The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

Two novel LEM-domain proteins are splice products of the annotated Drosophila melanogaster gene CG9424 (Bocksbeutel)

The LEM motif is a sequence of 40-50 amino acids that has been identified in a number of non-related proteins of the inner nuclear membrane including the lamina-associated polypeptides 2 (LAP2), emerin, MAN1 and the Drosophila protein otefin. This evolutionary conserved sequence motif can mediate via the interaction with the small protein BAF the binding of LEM-domain proteins to DNA. Taking advantage of its sequenced genome we analyzed whether Drosophila possesses beside otefin additional genes coding for proteins with a LEM motif. A putative candidate gene was the annotated gene CG9424 which we named Bocksbeutel. Of all putative Drosophila LEM-domain proteins, otefin and Bocksbeutel exhibited the highest similarity in the LEM motif (53% identical amino acids). The Bocksbeutel gene can code for two isoforms of 399 and 351 amino acids that are produced by alternative splicing. In the alpha-isoform a transmembrane domain is localized close to the carboxyterminus. This segment is absent in the shorter beta-isoform. By RT-PCR we could show that in the embryo the mRNA coding for the alpha-isoform and in significantly lower amounts the mRNA coding for the beta-isoform are expressed. When expressed in transfected cells as GFP fusion proteins, the beta-isoform is localized predominantly in the nucleoplasm and the alpha-isoform is targeted to the nuclear envelope, indicating that Bocksbeutel-alpha is localized in the inner nuclear membrane. Bocksbeutel-alpha is the predominant isoform expressed in cells, larvae, and flies. Indirect immunofluorescence with Bocksbeutel-specific antibodies on tissues and cultured cells revealed that Bocksbeutel proteins are localized in the nuclear envelope and in the cytoplasm. By RNA interference we have down-regulated the expression of Bocksbeutel, BAF, otefin, and lamin DmO in Drosophila Kc167 cells. The down-regulation of Bocksbeutel and otefin had no influence on the viability of Kc167 cells and the intracellular localization of all other nuclear and nuclear envelope proteins analyzed. In contrast, when lamin DmO was reduced by RNAi the distribution of Bocksbeutel and otefin in the nuclear envelope of Kc167 cells was significantly altered. We conclude that the two LEM-domain proteins Bocksbeutel and otefin are no limiting components for the maintenance of the nuclear architecture in cultured Drosophila cells at interphase.     

Primary structure of a novel 4-acetamido-4''-isothiocyanostilbene-2,2''-disulphonic acid (SITS)-binding membrane protein highly expressed in Torpedo californica electroplax.

Polyclonal rabbit antibodies were raised against 4-acetamido-4'-isothiocyanostilbene-2,2'-disulphonic acid (SITS), an inhibitor of a variety of anion transport proteins. These antibodies specifically recognize SITS-reacted erythrocyte band 3 in immunoprecipitations and Western blots. In Western blots of SITS-reacted membrane proteins derived from vesicles of the electric organ of Torpedo californica (known to express a SITS-sensitive Cl- channel) the antibodies recognized two major species of approximately 93 kDa and approximately 105 kDa. The approximately 93 kDa protein was identified as the alpha-subunit of the Na,K-ATPase. The approximately 105 kDa protein (designated sp105) is a glycoprotein which binds to wheat-germ agglutinin and concanavalin A and is present as a disulphide-linked homodimer under non-reducing conditions. A partial amino acid sequence and a polyclonal antibody were used to clone the corresponding cDNA. sp105 is encoded in electroplax by two abundant mRNAs of approximately 6 and approximately 6.8 kb. A hybridizing mRNA of approximately 5 kb was over 200-fold and over 500-fold less abundant in brain and heart respectively. Sequence analysis of the cDNA predicted a novel protein of 697 amino acids containing eight potential N-linked glycosylation sites. Analysis of hydrophobicity indicated the presence of at least one, and possibly three, putative membrane-spanning domains. When expressed from the Sp6 message in Xenopus laevis oocytes, the protein was inserted into membranes, glycosylated and processed to form a dimer. However, no increase in 36Cl uptake or in membrane conductance could be detected. We found no effect of hybrid depleting the specific message on expression of the Torpedo electroplax Cl- channel in oocytes. Thus we conclude that this novel electroplax membrane protein is probably not a functional part of the chloride channel.     

Antipeptide Antibodies Against a Torpedo Cysteine-String Protein

Abstract: An antipeptide antiserum was raised against the C-terminal undecapeptide of a Torpedo cysteine-string protein (csp), a putative subunit or modulator of presynaptic calcium channels. This antiserum was shown to identify selectively the 27-kDa in vitro translation product of the csp cRNA both by immunoprecipitation and on immunoblots. When affinity-purified anti-csp antibodies were used to probe immunoblots of membrane proteins from Torpedo electric organ or liver, specific immunoreactivity was detected only in electric organ. This immunoreactivity was associated principally with a single protein species of about 34 kDa. These results indicate that csp immunoreactivity is detectably expressed in electroplax, a heavily innervated tissue, but not in liver, which should have an appreciably lower abundance of presynaptic calcium channel proteins. Moreover, the increased relative molecular mass of csp in electric organ (compared with in vitro translated material) implies that csp is posttranslationally modified. Finally, immunoblot analysis of either intact, alkali-treated, or solubilized membrane fractions of electric organ reveals that csp is predominantly a membrane protein.     

Lipin-1γ isoform is a novel lipid droplet-associated protein highly expressed in the brain

Lipin-1 proteins are phosphatidic acid phosphatases (PAPs) catalyzing the conversion from phosphatidic acid (PA) to diacylglycerol (DG). Two alternative splicing isoforms, lipin-1α and -1β, are localized at different subcellular compartments. A third splicing isoform, lipin-1γ was recently cloned and its subcellular localization is unknown. Here, we demonstrate that lipin-1γ is localized to lipid droplets (LDs), an association mediated by a hydrophobic, lipin-1γ-specific domain. Additional expression of lipin-1γ altered LD morphology without affecting the triacylglycerol (TG) level. In human tissues, lipin-1γ is the main lipin-1 isoform expressed in normal human brain, suggesting a specialized role in regulating brain lipid metabolism.     

Physical interactions of Dmnk with Orb: implications in the regulated localization of Orb by Dmnk during oogenesis and embryogenesis.

The Dmnk (Drosophila maternal nuclear kinase) gene, encoding a nuclear protein serine/threonine kinase, is expressed predominantly in the germline cells during embryogenesis, suggesting its possible role in the establishment of germ cells. We report here that Dmnk interacts physically with Drosophila RNA binding protein Orb, which plays crucial roles in the establishment of Drosophila oocyte by regulating the distribution and translation of several maternal mRNAs. Considering similar spatiotemporal expression pattern of Dmnk and orb during oogenesis and early embryogenesis, it is suggested that Dmnk plays a role in establishment of germ cells by interacting with Orb. Although there are two forms of Dmnk proteins, Dmnk-L (long) and Dmnk-S (short) via the developmentally regulated alternative splicing, Orb can associate with both forms of Dmnk proteins when expressed in culture cells. However, immunohistochemical analysis revealed that Dmnk-S, but not Dmnk-L, can affect the subcellular localization of Orb in a kinase activity-dependent manner, suggesting differential functions of Dmnk-S and Dmnk-L in the regulation of Orb.     

Overlapping domains of the heterochromatin-associated protein HP1 mediate nuclear localization and heterochromatin binding

The Drosophila protein HP1 is a 206 amino acid heterochromatin- associated nonhistone chromosomal protein. Based on the characterization of HP1 to date, there are three properties intrinsic to HP1: nuclear localization, heterochromatin binding, and gene silencing. In this work, we have concentrated on the identification of domains responsible for the nuclear localization and heterochromatin binding properties of HP1. We have expressed a series of beta- galactosidase/HP1 fusion proteins in Drosophila embryos and polytene tissue and have used beta-galactosidase enzymatic activity to identify the subcellular localization of each fusion protein. We have identified two functional domains in HP1: a nuclear localization domain of amino acids 152-206 and a heterochromatin binding domain of amino acids 95- 206. Both of these functional domains overlap an evolutionarily conserved COOH-terminal region.     

pasilla, the Drosophila homologue of the human Nova-1 and Nova-2 proteins, is required for normal secretion in the salivary gland.

From a screen for genes expressed and required in the Drosophila salivary gland, we identified pasilla (ps), which encodes a set of proteins most similar to human Nova-1 and Nova-2. Nova-1 and Nova-2 are nuclear RNA-binding proteins normally expressed in the CNS where they directly regulate splicing. In patients suffering from paraneoplastic opsoclonus myoclonus ataxia (POMA), Nova-1 and Nova-2 proteins are present as auto-antigens. Consistent with a role in splicing, PS is localized to nuclear puncta. The salivary glands of ps mutants internalize normally and maintain epithelial polarity. However, the mutant salivary glands develop irregularities in overall morphology and have defects in apical secretion. The secretory defects in ps mutants provide a potential mechanism for the loss of motor function observed in POMA patients.     

C6ORF32 is upregulated during muscle cell differentiation and induces the formation of cellular filopodia

We have identified a gene by microarray analysis that is located on chromosome 6 (c6orf32), whose expression is increased during human fetal myoblast differentiation. The protein encoded by c6orf32 is expressed both in myogenic and non-myogenic primary cells isolated from 18-week old human fetal skeletal muscle. Immunofluorescent staining indicated that C6ORF32 localizes to the cellular cytoskeleton and filopodia, and often displays polarized expression within the cell. mRNA knockdown experiments in the C2C12 murine myoblast cell line demonstrated that cells lacking c6orf32 exhibit a myogenic differentiation defect, characterized by a decrease in the expression of myogenin and myosin heavy chain (MHC) proteins, whereas MyoD1 was unaltered. In contrast, overexpression of c6orf32 in C2C12 or HEK293 cells (a non-muscle cell line) promoted formation of long membrane protrusions (filopodia). Analysis of serial deletion mutants demonstrated that amino acids 55-113 of C6ORF32 are likely involved in filopodia formation. These results indicate that C6ORF32 is a novel protein likely to play multiple functions, including promoting myogenic cell differentiation, cytoskeletal rearrangement and filopodia formation.     

Nuclear localization of C-terminal domains of the kinesin-like protein MKLP-1.

The successful execution of mitosis in mammalian cells requires the activities of numerous kinesin-like proteins. The Mitotic Kinesin-Like Protein-1 (MKLP-1) localizes to the spindle equator and is believed to participate in the separation of spindle poles during anaphase B. Injection of antibodies against MKLP-1 into dividing cells results in cell cycle arrest, suggesting that MKLP-1 is essential for mitosis. To further characterize MKLP-1, constructs encoding C-terminal domains of MKLP-1 were expressed as fusions to the green fluorescent protein and localized in HeLa cells. All constructs localized to the nucleus indicating the presence of at least one nuclear localization sequence in the C-terminus of the protein. C-terminal domains of MKLP-1 expressed in insect cells also localized to the nucleus as shown by subcellular fractionation. These proteins remained tightly associated with the nucleus following both detergent and salt extraction, suggesting a tight interaction with a component of the nucleus.     

The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties.

Neurotactin (Nrt), a Drosophila transmembrane glycoprotein which is expressed in neuronal and epithelial tissues during embryonic and larval stages, exhibits heterophilic adhesive properties. The extracellular domain is composed of a catalytically inactive cholinesterase-like domain. A three-dimensional model deduced from the crystal structure of Torpedo acetylcholinesterase (AChE) has been constructed for Nrt and suggests that its extracellular domain is composed of two sub-domains organized around a gorge: an N-terminal region, whose three-dimensional structure is almost identical to that of Torpedo AChE, and a less conserved C-terminal region. By using truncated Nrt molecules and a homotypic cell aggregation assay which involves a soluble ligand activity, it has been possible to show that the adhesive function is localized in the N-terminal region of the extracellular domain comprised between His347 and His482. The C-terminal region of the protein can be removed without impairing Nrt adhesive properties, suggesting that the two sub-domains are structurally independent. Chimeric molecules in which the Nrt cholinesterase-like domain has been replaced by homologous domains from Drosophila AChE, Torpedo AChE or Drosophila glutactin (Glt), share similar adhesive properties. These properties may require the presence of Nrt cytoplasmic and transmembrane domains since authentic Drosophila AChE does not behave as an adhesive molecule when transfected in S2 cells.