP-,Q-S-------------Q---Y----------Q--R---L------D-------------SV-P-----V-------------- 3 9 8 NQN--N-LKNI-A-P-AIS-I--.--ST"-K---K-----E-TI--A----Q-DR---F------D----------I--PF-T---- AC----GAHA---S-L

494 AETGQFQK1;LYAKKVNYTPDYVFLLRSVhSNPEQGAG~ASMLVAEEE~LA. .DINQI;DIFMEHSMV~QCTAFLLDALKHNRPAEGAiQTRLLEMNLM 493 -----V----------G----WI----N---IS"-QQ--Q---QD----- --T----V---YNLI------------N--- 497 --LQ--E--IP-CQ--G-Q-NFLV-IS-LI--S-DRASE--VS-LQNP-TASQI--EK-A-L-FSQNHI--G-SL------GDT-DQ-H----V--V--L

.. S"p-"""""

592 SRPQVADAILGNAMFTHYDIAHIAQLCEKAGLLQRALEH;TDLYDIKRA;VHTHMLNAEWLVSFFGTLS;IEDSLECLKA~;ILTANLRQNL~ICVQIATKY~ 5 9 1 H-----------Q--------------------------F-------------- L--P----NY--S----------R---S"I--I------~--V-s--- 597 H-----------NI-S---KPT-----Y"----Y-----N---IK----C----NA-PID---GY--K-N--Q--A----LMDN-IQA-I-TV--V---F~

- A - 3 . 692 EQLTNKALIDLFEGFKSYD~L~YFLSSIVNFSQDPEVHFKYIQAACKTNQIKEVERICRESNCYNPERVKNFLKEALTDQLPLIIVCDRFDFVHDLVLY 6 9 1 ---sTQs--E---s---FE------G---------D------------G---------------D----------------------------------- 697 DLIGPST--K---DYNATE--Y-Y-A-L--LTE-KD-VY---E--A-MK-YR-I---VKDN-V-D--------- D-N-E-----V---------- EMI--

3 . 4- 792 LYRNNLQKYIEIYVQKVNPSRLPWVGGLLDVDCSEOIIKNLILVVKGQFSTDELVEEVEKRNRLKLLLPWLESRVHEGCVEPATHNAL~IYIDSNNNP 7 9 1 ----~--------------------I-----------~--------R---------A----------------A-I----E------------------- 797 --KSQNL-F--T---Q----KTAQ---A---M--D-~-QS-LQS-L--~IN--TT---------I---F--QSLSQ-IQDQ-VY------------ S-

892 ERYLKENQY;DSRWGRYCEKRDPHLACVAYERGLCDREiIAVCNENSLFKSERRYLVGRRDAELWAEVLSESNPYKRQ~IDQWQTAL~ETQDPDDIS; 8 9 1 --F-R--P--------K-----------------Q--L---N----------LS----R-K-P---GS--L-----R-P---------------- 897 -KF----DQ--TLD--H-------Y--YI---K-QN-DD--RIT----MY-YQ----LE-S-LD--NK--NQE-IHR-----S-ISVGIP-LT--EPV-L

EEV--

992 TVKAFMTADLPNELIELLE~IILDSSVFS;)HRNLQNLLI;TAIKADRTR;MDYINRLEN~DAPDIANIA;SNQLYEEAFAIFKKFDVT~AIQVLIDQVN 9 9 1 -"---"""""""- 997 --Q----NG-KL-----------EP-P-NENVA--G--L-S---YEP-K-SS--EK-D----DE--PLC-EHD-K----E-YD-HEMYGK-LK---EDIM

V--N----E---------------------E- ---D--------------E-F-------R-------- V----EHIG

1092 N L E R A N E F A E R C N E P A V W S Q L A K A Q L Q Q G ~ V K E A I D S Y I K G R L A D L E E F ~ 1 0 9 1 --D--Y---------------------K-M---------------S-~--QA-NTSGN-EE--K------------- 1097 S-D--ASY-DKI-T-EL---IGT~--DGLRIPD--E-----E---N-EN-I-I-EQAGKYEE-IPF-L----TLK-PK-DGA--L---ELNKIHEI-NLL

V-T---F-L-K-N---E-----

1192 SGPNHADIQKIGNRCFSDGMYDAAKLLYNNVSNFARLAITLVYLKEFQGAVDSRRKANS~RTWKEVCFA~VDAEEFRLA~MCGLHIWH~DELEDLINY~ 1 1 9 1 N---N-H--QV-D--YDEK---------------G---S---H-G-Y-A---G-------------------GK--------------------E----- 1197 A-S-V-NLDHV-DKL-ENKE-K--R-C-SA---YSK--S-----GDY-A---T----SNIKV--L-ND--IEKK--K---I---NLI---E--DE-VER-

1292 QNRGYFDEL;ALLESALGLERAHMGMFTELAILYSKFKPSKMREHLELFWSRVNIPKVL~AAESAHLWSELVFLYDKYEEYDNAVL~HPTEAWREGH 1 2 9 1 -D----E---TM--A------------------------Q-----------------------Q----A---------------IIT--N---D--K--Q 1297 ESN---E---S-F-AG-------------------- YE-D-TF---K-----I-----I--V-Q----------- AH-D-W---A-TLIEKS-KDLDHAY

1 3 9 2 FKDIITKVANIELYYKAIEFYLDFKPLLLNDMLLVLAPRMDHTRAVSYFSKTGYLPLV~PYLRSVQSLNNKAINEALNGLLIDEEDYQGLRNSIDGFDNF 1 3 9 1 ----------v-------Q---E-------- L-~..-S--LA-----N----VKQ------------NH---sV--s--N-F-T-----A--T---Ay--- 1397 --EVW--S-L-I-----N--VK"S--V-L-TS--V-L-TS-T--L-IP-T-KI---SDN---I--F-IN-LPK--SW-Q-YHD-M-E----KA-QDAV-SY-K~

1492 DNIALAQKLEKHELTEFRRIAAYLYKGNNRWKQSVELCKKDKLYKD~Y~ESCKQDI~ELLG~LERDAYDCF~CLYQCYDLLRPDVILELA~HK

1497 -QLG--SR--S-K-IF-KK-G-L--RR-KK-RK-LSIL-EE--W---I-T--I-QDPKVV-A--TY-V-TGNREG-V-L--~-N-V-IEFV--IS-MNS

1592 IVDFAMPYL;QVLREYTTK;DKLELNEAQREKEDDSTEH~NIIQ~PQLMITAGP~GI~PQYAQNYPP~AATVT~GG~NMGYPYL. 1678

1 4 9 1 ---S---R------I---------F----------------s------Q--s--KDTEL-----Q---QEEKRE--G---FT---------v--T--R-N

1 5 9 1 -M------F---MK--L------DAS-SL-KE-EQA--TQP-VYGQ----L----SVAV---APFG-...GY-APPY-QPQP-FG-SM 1 6 7 5 1 5 9 7 LE-YIK-FE-SIKK-QNDSIK-ITEEL-K ..- SGSNE--- .... DGQP--L.MNS"NVQ-TGF ....................... 1653

FIGURE 4.-Ali~ment of the Drosophila clathrin HC amino acid sequence with the rat and yeast HCs. Rat and yeast residues identical to the Drosophila residue above them are indicated by a dash (-). Periods (.) indicate gaps in the sequence. Arrows indicate the location of the four sequences used for design of PCR primer pools used in cloning.

KATZEN, T. KORNBERG and M. BISHOP, in preparation) were tested in this way. One of these groups, designated group VI, showed unambiguous complementation by the longer of the two P[w+;Chc] elements tested (pCHC3). The smaller construct (pCHC9) failed to rescue the same mutants. Results of some representative crosses demonstrating complementation of group VI alleles by the pCHC3 element are presented in Table 1. As indicated in Figure 6 ,

several independent autosomal insertions of each P element construct were tested. In most test crosses, a considerably smaller number of progeny were scored, but in every case the results were entirely consistent with those presented in more detail in the table; only the pCHC3 element complemented any of the tested alleles, and all alleles complemented by the element had been previously assigned to the same complementation group by pairwise crosses.


FIGURE 5.-In situ hybridization of the cloned Chc gene to larval polytene chromosomes. The location of the gene at position 13F2 on the X chromosome is indicated by the arrowhead.

The difference between the pCHC3 and pCHC9 elements that accounts for the complementation of the group VI lethal mutations is represented by material that lies outside of (upstream of) the transcribed material present in the cDNAs (Figure 6). This may indicate that cis-acting control elements required for the expression of the Chc gene are located further upstream of the region included in the pCHC9 construct. Alternatively, there is a possibility that the group VI mutants actually lie in a small gene just proximal to the Chc gene, and that the complemen-

tation observed with the pCHC3 element represents complementation of mutations in this neighboring gene rather than complementation of clathrin HC mutations. T o test this possibility, an additional germline transformation experiment was carried out, in which all of the upstream 5' material carried by the pCHC3 construct and the 5' end of the Chc transcript were present, but most of the clathrin HC gene was removed (Figure 6). Five independent autosomal insertion isolates of this construct (pCHC2) failed to complement the group VI mutations (see Table 1 for data from a representative pCHC2 insertion). This shows that the complementation of Group VI mutants by the pCHC3 element requires DNA sequences including the clathrin HC gene, and argues strongly against the hypothesis that the gene complemented by the pCHC3 element lies proximal of the Chc gene. On the basis of these experiments, we have designated the group VI mutations as Chc mutants.

Phenotypes of Chc mutants: We have identified three lethal alleles of the Chc gene, and a fourth with considerably reduced viability. All four alleles were complemented by the pCHC3 construct, although the frequency with which this occurred and the time elapsed before hemizygous individuals carrying the complementing P element eclosed varied considerably. In general, the Chc', Chc' and Chc3 alleles were rescued with comparable efficiencies, at frequencies approaching that expected for complete rescue of the lethal phenotype. The Chc' allele was rescued at the lowest frequencies (Table 1). From the observed leak- iness of the Chc' allele, one might have expected relatively efficient complementation by the P[w+;Chc] elements, since some residual Chc function is already present in the Chc'background. The consistent observation of low efficiency rescue of this allele suggests that a partially dominant lethal effect is conferred by the Chc' mutant polypeptide.

Hemizygous Chc' males that survived to adulthood lived for several weeks after eclosion, but were invariably sterile. The sterility of these males was rescued by P[w+;Chc] elements that complement the Chc lethal alleles, showing that the sterility is a result of the Chc mutation and not some other mutation in the genetic background of the flies used in these experiments. Fertility of Chc' males correlated perfectly with the segregation of the P[w+;Chc] element. Of 99 Chc' males individually tested, all were sterile. In contrast, 82 of 84 Chc' males carrying the autosomal pCHC3- A2 insertion and 40/40 Chc' males carrying the pCHC3-A20 insertion were fertile. Chc' males carrying the pCHC2-T2 1 insert were sterile (n = 14), again arguing that the effect is due to the Chc gene and not a proximal neighboring gene on the pCHC3 element.

Occasionally, we also recovered homozygous Chc' females from backcrosses of Chc4/Y;pCHC3/+ males


sd 3 ' 3' Ch c 5 ' - -* """"

B x

e c 9

S A C 2

I X R

I 0/5

X R

14/19

X

FIGURE 6,"Restriction map of the Chc region and complementation by P[w+;Chc] constructs. The two X genomic clones used for subcloning into P element vectors are shown below the map. The approximate location of the Chc transcript, indicated above the map by a dashed arrow, was determined by hybridizing oligonucleotides corresponding to the 5' and 3' ends of Chc cDNA clone 6 to cloned genomic restriction fragments. The location of the 3' end of the scalloped transcript (CAMPBELL et al. 1992) is also indicated. Below the X clones are the fragments reintroduced into Drosophila via P element-mediated germline transformation on the pCasper4 vector. The number of independent insertions tested and found to complement the group VI mutations are indicated. Restriction sites indicated are EcoRI = R; XbaI = X. The EcoRI site in parentheses is a cloning junction from clone XCHC2 and is absent in genomic DNA.

TABLE 1

Complementation of X-linked mutations by the clathrin heavy chain gene P[w+;CLc] insertions

No. of progeny of each genotype class

0 P 0 6 6 6 Relative rescue

Lethal allele y w/FM?Cb,' y wlw Chc;pCHCl+ y w f w Chc;+f+ F M 7 ~ f y 6 , ~ w Chcfr;pCHCf+ w Chc'fy;+f+ efficiency'

Cross 1 : 99 ChclFM7c X 86 y W / Y ; ~ C H C I - A Z / + ~ Chc' 102 67 62 15 43 0 0.84 C h 2 80 51 40 34 29 0 0.72 Chc3 399 197 191 202 161 0 0.81 Chc' 220 141 130 136 52 31 0.19

Chc' 407 239 182 133 0 0 0.0 a The P[w+;Chc] elements, also referred to as pCHC, carry the w+ marker, which rescues the mutant w eye color phenotype. The FM7c

balancer X chromosome carries the dominant B (Bar) marker. The presence of the Chc'/Y;pCHC/+ class among the progeny indicates complementation of the lethal mutation carried on the X chromosome by the pCHC element. The presence of the Chc'/Y;+/+ class among the progeny indicates ''leakage'' survival of an individual carrying the lethal mutation, and is a measure of background survival in the absence of complementing sequences.

Because of the weak w+ activity of most pCasPer4 constructs, no attempt was made to distinguish W + ( i e . , PCHC) from the Wn (apricot) background of the FM7c balancer; w+ and Up FM7c classes were combined.

Relative rescue efficiency is defined as (no. of w+ males - no. of w males)/(l/2 no. balancer FM7c females). Relative rescue efficiency was calculated based on the number of FM7c females because of the relatively poor viability of males carrying the FM7c balancer.

For pCHC3, data are shown for the A2 insertion, which is on chromosome III . For pCHCZ, data are shown for the T21 insertion (autosome location undetermined).

Cross 2: 99 ChclFM7c X 88y W / Y ; ~ C H C ~ - A Z I / + ~


TABLE 2

Larval hatching frequencies of clathrin mutants ~ ~ ~

Total Genotype" eggs counted No. hatched Percent hatched wildtype'

Percent of

99 w/w x w w/Y 219 167 7 6c 100 99 Chc'lw x W w/Y 590 316 54 70 99 Chc'/w x 88 w/Y 517 297 57 76 99 C h 2 / w x W w/Y 330 180 55 72 99 Chc4/w x 88 w/Y 625 397 64 a4

a Virgin females heterozygous for the X-linked Chc mutations were crossed to males carrying the parental w chromosome used in EMS mutagenesis [referred to as W(K) in CAMPBELL et al. (1991)l.

Fertilized and unfertilized eggs were not distinguished in this study. The larval hatching rate for the parental chromosome (w) was consistently lower than would be normally expected. Thus the data were

normalized to the hatch rate of the wild-type control cross (w/w X w/Y). ~. . I

to Chc4/FM7c females. Chc4/Chc4 females represented 0.2-0.3% of the progeny and took 1.5-2 times longer to emerge. In subsequent experiments where Chc4/ FM7c females were crossed to males carrying a duplication of the X including the Chc gene (Chc'/Dp(l;Y) B' y+ sd+ shi+ Y'), the survival of Chc' homozygotes was sometimes considerably greater, accounting for 30- 40% of total female progeny; viability of flies carrying the Chc' allele may be sensitive to subtle differences in genetic background which are not yet understood. Of 20 homozygous Chc' females tested, half were fertile, but showed considerably delayed and reduced egg production.

T o assess the phenotype of the Chc mutations, each of the mutant alleles was crossed away from the FM7c balancer to eliminate potential confusion arising from the poor viability of the balancer chromosome itself. In crosses where 1/2 of the males, or 1/4 of the total cross progeny, were expected to express the mutant phenotype, the rate of hatching at the end of embryogenesis was compared to that of parallel stocks of the parent w(K) chromosome (Table 2). For three of the four mutant alleles (Chc', C h 2 , Chc?), the rate of hatching was 70-76% of the wild-type w(K) control. The Chc' allele consistently exhibited a higher rate of hatching, consistent with our observations of occa- sional survival of hemizygous Chc' males to adulthood.

The cuticles from unhatched embryos collected from Chc'/+ and Chc2/+ females crossed to wild-type Oregon-R males (n = 79 and 253, respectively), and from Chc2/+ females crossed to Chc'/Dp(l;Y) B" y+ sd+ shi+ Y' males (n = 52), were examined to determine at which stage(s) of embryonic development the Chc lethal mutations cause arrest, and if they induce any obvious morphological defects. In the cross to Ore- gon-R males, only male progeny could be Chc'; in the cross to Chc'/Dp(l;Y) Bs y+ sd+ shi+ males, only female progeny could be Chc', due to the presence of a duplication of the wild-type Chc region translocated to the Y chromosome. No regular cuticle defect was observed in the unhatched embryos. Although a few abnormal embryos were observed, in most cases the

cuticle patterns were normal for late stage embryos. This suggests that maternally derived clathrin allows the survival of Chc mutants through most of embryonic development, but is insufficient for hatching to the larval stages.

Chc mutations differentially block oogenesis: To examine the effects of the Chc mutations on oogenesis, we induced homozygous germline clones of the mutant Chc alleles using the dominant female-sterile tech- nique of PERRIMON, ENCSTROM and MAHOWALD (1 984). Chc/FM7c females were crossed to males carrying the dominant female-sterile mutation ovoD', and the first instar larvae were irradiated to induce mitotic recombination between the ovoD' and Chc chromosomes. The Chc/ovoD' female progeny were mated to Chc+ males and screened for the presence of Chc/Chc germline clones by their ability to lay eggs. Since the presence of the dominant ovoD' mutation results in a complete block of oogenesis prior to the initiation of vitellogenesis (OLIVER, PAULI, and MAHOWALD 1990), most females capable of producing mature eggs are expected to be those in which mitotic recombination has occurred in the germline between the ovoD' and Chc chromosomes, to produce (ova+) Chc/Chc germline cells.

In our initial experiment we screened for germline clones of the lethal alleles Chc' and C h 2 , but we were unable to obtain any fecund females. Out of 409 Chc'/ ovoD' females and 369 Chc2/ovoD' females screened, we obtained two and one fecund females, respectively. The progeny of these females, however, were found to be Chc+, indicating that they arose from a mitotic recombination event that occurred between the ovo and Chc loci, rather than proximal to both, thereby resulting in the induction of ovo+ Chc+ germline clones.

The number of Chc'/ovoD' and Chc2/ovoD' females which were screened in this experiment is well above the minimum number of females (200) which must be screened to conclude that homozygous clones were induced but were unable to undergo normal oogenesis [see PERRIMON, ENGSTROM and MAHOWALD (1 984)].


TABLE 3

Germline clonal analysis of Che lethal mutations

ovaD' females No. of Chcl

No. (frequency) of Chc+ Chc allele

No. (frequency) of Chc tested clones recovered clones recovered viable progeny

No. of Chc clones giving

Chc' 588 2 + 3a (0.9%) 1 (0.2%) 0 Chc' 366 2 (0.6%) 2 (0.6%) 0 Chc3 356 2 + 2U(1.1%) 4 (1.1%) 0 Chc' 321 3 (0.9%) 14 (4.4%) 14

a The Chc' and Chc' alleles yielded 3 and 2 clones, respectively, in which small but normal appearing, presumably wild-type eggs were present, but from which no viable progeny were recovered to test for the presence or absence of the X-linked lethal mutation. The background level at which wild-type recombinant clones were observed in irradiated Chc'/ovoD' individuals is sufficiently high to account for these as probable wild-type recombinants.

Furthermore, in a concurrent experiment, germline clones of the Z(Z)6P6 allele of the brainiac locus (GOODE, WRIGHT and MAHOWALD 1992) were induced at a frequency of approximately 5%. This dem- onstrated that the irradiation treatment used induced a sufficient level of mitotic recombination to produce germline clones at the normally expected frequencies (5-8%), based on the extensive germline clone analysis previously carried out in this laboratory.

When we screened irradiated Chc4/ovoD' females, fecund individuals were recovered at a frequency of 4.4% (again excluding Chc+ clones; Table 3). When the ovaries containing true Chc4/Chc4 clones were dissected and examined, oogenesis in these clones appeared to be completely normal.

Altogether, the results suggested that the lethal Chc alleles are either cell lethal in the germline, or that germline clones of these alleles are unable to complete normal oogenesis. To investigate these alternate pos- sibilities, we rescreened for germline clones of the three lethal Chc alleles (Chc', ChZ and Chc') by dissecting the ovaries from irradiated Chc'/ovoD' females and examining them for the presence of vitellogenic egg chambers. The recovery of vitellogenic ovaries would indicate that the Chc' germline clones develop to a late stage of oogenesis but arrest prior to the production or oviposition of mature eggs.

As expected, for each of the lethal alleles we recovered a few fecund females with apparently wild- type germline clones. These produced viable progeny which when tested proved to be Chc+ recombinants (Table 3). In addition, we recovered two fecund Chc3/ ovoD' females which laid a small number of eggs (<20) that were approximately one half to two thirds of normal length, with short paddle-like chorionic filaments and which failed to hatch. The ovaries dissected from these females contained vitellogenic ovarioles which appeared normal, but the mature eggs present in these ovarioles had the same abnormal appearance as those which had been oviposited.

For all three lethal alleles, ovaries containing vitellogenic ovarioles and/or eggs were recovered at ex- tremely low frequencies (Table 3); the majority of

those dissected appeared typical of ovoD'. Among those recovered which contained vitellogenic egg chambers, we found one from a Chc'/ovoD' female, two from Chc2/ovoD' females and another two from Chc3/ovoD' females which contained only very small clones. They consisted of only one or two vitellogenic ovarioles containing a small number of eggs (1- 10) which again were shorter than normal with short paddle-like filaments. We interpret these clones as being true Chcl clones, given that they were recovered at low frequencies for each of the lethal alleles (Table 3), and that the abnormal eggs produced by the clones of the three lethal alleles had similar mutant phenotypes.

Taken together, these observations argue that the lethal Chc alleles block oogenesis prior to vitellogenesis, except in a few rare instances where an escape beyond this block results in the formation of abnor- mally short, inviable eggs. In contrast, we were able to recover Chc' germline clones, which produced normal eggs and viable offspring. This is consistent with our observation that viable Chc4/Chc4 females can be recovered, of which about half are fertile. Therefore, the Chc' allele is sufficient for viability in both males and females, and for fertility in females, but it is not sufficient for fertility in males.

DISCUSSION

We have identified the genetic locus encoding the clathrin heavy chain of D. melanogaster. The nucleotide sequence of cDNAs reveals a predicted polypeptide that is 80% identical to the mammalian HC. Four mutations mapping to the same chromosomal location are complemented by a cloned copy of the wild-type gene reintroduced into mutant flies on a P transposable element. Three of these mutations block development late in embryogenesis. Males carrying the fourth allele sometimes survive to adulthood, but are sterile. These sterility defects are rescued by a single copy of the cloned wild-type gene.

The mapping of the Drosophila Chc gene to the 13F region of the X chromosome initially suggested to us that the shibire gene, which maps near this region


and displays mutant phenotypes affecting endocytosis (KOSAKA and IKEDA 1983), might encode the clathrin HC of D. melanogaster. Further mapping showed that the Chc gene is distinct from the shibire gene, which has recently been shown to encode a Drosophila homolog of the microtubule-stimulated GTPase, dynamin (VAN DER BLIEK and MEYEROWITZ 199 1 ; CHEN et al. 1991). We do not know if the location of these two genes of related function within 100 kb of each other is simple coincidence or indicative of a clustering of endocytic functions in this area of the Drosophila genome. Each of the four Chc alleles complements the canonical s W ’ mutation fully at the nonpermissive temperature (30”).

The survival of embryos lacking the Chc gene product is not surprising since a copious store of maternally derived clathrin is probably required during oogenesis, especially for vitellogenesis (ROTH and PORTER 1964; BRENNAN et al. 1982; DIMARIO and MAHOWALD 1987). Such a large maternal store of clathrin trans- ported into the oocyte, might mask the effects of the absence of new wild-type clathrin production in Chc lethal embryos and suffice for development to a late stage of embryogenesis, which is normally completed within 24 hr at 25 O . The clathrin HC is known to be relatively stable, having a measured half-life of -60 hr in mammalian tissue culture cells (ACTON and BRODSKY 1990). Normally, germline clonal analysis can be used to unmask such maternal effects. Unfor- tunately, the germline clonal analysis of some Chc mutations is not informative because clathrin is apparently essential for survival or development of germ cells until they can be detected in ovaries [stage 5 , according to KING (1 970)]. Somatic cell mosaic analysis could be useful for determining the specific clathrin requirements of particular cell types in other tissues. A preliminary study of Chc somatic eye cell clones using a twin-spot analysis suggests that Chc‘ clones are either cell lethal, or have a significantly reduced viability (<lo%) compared to Chc+ clones (M. MORGAN, unpublished observations). Consequently, the isolation or construction of temperature-sensitive Chc alleles may be required for studies of the effects of blocks in clathrin function in different tissues and developmental stages.

We suggest that the three lethal Chc alleles block development late in embryogenesis when the nervous system is constructed and begins to function. In- creased neuromotor function at hatching would be expected to require substantial amounts of clathrin for the membrane recycling associated with increased synaptic activity. This may define a developmental step particularly sensitive to perturbations in clathrin function.

The fourth allele, Chc4, consistently exhibited a higher rate of hatching to the larval stage. Occasion-

ally, hemizygous Chc4 adult males were observed in the balanced Chc4 stock. They appeared healthy, living for several weeks and exhibiting apparently normal walking, flying, feeding and mating behaviors. How- ever, they were sterile. The sterility was rescued by P[w+;Chc] insertions that complement the Chc lethal mutations. This demonstrates unambiguously that the sterility is a consequence of the Chc4 mutation. Prelim- inary evidence (C. BAZINET, unpublished observations) suggests a defect in the individualization process, during which spermatids, assembled in a syncytial cyst cell, are invested in their own individual cell membranes and large amounts of cytoplasmic material are extruded from them (TOKAYASU, PEACOCK and HARDY 1972). Sperm individualization requires a very active and coordinated assembly and/or reorganization of membranes, which might be extraordinarily sensitive to defects in vesicular transport processes.

Because Chc4 males sometimes survive to adulthood, one might expect that this apparently weak allele would be easier to rescue than the other alleles. Sur- prisingly, we observed that it is the most difficult of the four alleles to complement with the wild-type gene reintroduced on a P element. When background “leakage” frequencies are taken into account (the frequency at which w males carrying the Chc mutation emerged from the appropriate rescue crosses), the efficiency by which the Chc4 viability defect was complemented was always considerably lower than observed for the other Chc lethal alleles. This suggests a partial dominance of the Chc4 mutant polypeptide. The persistence of a partially defective HC in various clathrin structures would account for the observed dominant effect of the Chc4 mutant allele. Isolation of suppressors of the Chc‘ mutation may be an effective strategy for the identification of other Drosophila gene products interacting with clathrin itself or with clathrin-dependent physiological pathways.

The fertility defect of surviving Chc4 males appears to be completely penetrant but recessive. Fertility was very efficiently restored to Chc4 males by a wild-type copy of the gene. Since hemizygous Chc4 males occasionally survive to adulthood but are invariably sterile, more clathrin function may be required during ga- metogenesis than in some critical process(es) of the lethal embryonic phase. However, the results of the Chc‘ germline clone analysis and the fertility tests of Chc4/Chc4 females argue that the Chc‘ polypeptide is sufficient for successful oogenesis, during which the endocytosis of yolk protein precursor is expected to require large amounts of clathrin. Alternatively, the Chc4 mutation may impair a specialized clathrin function required in spermatogenesis but not in other tissues. That distinct functions of clathrin may be genetically distinguished was suggested by LEMMON et ul. (1 99 l), who observed that a carboxy-terminal dele-


tion of the yeast clathrin HC efficiently rescues the viability defect of null mutations but fails to complement defects in a-factor processing.

Extreme demands on the endocytic and secretory machinery in essential differentiated cell types of higher eukaryotes with complex body plans probably accounts for the lethal mutability of the Drosophila Chc gene. Significantly, even though unicellular organisms such as yeast (PAYNE and SCHEKMAN 1985; LEMMON and JONES 1987) and Dictyostelium (O’HAL- LORAN and ANDERSON 1992b) can grow vegetatively in the absence of clathrin, the simple developmental programs of these organisms, such as sporulation, mating and fruiting body formation, which require more complex cellular morphogenesis, are abolished or significantly reduced in the absence of clathrin function.

Careful analysis of clathrin mutant phenotypes in Drosophila should provide a fruitful approach to understanding the molecular basis of membrane orga- nization in specialized cell types. The 90% similarity of the Drosophila and mammalian heavy chains suggests that the molecules with which the HC interacts in Drosophila cells are very similar to their mammalian counterparts. Genetic analysis of the Drosophila HC and such interacting gene products could inform our understanding of their roles in mammalian cell and developmental processes and the diseases affecting them. The biochemical characterization of many mammalian clathrin-associated proteins and a growing catalogue of their yeast homologs (KIRCHHAUSEN 1990; KIRCHHAUSEN et al. 1991 ; NAKAYAMA et al. 199 1 ; SILVEIRA et al. 1990) provide convenient probes for their identification in Drosophila .

We thank BETH ALBRECHT for instructions on embryo injections. DANIEL PAULI, ALLAN LOHE, PETER HARTE, HELEN SALZ and MARCELO JACOBS-LORENA contributed materials, stocks, stimulating discussions and helpful suggestions, and generously shared equip ment and facilities that made this work possible. JEFF WEIGLE contributed mightily to the sequencing efforts. We thank DAN ODOM for Northern transfers of adult Drosophila RNA used to detect clathrin transcripts. This work was supported by a postdoc- toral fellowship from the American Heart Association, Northeast Ohio Affiliate (to C.B.), by the Markey Center for Developmental Genetics, Case Western Reserve University, by National Science Foundation grant DCB89-15360 (to S.K.L.), and by U.S. Public Health Services grant HD 17607 (to A.P.M.). A.L.K. was supported by U S . Public Health Services grant CA 44338 and the G. W. Hooper Research Foundation.

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Communicating editor: M. T. FULLER

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