The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40 Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.     

Patterns of molecular evolution of the germ line specification gene oskar suggest that a novel domain may contribute to functional divergence in Drosophila

In several metazoans including flies of the genus Drosophila, germ line specification occurs through the inheritance of maternally deposited cytoplasmic determinants, collectively called germ plasm. The novel insect gene oskar is at the top of the Drosophila germ line specification pathway, and also plays an important role in posterior patterning. A novel N-terminal domain of oskar (the Long Oskar domain) evolved in Drosophilids, but the role of this domain in oskar functional evolution is unknown. Trans-species transgenesis experiments have shown that oskar orthologs from different Drosophila species have functionally diverged, but the underlying selective pressures and molecular changes have not been investigated. As a first step toward understanding how Oskar function could have evolved, we applied molecular evolution analysis to oskar sequences from the completely sequenced genomes of 16 Drosophila species from the Sophophora subgenus, Drosophila virilis and Drosophila immigrans. We show that overall, this gene is subject to purifying selection, but that individual predicted structural and functional domains are subject to heterogeneous selection pressures. Specifically, two domains, the Drosophila-specific Long Osk domain and the region that interacts with the germ plasm protein Lasp, are evolving at a faster rate than other regions of oskar. Further, we provide evidence that positive selection may have acted on specific sites within these two domains on the D. virilis branch. Our domain-based analysis suggests that changes in the Long Osk and Lasp-binding domains are strong candidates for the molecular basis of functional divergence between the Oskar proteins of D. melanogaster and D. virilis. This molecular evolutionary analysis thus represents an important step towards understanding the role of an evolutionarily and developmentally critical gene in germ plasm evolution and assembly.     

Evolution of a Distinct Genomic Domain in Drosophila: Comparative Analysis of the Dot Chromosome in Drosophila melanogaster and Drosophila virilis

The distal arm of the fourth (“dot”) chromosome of Drosophila melanogaster is unusual in that it exhibits an amalgamation of heterochromatic properties (e.g., dense packaging, late replication) and euchromatic properties (e.g., gene density similar to euchromatic domains, replication during polytenization). To examine the evolution of this unusual domain, we undertook a comparative study by generating high-quality sequence data and manually curating gene models for the dot chromosome of D. virilis (Tucson strain 15010–1051.88). Our analysis shows that the dot chromosomes of D. melanogaster and D. virilis have higher repeat density, larger gene size, lower codon bias, and a higher rate of gene rearrangement compared to a reference euchromatic domain. Analysis of eight “wanderer” genes (present in a euchromatic chromosome arm in one species and on the dot chromosome in the other) shows that their characteristics are similar to other genes in the same domain, which suggests that these characteristics are features of the domain and are not required for these genes to function. Comparison of this strain of D. virilis with the strain sequenced by the Drosophila 12 Genomes Consortium (Tucson strain 15010–1051.87) indicates that most genes on the dot are under weak purifying selection. Collectively, despite the heterochromatin-like properties of this domain, genes on the dot evolve to maintain function while being responsive to changes in their local environment.EUKARYOTIC genomes are packaged into two major types of chromatin: euchromatin is gene rich and has a diffuse appearance during interphase, while heterochromatin is gene poor and remains densely packaged throughout the cell cycle (Grewal and Elgin 2002). The distal 1.2 Mb of the fourth chromosome of Drosophila melanogaster, known as the dot chromosome or Muller F element, is unusual in exhibiting an amalgamation of heterochromatic and euchromatic properties. This domain has a gene density that is similar to the other autosomes (Bartolomé et al. 2002; Slawson et al. 2006). However, it appears heterochromatic by many criteria, including late replication and very low levels of meiotic recombination (Wang et al. 2002; Arguello et al. 2010). It exhibits high levels of association with heterochromatin protein 1 (HP1) and histone H3 di- and trimethylated at lysine 9 (H3K9me2/3), as shown by immunofluorescent staining of the polytene chromosomes (Riddle and Elgin 2006; Slawson et al. 2006). This association with heterochromatin marks has recently been confirmed by the modENCODE Project [N. C. Riddle, A. Minoda, P. V. Kharchenko, A. A. Alekseyenko, Y. B. Schwartz, M. Y. Tolstorukov, A. A. Gorchakov, C. Kennedy, D. Linder-Basso, J. D. Jaffe, G. Shanower, M. I. Kuroda, V. Pirrotta, P. J. Park, S. C. R. Elgin, G. H. Karpen, and the modENCODE Consortium (http://www.modencode.org), unpublished results]. To understand this unique domain and to examine the evolution of a region with very low levels of recombination, we have undertaken a comparative study using the dot chromosome of D. virilis, a species that diverged from D. melanogaster 40–60 million years ago (Powell and Desalle 1995). We sequenced and improved the assembly of the D. virilis dot chromosome and created a manually curated set of gene models to ensure that both the assembly and the gene annotations are at a quality comparable to those in D. melanogaster. We then compared the sequence organization and gene characteristics of the distal portion of the D. virilis dot chromosome with the corresponding region from the D. melanogaster dot chromosome.In addition to examining the long-term dot chromosome evolution, we also investigated the short-term dot chromosome evolution by comparing the genomic sequences from two different strains of D. virilis. Agencourt Biosciences (AB) has previously produced a whole genome shotgun assembly of Tucson strain 15010–1051.87, while we have sequenced Tucson strain 15010–1051.88 of D. virilis [the Genomics Education Partnership (GEP) assembly]. The AB assembly has been improved by the Drosophila 12 Genomes Consortium and released as part of the comparative analysis freeze 1 (CAF1) assembly (Drosophila 12 Genomes Consortium et al. 2007).Using the GEP and CAF1 assemblies from D. virilis, and the high-quality D. melanogaster assembly and its gene annotations from FlyBase (Crosby et al. 2007), we compared the gene properties and sequence organization of the dot chromosomes and reference euchromatic and heterochromatic domains. The dot chromosomes from D. melanogaster and D. virilis are distinct from the heterochromatic and euchromatic regions of the two genomes, both in organization (e.g., repeat density) and in characteristics of the genes (e.g., size, codon bias). The two dot chromosomes resemble each other by most criteria and differ only in the types of repetitive sequences present and in relative gene order and orientation. Despite the very low rate of meiotic recombination, comparison of the two D. virilis strains shows that dot chromosome genes are under weak purifying selection. Our analysis of genes that are present in a euchromatic chromosome arm in one species and on the dot chromosome in the other (the “wanderer” genes) shows that this set of genes evolves to maintain function while responding to the changes in the local chromosomal environment.     

Molecular Cloning, Sequencing, and Expression of a Chemoreceptor Gene from Leptospirillum ferrooxidans

We have cloned and sequenced a 2,262-bp chromosomal DNA fragment from the chemolithoautotrophic acidophilic bacterium Leptospirillum ferrooxidans. This DNA contained an open reading frame for a 577-amino-acid protein showing several characteristics of the bacterial chemoreceptors and, therefore, we named this gene lcrI for Leptospirillum chemotaxis receptor I. This is the first sequence reported for a gene from L. ferrooxidans encoding a protein. The lcrI gene showed both ς²⁸-like and ς⁷⁰-like putative promoters. The LcrI deduced protein contained two hydrophobic regions most likely corresponding to the two transmembrane regions present in all of the methyl-accepting chemotaxis proteins (MCPs) which make them fold with both periplasmic and cytoplasmic domains. We have proposed a cytoplasmic domain for LcrI, which also contains the highly conserved domain (HCD region), present in all of the chemotactic receptors, and two probable methylation sites. The in vitro expression of a DNA plasmid containing the 2,262-bp fragment showed the synthesis of a 58-kDa protein which was immunoprecipitated by antibodies against the Tar protein (an MCP from Escherichia coli), confirming some degree of antigenic conservation. In addition, this 58-kDa protein was expressed in E. coli, being associated with its cytoplasmic membrane fraction. It was not possible to determine a chemotactic receptor function for LcrI expressed in E. coli. This was most likely due to the fact that the periplasmic pH of E. coli, which differs by 3 to 4 pH units from that of acidophilic chemolithotrophs, does not allow the right conformation for the LcrI periplasmic domain.     

Rapid divergence in the course of Drosophila evolution reveals structural important domains of the Notch antagonist Hairless

 Hairless is a member of the Notch signalling pathway, where it acts as antagonist by binding to Suppressor of Hairless [Su(H)], thereby inhibiting Notch target gene activation. The pathway and its members are highly conserved in metazoans from worms to humans. However, a Hairless orthologue from another species has not yet been identified. The identification of Hairless in largely diverged species by cross-hybridization has failed so far probably due to a low degree of conservation. Therefore, we turned to D. hydei where a Hairless mutation has been described before. The D. hydei Hairless orthologue is reasonably well conserved with regard to gene structure and expression. The prospective Hairless protein orthologues share several highly conserved regions which are separated by quite diverged stretches. As to be expected, the largest region of high conservation corresponds to the Su(H) binding domain. This region is also functionally conserved, since this D. hydei protein domain binds very strongly to the D. melanogaster Su(H) protein. The other conserved regions support our earlier structure-function analysis since they nicely correspond to previously defined, functionally important protein domains. Most notably, the very C-terminal domain which is very sensitive to structural alterations, is nearly identical between the two species. In summary, this evolutionary study improves the knowledge on functionally significant domains of the Hairless protein, and may be helpful for the future identification of homologues in other animals, especially in vertebrates. Received: 26 August 1998 / Accepted: 9 November 1998     

A Conserved Functional Domain of Drosophila Coracle Is Required for Localization at the Septate Junction and Has Membrane-organizing Activity

The protein 4.1 superfamily is comprised of a diverse group of cytoplasmic proteins, many of which have been shown to associate with the plasma membrane via binding to specific transmembrane proteins. Coracle, a Drosophila protein 4.1 homologue, is required during embryogenesis and is localized to the cytoplasmic face of the septate junction in epithelial cells. Using in vitro mutagenesis, we demonstrate that the amino-terminal 383 amino acids of Coracle define a functional domain that is both necessary and sufficient for proper septate junction localization in transgenic embryos. Genetic mutations within this domain disrupt the subcellular localization of Coracle and severely affect its genetic function, indicating that correct subcellular localization is essential for Coracle function. Furthermore, the localization of Coracle and the transmembrane protein Neurexin to the septate junction display an interdependent relationship, suggesting that Coracle and Neurexin interact with one another at the cytoplasmic face of the septate junction. Consistent with this notion, immunoprecipitation and in vitro binding studies demonstrate that the amino-terminal 383 amino acids of Coracle and cytoplasmic domain of Neurexin interact directly. Together these results indicate that Coracle provides essential membrane-organizing functions at the septate junction, and that these functions are carried out by an amino-terminal domain that is conserved in all protein 4.1 superfamily members.     

Genetic analysis of the cytoplasmic domain of the PfRh2b merozoite invasion protein of Plasmodium falciparum

Apicomplexan parasites employ multiple adhesive ligands for recognition and entry into host cells. The Duffy binding-like (DBL) and the reticulocyte binding protein-like (RBL) families are central to the invasion of erythrocytes by the malaria parasite. These type-1 transmembrane proteins are composed of large ectodomains and small conserved cytoplasmic tail domains. The cytoplasmic tail domain of the micronemal DBL protein EBA-175 is required for a functional ligand-receptor interaction, but not for correct trafficking and localisation. Here we focus on the cytoplasmic tail domain of the rhoptry-localised Plasmodium falciparum RBL PfRh2b. We have identified a conserved sequence of six amino acids, enriched in acidic residues, in the cytoplasmic tail domains of RBL proteins from Plasmodium spp. Genetic analyses reveal that the entire cytoplasmic tail and the conserved motif within the cytoplasmic tail are indispensable for invasion P. falciparum. Site-directed mutagenesis of the conserved moiety reveals that changes in the order of the amino acids of the conserved moiety, but not the charge of the sequence, can be tolerated. Shuffling of the motif has no effect on either invasion phenotype or PfRh2b expression and trafficking. Although the PfRh2b gene can be readily disrupted, our results suggest that modification of the PfRh2b cytoplasmic tail results in strong dominant negative activity, highlighting important differences between the PfRh2b and EBA-175 invasion ligands.     

The Platform Protein Is Essential for Type IV Pilus Biogenesis

A systematic genetic analysis was performed to identify the inner membrane proteins essential for type IV pilus (T4P) expression in Pseudomonas aeruginosa. By inactivating the retraction aspect of pilus function, genes essential for T4P assembly were discriminated. In contrast to previous studies in the T4P system of Neisseria spp., we found that components of the inner membrane subcomplex consisting of PilMNOP were not essential for surface pilus expression, whereas the highly conserved inner membrane protein PilC was essential. Here, we present data that PilC may coordinate the activity of cytoplasmic polymerization (PilB) and depolymerization (PilT) ATPases via their interactions with its two cytoplasmic domains. Using in vitro co-affinity purification, we show that PilB interacts with the N-terminal cytoplasmic domain of PilC. We hypothesized that PilT similarly interacts with the PilC C-terminal cytoplasmic domain. Overexpression of that domain in the wild-type protein reduced twitching motility by ∼50% compared with the vector control. Site-directed mutagenesis of conserved T4P-specific residues in the PilC C-terminal domain yielded mutant proteins that supported wild-type pilus assembly but had a reduced capacity to support twitching motility, suggesting impairment of putative PilC-PilT interactions. Taken together, our results show that PilC is an essential inner membrane component of the T4P system, controlling both pilus assembly and disassembly.     

Highly conserved ENY2/Sus1 protein binds to Drosophila CTCF and is required for barrier activity

Chromatin insulators affect interactions between promoters and enhancers/silencers and function as barriers for the spreading of repressive chromatin. Drosophila insulator protein dCTCF marks active promoters and boundaries of many histone H3K27 trimethylation domains associated with repressed chromatin. In particular, dCTCF binds to such boundaries between the parasegment-specific regulatory domains of the Bithorax complex. Here we demonstrate that the evolutionarily conserved protein ENY2 is recruited to the zinc-finger domain of dCTCF and is required for the barrier activity of dCTCF-dependent insulators in transgenic lines. Inactivation of ENY2 by RNAi in BG3 cells leads to the spreading of H3K27 trimethylation and Pc protein at several dCTCF boundaries. The results suggest that evolutionarily conserved ENY2 is responsible for barrier activity mediated by the dCTCF protein.     

The N-terminal Domain of the Drosophila Mitochondrial Replicative DNA Helicase Contains an Iron-Sulfur Cluster and Binds DNA

The metazoan mitochondrial DNA helicase is an integral part of the minimal mitochondrial replisome. It exhibits strong sequence homology with the bacteriophage T7 gene 4 protein primase-helicase (T7 gp4). Both proteins contain distinct N- and C-terminal domains separated by a flexible linker. The C-terminal domain catalyzes its characteristic DNA-dependent NTPase activity, and can unwind duplex DNA substrates independently of the N-terminal domain. Whereas the N-terminal domain in T7 gp4 contains a DNA primase activity, this function is lost in metazoan mtDNA helicase. Thus, although the functions of the C-terminal domain and the linker are partially understood, the role of the N-terminal region in the metazoan replicative mtDNA helicase remains elusive. Here, we show that the N-terminal domain of Drosophila melanogaster mtDNA helicase coordinates iron in a 2Fe-2S cluster that enhances protein stability in vitro. The N-terminal domain binds the cluster through conserved cysteine residues (Cys⁶⁸, Cys⁷¹, Cys¹⁰², and Cys¹⁰⁵) that are responsible for coordinating zinc in T7 gp4. Moreover, we show that the N-terminal domain binds both single- and double-stranded DNA oligomers, with an apparent K_d of ∼120 nm. These findings suggest a possible role for the N-terminal domain of metazoan mtDNA helicase in recruiting and binding DNA at the replication fork.     

Identification of peptide inhibitors of pre-mRNA splicing derived from the essential interaction domains of CDC5L and PLRG1

CDC5L and PLRG1 are both spliceosomal proteins that are highly conserved across species. They have both been shown to be part of sub- spliceosomal protein complexes that are essential for pre-mRNA splicing in yeast and humans. CDC5L and PLRG1 interact directly in vitro. This interaction is mediated by WD40 regions in PLRG1 and the C-terminal domain of CDC5L. In order to determine whether this interaction is important for the splicing mechanism, we have designed peptides corresponding to highly conserved sequences in the interaction domains of both proteins. These peptides were used in in vitro splicing experiments as competitors to the cognate sequences in the endogenous proteins. Certain peptides derived from the binding domains of both proteins were found to inhibit in vitro splicing. This splicing inhibition could be prevented by preincubating the peptides with the corresponding partner protein that had been expressed in Escherichia coli. The results from this study indicate that the interaction between CDC5L and PLRG1 is essential for pre-mRNA splicing and further demonstrate that small peptides can be used as effective splicing inhibitors.     

Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily

Protein-interaction domains can create unique macromolecular complexes that drive evolutionary innovation. By combining bioinformatic and phylogenetic analyses with structural approaches, we have discovered that the docking and dimerization (D/D) domain of the PKA regulatory subunit is an ancient and conserved protein fold. An archetypal function of this module is to interact with A-kinase-anchoring proteins (AKAPs) that facilitate compartmentalization of this key cell-signaling enzyme. Homology searching reveals that D/D domain proteins comprise a superfamily with 18 members that function in a variety of molecular and cellular contexts. Further in silico analyses indicate that D/D domains segregate into subgroups on the basis of their similarity to type I or type II PKA regulatory subunits. The sperm autoantigenic protein 17 (SPA17) is a prototype of the type II or R2D2 subgroup that is conserved across metazoan phyla. We determined the crystal structure of an extended D/D domain from SPA17 (amino acids 1–75) at 1.72 Å resolution. This revealed a four-helix bundle-like configuration featuring terminal β-strands that can mediate higher order oligomerization. In solution, SPA17 forms both homodimers and tetramers and displays a weak affinity for AKAP18. Quantitative approaches reveal that AKAP18 binding occurs at nanomolar affinity when SPA17 heterodimerizes with the ropporin-1-like D/D protein. These findings expand the role of the D/D fold as a versatile protein-interaction element that maintains the integrity of macromolecular architectures within organelles such as motile cilia.     

Vps15 is required for stress induced and developmentally triggered autophagy and salivary gland protein secretion in Drosophila

Autophagy is a catabolic process used to deliver cellular material to the lysosome for degradation. The core Vps34/class III phosphatidylinositol 3-kinase (PI3K) complex, consisting of Atg6, Vps15, and Vps34, is highly conserved throughout evolution, critical for recruiting autophagy-related proteins to the preautophagosomal structure and for other vesicular trafficking processes, including vacuolar protein sorting. Atg6 and Vps34 have been well characterized, but the Vps15 kinase remains poorly characterized with most studies focusing on nutrient deprivation-induced autophagy. Here, we investigate the function of Vps15 in different cellular contexts and find that it is necessary for both stress-induced and developmentally programmed autophagy in various tissues in Drosophila melanogaster. Vps15 is required for autophagy that is induced by multiple forms of stress, including nutrient deprivation, hypoxia, and oxidative stress. Furthermore, autophagy that is triggered by physiological stimuli during development in the fat body, intestine, and salivary gland also require the function of Vps15. In addition, we show that Vps15 is necessary for efficient salivary gland protein secretion. These data illustrate the broad importance of Vps15 in multiple forms of autophagy in different animal cells, and also highlight the pleiotropic function of this kinase in multiple vesicle-trafficking pathways.Autophagy is an evolutionarily conserved process in which cytoplasmic proteins or organelles are packaged into lysosomes for degradation. This process can be initiated by a variety of stimuli, such as high levels of starvation or stress, to provide nutrients to the cells or to clear the cell of damaged organelles or protein aggregates.¹ In some circumstances, autophagy can promote an alternative form of cell death, such as in the clearance of larval tissues in Drosophila melanogaster.² As defects in autophagy have been implicated in several physiological and pathological conditions, such as cancer, neurodegenerative diseases, and aging,^3,4 it is important to obtain a complete understanding of the molecular mechanisms controlling autophagy.The induction of autophagy is regulated by the Atg1/Ulk1 complex, and this complex is regulated by mechanistic target of rapamycin (mTOR).⁵ Vesicle nucleation is controlled by the class III phosphoinositide 3-kinase (PI3K) complex that generates phosphatidylinositol 3-phosphate (PI3P).⁶ This conserved complex consists of vacuolar protein sorting 34 (Vps34; also known as Pik3c3), Atg6/Becn1 (also known as Vps30 in yeast), and the serine-threonine kinase Vps15/ird1 (p150 in mammals; also known as Pik3r4).^7,8 Localized production of PI3P by Vps34 can act to recruit proteins containing PX or FYVE domains to membrane compartments, such as the autophagosome isolation membrane.⁹ Vps34 is also required more broadly for several vesicular trafficking processes such as the sorting of hydrolytic enzymes to the yeast vacuole and mammalian lysosome, and endocytic trafficking.^{10, 11, 12} There is mounting evidence demonstrating the pleiotropic function of the PI3K/Vps34 complex, but this has not been well studied in the context of autophagy under different physiological and cell contexts in animals.Of the three core PI3K complex proteins, Vps15 remains an understudied kinase, and its function has not been rigorously investigated in multicellular organisms in vivo. Most of the focus on the role of this complex in autophagy regulation has been on nutrient deprivation-initiated autophagy. Indeed, previous studies determined Vps15 to be necessary for starvation-induced autophagy in the Drosophila larval fat body.^13,14 However, its role in hormone-regulated autophagy, a process that occurs in the intestine,¹⁵ salivary glands,¹⁶ and fat body¹⁷ of developing Drosophila, as well as its role in other stress-induced conditions have not yet been examined. In order to address the role of Vps15 in these and other processes regulated by autophagy, we utilized Vps15 knockdown as well as a previously described null mutant¹⁴ to examine its role in a multicellular organism in vivo. We found that Vps15 is required not only for stress-induced autophagy in multiple tissues, but it is also a broad regulator of developmentally programmed autophagy in Drosophila. In addition, Vps15 is necessary for efficient protein secretion, as indicated by its role in the secretion of glue proteins from the Drosophila salivary gland. Together, these results highlight the importance of Vps15 in multiple processes in vivo.     

Mutations in Drosophila Enabled and Rescue by Human Vasodilator-stimulated Phosphoprotein (VASP) Indicate Important Functional Roles for Ena/VASP Homology Domain 1 (EVH1) and EVH2 Domains

Drosophila Enabled (Ena) was initially identified as a dominant genetic suppressor of mutations in the Abelson tyrosine kinase and, more recently, as a member of the Ena/human vasodilator-stimulated phosphoprotein (VASP) family of proteins. We have used genetic, biochemical, and cell biological approaches to demonstrate the functional relationship between Ena and human VASP. In addition, we have defined the roles of Ena domains identified as essential for its activity in vivo. We have demonstrated that VASP rescues the embryonic lethality associated with loss of Ena function in Drosophila and have shown that Ena, like VASP, is associated with actin filaments and focal adhesions when expressed in cultured cells. To define sequences that are central to Ena function, we have characterized the molecular lesions present in two lethal ena mutant alleles that affected the Ena/VASP homology domain 1 (EVH1) and EVH2. A missense mutation that resulted in an amino acid substitution in the EVH1 domain eliminated in vitro binding of Ena to the cytoskeletal protein zyxin, a previously reported binding partner of VASP. A nonsense mutation that resulted in a C-terminally truncated Ena protein lacking the EVH2 domain failed to form multimeric complexes and exhibited reduced binding to zyxin and the Abelson Src homology 3 domain. Our analysis demonstrates that Ena and VASP are functionally homologous and defines the conserved EVH1 and EVH2 domains as central to the physiological activity of Ena.