Unique N-terminal Domain Research Articles

Evolutionary pathways of an ancient gene recX

RecX is a regulator of RecA activity by interacting with RecA protein or RecA filaments. Genes encoding RecX were found in genomes of a wide diversity of bacteria and some plants ( e.g., Arabidopsis thaliana and Oryza sativa). Our comparative genome analysis showed that although members of the RecX family are found in many bacterial species, they are not found in archaea and the only gene found in eukaryotes is likely derived from bacteria genomes. It is therefore proposed that RecX is of bacterial origin, and the gene had presented in the common ancestor of bacteria. Moreover, bacterial RecX and plant RecX domain are homologues, and RecX domain in plants may have derived from bacteria via unknown pathways. Plant RecX-like protein was formed by a gene fusion event between a unique N-terminal domain of unknown origin and RecX domain within plant cells. Finally, three possible evolutionary pathways from bacteria to plant were discussed.

Highly Conserved Regions within the Spike Proteins of Human Coronaviruses 229E and NL63 Determine Recognition of Their Respective Cellular Receptors

We have recently demonstrated that the severe acute respiratory syndrome coronavirus (SARS-CoV) receptor angiotensin converting enzyme 2 (ACE2) also mediates cellular entry of the newly discovered human coronavirus (hCoV) NL63. Here, we show that expression of DC-SIGN augments NL63 spike (S)-protein-driven infection of susceptible cells, while only expression of ACE2 but not DC-SIGN is sufficient for entry into nonpermissive cells, indicating that ACE2 fulfills the criteria of a bona fide hCoV-NL63 receptor. As for SARS-CoV, murine ACE2 is used less efficiently by NL63-S for entry than human ACE2. In contrast, several amino acid exchanges in human ACE2 which diminish SARS-S-driven entry do not interfere with NL63-S-mediated infection, suggesting that SARS-S and NL63-S might engage human ACE2 differentially. Moreover, we observed that NL63-S-driven entry was less dependent on a low-pH environment and activity of endosomal proteases compared to infection mediated by SARS-S, further suggesting differences in hCoV-NL63 and SARS-CoV cellular entry. NL63-S does not exhibit significant homology to SARS-S but is highly related to the S-protein of hCoV-229E, which enters target cells by engaging CD13. Employing mutagenic analyses, we found that the N-terminal unique domain in NL63-S, which is absent in 229E-S, does not confer binding to ACE2. In contrast, the highly homologous C-terminal parts of the NL63-S1 and 229E-S1 subunits in conjunction with distinct amino acids in the central regions of these proteins confer recognition of ACE2 and CD13, respectively. Therefore, despite the high homology of these sequences, they likely form sufficiently distinct surfaces, thus determining receptor specificity.

A Novel Route for F-box Protein-mediated Ubiquitination Links CHIP to Glycoprotein Quality Control

In SCF (Skp1/Cullin/F-box protein) ubiquitin ligases, substrate specificity is conferred by a diverse array of F-box proteins. Only in fully assembled SCF complexes, it is believed, can substrates bound to F-box proteins become ubiquitinated. Here we show that Fbx2, a brain-enriched F-box protein implicated in the ubiquitination of glycoproteins discarded from the endoplasmic reticulum, binds the co-chaperone/ubiquitin ligase CHIP (C terminus of Hsc-70-interacting protein) through a unique N-terminal PEST domain in Fbx2. CHIP facilitates the ubiquitination and degradation of Fbx2-bound glycoproteins, including unassembled NMDA receptor subunits. These findings indicate that CHIP acts with Fbx2 in a novel ubiquitination pathway that links CHIP to glycoprotein quality control in neurons. In addition, they expand the repertoire of pathways by which F-box proteins can regulate ubiquitination and suggest a new role for PEST domains as a protein interaction motif.

The first N-terminal transmembrane helix of each subunit of the antigenic peptide transporter TAP is essential for independent tapasin binding

The heterodimeric ABC transporter TAP translocates proteasomal degradation products from the cytosol into the lumen of the endoplasmic reticulum, where these peptides are loaded onto MHC class I molecules by a macromolecular peptide-loading complex (PLC) and subsequently shuttled to the cell surface for inspection by cytotoxic T lymphocytes. Tapasin recruits, as a central adapter protein, other components of the PLC at the unique N-terminal domains of TAP. We found that the N-terminal domains of human TAP1 and TAP2 can independently bind to tapasin, thus providing two separate loading platforms for PLC assembly. Moreover, tapasin binding is dependent on the first N-terminal transmembrane helix of TAP1 and TAP2, demonstrating that these two helices contribute independently to the recruitment of tapasin and associated factors.

Structure of human tryptophanyl-tRNA synthetase in complex with tRNATrp reveals the molecular basis of tRNA recognition and specificity

Aminoacyl-tRNA synthetases (aaRSs) are a family of enzymes responsible for the covalent link of amino acids to their cognate tRNAs. The selectivity and species-specificity in the recognitions of both amino acid and tRNA by aaRSs play a vital role in maintaining the fidelity of protein synthesis. We report here the first crystal structure of human tryptophanyl-tRNA synthetase (hTrpRS) in complex with tRNATrp and Trp which, together with biochemical data, reveals the molecular basis of a novel tRNA binding and recognition mechanism. hTrpRS recognizes the tRNA acceptor arm from the major groove; however, the 3′ end CCA of the tRNA makes a sharp turn to bind at the active site with a deformed conformation. The discriminator base A73 is specifically recognized by an α-helix of the unique N-terminal domain and the anticodon loop by an α-helix insertion of the C-terminal domain. The N-terminal domain appears to be involved in Trp activation, but not essential for tRNA binding and acylation. Structural and sequence comparisons suggest that this novel tRNA binding and recognition mechanism is very likely shared by other archaeal and eukaryotic TrpRSs, but not by bacterial TrpRSs. Our findings provide insights into the molecular basis of tRNA specificity and species-specificity.

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to ∼200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation.

Characterization of TccP-mediated N-WASP activation during enterohaemorrhagic Escherichia coli infection

Subversion of the host cell cytoskeleton is the hallmark of enterohaemorrhagic Escherichia coli (EHEC) infection. EHEC translocates the trans-membrane receptor protein Tir (translocated intimin receptor), which links the extracellular bacterium to the eukaryotic cell actin cytoskeleton, triggering formation of actin-rich pedestals beneath adherent bacteria. Tir-mediated actin accretion by EHEC requires TccP (Tir cytoskeleton coupling protein), a recently discovered type III secretion system effector protein which, following translocation, binds and activates Wiskott-Aldrich syndrome protein (N-WASP), which in turn activates the actin-related protein 2/3 complex leading to localized polymerization of actin. In this study, truncated N-WASP and TccP derivatives were generated and tested in in vitro actin polymerization and epithelial cell infection assays. The C-terminal amino acids 253-276 of the GTPase binding domain (GBD) of N-WASP were identified as essential, although not sufficient, for TccP:N-WASP protein:protein interaction, TccP-mediated N-WASP activation and induction of actin polymerization. TccP from EHEC O157:H7 strain EDL933 consists of a unique N-terminal domain and six proline-rich repeats. Progressive deletions within the N-terminus of TccP revealed that residues 1-21 are necessary and sufficient for its translocation, while amino acids 1-181, encompassing the N-terminal translocation signal and two proline-rich repeats, are sufficient for triggering actin polymerization in EHEC-infected epithelial cells and in in vitro actin polymerization assays. This study defines the modular domain structure of TccP and the molecular basis of TccP-mediated N-WASP activation and EHEC-induced remodelling of the host actin cytoskeleton.

A Chlorophyll a/b-binding Protein Homolog That Is Induced by Iron Deficiency Is Associated with Enlarged Photosystem I Units in the Eucaryotic Alga Dunaliella salina

Adaptation of the halotolerant alga Dunaliella salina to iron deprivation involves extensive changes of chloroplast morphology, photosynthetic activities, and induction of a major 45-kDa chloroplast protein termed Tidi. Partial amino acid sequencing of proteolytic peptides suggested that Tidi resembles chlorophyll a/b-binding proteins which compose light-harvesting antenna complexes (LHC) (Varsano, T., Kaftan, D., and Pick, U. (2003) J. Plant Nutr. 26, 2197-2210). Here we show that Tidi shares the highest amino acid sequence similarity with light-harvesting I chlorophyll a/b-binding proteins from higher plants but has an extended proline-rich N-terminal domain. The accumulation of Tidi is reversed by iron supplementation, and its level is inversely correlated with photosystem I (PS-I) reaction center proteins. In native gel electrophoresis, Tidi co-migrates with enlarged PS-I-LHC-I super-complexes. Single particle electron microscopy analysis revealed that PS-I units from iron-deficient cells are larger (31 and 37 nm in diameter) than PS-I units from control cells (22 nm). The 77 K chlorophyll fluorescence emission spectra of isolated complexes suggest that the Tidi-LHC-I antenna are functionally coupled to the reaction centers of PS-I. These findings indicate that Tidi acts as an accessory antenna of PS-I. The enlargement of PS-I antenna in algae and in cyanobacteria under iron deprivation suggests a common limitation that requires rebalancing of the energy distribution between the two photosystems.

Membrane Topology of the Transporter Associated with Antigen Processing (TAP1) within an Assembled Functional Peptide-loading Complex

The transporter associated with antigen processing (TAP) translocates antigenic peptides from the cytosol into the endoplasmic reticular lumen for subsequent loading onto major histocompatibility complex (MHC) class I molecules. These peptide-MHC complexes are inspected at the cell surface by cytotoxic T-lymphocytes. Assembly of the functional peptide transport and loading complex depends on intra- and intermolecular packing of transmembrane helices (TMs). Here, we have examined the membrane topology of human TAP1 within an assembled and functional transport complex by cysteine-scanning mutagenesis. The accessibility of single cysteine residues facing the cytosol or endoplasmic reticular lumen was probed by a minimally invasive approach using membrane-impermeable, thiol-specific fluorophores in semipermeabilized "living" cells. TAP1 contains ten transmembrane segments, which place the N and C termini in the cytosol. The transmembrane domain consists of a translocation core of six TMs, a building block conserved among most ATP-binding cassette transporters, and a unique additional N-terminal domain of four TMs, essential for tapasin binding and assembly of the peptide-loading complex. This study provides a first map of the structural organization of the TAP machinery within the macromolecular MHCI peptide-loading complex.

Four and a Half LIM Protein 1 Binds Myosin-binding Protein C and Regulates Myosin Filament Formation and Sarcomere Assembly

Four and a half LIM protein 1 (FHL1/SLIM1) is highly expressed in skeletal and cardiac muscle; however, the function of FHL1 remains unknown. Yeast two-hybrid screening identified slow type skeletal myosin-binding protein C as an FHL1 binding partner. Myosin-binding protein C is the major myosin-associated protein in striated muscle that enhances the lateral association and stabilization of myosin thick filaments and regulates actomyosin interactions. The interaction between FHL1 and myosin-binding protein C was confirmed using co-immunoprecipitation of recombinant and endogenous proteins. Recombinant FHL2 and FHL3 also bound myosin-binding protein C. FHL1 impaired co-sedimentation of myosin-binding protein C with reconstituted myosin filaments, suggesting FHL1 may compete with myosin for binding to myosin-binding protein C. In intact skeletal muscle and isolated myofibrils, FHL1 localized to the I-band, M-line, and sarcolemma, co-localizing with myosin-binding protein C at the sarcolemma in intact skeletal muscle. Furthermore, in isolated myofibrils FHL1 staining at the M-line appeared to extend partially into the C-zone of the A-band, where it co-localized with myosin-binding protein C. Overexpression of FHL1 in differentiating C2C12 cells induced "sac-like" myotube formation (myosac), associated with impaired Z-line and myosin thick filament assembly. This phenotype was rescued by co-expression of myosin-binding protein C. FHL1 knockdown using RNAi resulted in impaired myosin thick filament formation associated with reduced incorporation of myosin-binding protein C into the sarcomere. This study identified FHL1 as a novel regulator of myosin-binding protein C activity and indicates a role for FHL1 in sarcomere assembly.

Structural basis for the inhibition of activin signalling by follistatin

The secreted, multidomain protein follistatin binds activins with high affinity, inhibiting their receptor interaction. We have dissected follistatin's domain structure and shown that the minimal activin-inhibiting fragment of follistatin is comprised of the first and second Fs domains (Fs12). This protein can bind to activin dimer and form a stable complex containing two Fs12 molecules and one activin dimer. We have solved crystal structures of activin A alone and its complex with Fs12 fragment to 2 A resolution. The complex structure shows how Fs12 molecules wrap around the back of the 'wings' of activin, blocking the type II receptor-binding site on activin A. Arginine 192 in Fs2 is a key residue in this interaction, inserting itself in between activin's fingers. Complex formation imposes a novel orientation for the EGF- and Kazal-like subdomains in the Fs2 domain and activin A shows further variation from the canonical TGF-beta family fold. The structure provides a detailed description of the inhibitory mechanism and gives insights into interactions of follistatin with other TGF-beta family proteins.

Structure Function Analysis of Hematopoietic Specific Src Family Kinases Lyn, Hck and Fgr Reveal a Critical Role for Lyn's Unique Domain in Regulating Growth of Myeloid Cells That Is Distinct from the Role of Hck and Fgr's Unique Domain.

A flurry of recent publications have implicated hematopoietic specific Src family kinases (SFKs) in contributing to the pathogenesis of lymphoid and myeloid leukemia. However, little is known about the mechanism(s) by which these kinases regulate the growth of myeloid cells. SFKs share significant structural and amino acid sequence homology, particularly in the catalytic domain and in the SH2 and SH3 domains of the regulatory region. Therefore, it is generally believed that SFKs play a redundant role in regulating growth and actin based functions. Although, there are no apparent differences in the known effector domains of SFKs, significant differences exist between the amino terminus unique regions of Hck, Fgr and Lyn. Here, we show that the deficiency of Hck or Fgr or both in myeloid cells results in reduced cytokine (stem cell factor [SCF] & IL-3) induced proliferation. In contrast, deficiency of Lyn SFK results in enhanced growth and survival of myeloid cells. SCF induced hypersensitivity due to Lyn deficiency is observed in spite of the presence of Hck and Fgr in myeloid cells, suggesting that Lyn functions with specificity in negatively regulating cytokine signaling. To determine whether SCF induced hypersensitivity due to Lyn deficiency is contributed via its N-terminal unique domain, we cloned and retrovirally expressed a mutant version of Lyn that lacks its unique domain. As expected, reconstituting Lyn−/− myeloid cells with a cDNA encoding the wildtype version of Lyn completely restored cytokine induced hypersensitivity to wildtype levels. In contrast, expression of a Lyn mutant lacking its N-terminal unique domain, but consistent of all the other domains, including the myristolation and the palmitoylation acetylation sites behaved in a fashion similar to Lyn deficient myeloid cells. Furthermore, neither overexpression of Hck, nor Fgr in Lyn−/− cells affected cytokine induced hypersensitivity. To further characterize the unique nature of Lyn's N-terminal (unique) domain in cytokine induced hypersensitivity, we cloned and expressed several chimeric proteins in which the unique domain of Lyn was replaced with the unique domain of Hck or Fgr. When expressed in Lyn−/− cells, these chimeric proteins were unable to correct cytokine induced hypersensitivity due to Lyn deficiency. In contrast, swapping Hck's or Fgr's unique domain with Lyn's unique domain completely rescued cytokine induced hypersensitivity in Lyn−/− cells. These results suggest that Lyn's unique domain plays a critical role in negatively regulating cytokine induced growth in myeloid cells. Although Lyn contains a palmitoylation and an acetylation site, the specific role of these sites in membrane targeting and in cytokine-induced growth is not known. To assess the role of these sites, we generated three additional mutants of Lyn and expressed them in Lyn−/− cells. Mutant of Lyn lacking both the acetylation and the palmitoylation site demonstrated cytokine induced hypersensitivity similar to that seen in Lyn−/− cells. In contrast, mutating the acetylation site alone in Lyn did not affect cytokine induced hyperproliferation, although mutating the palmitoylation site did result in cytokine induced hypersensitivity. Taken together, our results provide compeling evidence for a novel role for Lyn's unique domain in negatively regulating cytokine signaling, which is distinct from the role of Hck or Fgr's unique domain.

Differential effects of chemoreceptor methylation‐domain mutations on swarming and development in the social bacterium Myxococcus xanthus

The soil bacterium Myxococcus xanthus is a model organism for the study of multicellular behaviour and development in bacteria. M. xanthus cells move on solid surfaces by gliding motility, periodically reversing their direction of movement. Motility is co-ordinated to allow cells to effectively feed on macromolecules or prey bacteria when nutrients are plentiful and to form developmental fruiting bodies when nutrients are limiting. The Frz signal transduction pathway regulates cellular movements by modulating cell reversal frequency. Input to the Frz pathway is controlled by the cytoplasmic receptor, FrzCD, a methyl-accepting chemotaxis protein (MCP). FrzCD lacks the transmembrane and periplasmic domains common to MCPs but contains a unique N-terminal domain, the predicted ligand-binding domain. As deletion of the N-terminal domain of FrzCD only results in minor defects in motility, we investigated the possibility that the methylation of the conserved C-terminal domain of FrzCD plays a central role in regulating the pathway. For this study, each of the potential methylation sites of FrzCD were systematically modified by site-directed mutagenesis, substituting glutamine/glutamate pairs for alanines. Four of the seven mutations produced dramatic phenotypes; two of the mutations had a stimulatory effect on the pathway, as evidenced by cells hyper-reversing, whereas another two had an inhibitory effect, causing these cells to rarely reverse. These four mutants displayed defects in vegetative swarming and developmental aggregation. These results suggests a model in which the methylation domain can both activate and inhibit the Frz pathway depending on which residues are methylated. The diversity of phenotypes suggests that specific modifications of FrzCD act to differentially regulate motility and developmental aggregation in M. xanthus.

Regulation of Rat Basophilic Leukemia-2H3 Mast Cell Secretion by a Constitutive Lyn Kinase Interaction with the High Affinity IgE Receptor (FcεRI)

Signaling through the high affinity IgE receptor is initiated by noncovalently associated Lyn kinase, resulting in the secretion of inflammatory mediators from mast cells. A fraction of the total cellular Lyn is associated via its N-terminal unique domain with the cytoplasmic domain of the Fc epsilonRI beta subunit before receptor aggregation. In the current study, we stably transfected the unique domain of Lyn into rat basophilic leukemia-2H3 mast cells and examined the consequences on Fc epsilonRI-induced signal transduction and mediator secretion to further define the role of the unique domain of Lyn in mast cell secretion. Tyrosine phosphorylation of Fc epsilonRI beta and gamma subunits was partially inhibited in the Lyn unique domain transfectants after Ag stimulation. Ag stimulation of Lyn unique domain transfectants was accompanied by enhanced phosphorylation of MEK and ERK-2, which are required for leukotriene C4 (LTC4) release, and production of LTC4 was increased 3- to 5-fold, compared with cells transfected with vector alone. Conversely, tyrosine phosphorylation of the adaptor protein Gab2, which is essential for mast cell degranulation, was inhibited after Ag stimulation of Lyn unique domain transfectants, and Ag-induced release of histamine was inhibited up to 48%. In rat basophilic leukemia-2H3 cells, Lyn thus plays a dual role by positively regulating Fc epsilonRI phosphorylation and degranulation while negatively regulating LTC4 production. This study provides further evidence that the constitutive interaction between the unique domain of Lyn and the Fc epsilonRI beta subunit is a crucial step in the initiation of Fc epsilonRI signaling and that Lyn is limiting for Fc epsilonRI-induced secretion of inflammatory mediators.

Role of the N-Terminal Domain of Endoinulinase from Arthrobacter sp. S37 in Regulation of Enzyme Catalysis

Endoinulinase from Arthrobacter sp. S37 (EnIA), a member of the glycoside hydrolase family 32, is unique in that, unlike other members of the family, it contains a 250-residue N-terminal domain including a "laminin-G like jelly-roll" fold. This unique N-terminal domain is here suggested to be involved in dimerization and catalysis. The essentially inactive nature of enzymes produced by N-terminal truncation (Delta15, Delta45, Delta70, and Delta250) supported the pivotal role of this unique domain in catalysis and the need for its structural integrity. Significant reductions in the enzyme efficiency (kcat/Km) were observed when mutations were introduced at highly conserved tryptophan residues (Trp75 and Trp141) in the laminin-G like jelly-roll fold, implying their involvement in catalysis. Results from size-exclusion chromatography of the native and chimeric enzymes in the presence and absence of the domain suggested that the N-terminal domain could mediate dimerization.

Cellular Distribution, Post-translational Modification, and Tumorigenic Potential of Human Group III Secreted Phospholipase A2

Human group III secreted phospholipase A(2) (sPLA(2)-III) consists of a central group III sPLA(2) domain flanked by unique N- and C-terminal domains. We found that the sPLA(2) domain alone was sufficient for its catalytic activity and for its prostaglandin E(2) (PGE(2))-generating functions in various cell types. In several if not all cell types, the N- and C-terminal domains of sPLA(2)-III were proteolytically removed, leading to the production of the form containing only the sPLA(2) domain, which could be further N-glycosylated at two consensus sites. Immunohistochemistry demonstrated that sPLA(2)-III was preferentially expressed in the microvascular endothelium in human tissues with inflammation, ischemic injury, and cancer. In support of this, sPLA(2)-III was induced in cultured microvascular endothelial cells after stimulation with proinflammatory cytokines. Expression of sPLA(2)-III was also associated with various tumor cells, and colorectal cancer cells transfected with sPLA(2)-III exhibited enhanced PGE(2) production and cell proliferation, which required sPLA(2)-III catalytic activity. When implanted into nude mice, the sPLA(2)-III-transfected cells formed larger solid tumors with increased angiogenesis compared with control cells. Moreover, small interfering RNA for sPLA(2)-III significantly reduced PGE(2) production and proliferation of colorectal cancer cells. Taken together, these results reveal unique cell type-specific processing and N-glycosylation of sPLA(2)-III and the potential role of this enzyme in cancer development by stimulating tumor cell growth and angiogenesis.

Generation and Characterization of Monoclonal Antibodies to Human PCTAIRE 3

Monoclonal antibodies specific for the unique N-terminal domain of the PCTAIRE 3 protein kinase have been produced and characterized. The specificity of these antibodies has been assessed by ELISA, Western blot, and immunoflorescence techniques. These reagents are specific for recombinant PCTAIRE 3 of human origin and do not cross-react with PCTAIRE 1 or PCTAIRE 2, despite the high homology between members of the PCTAIRE subfamily of cdc2-like kinases. Immunoblotting of transfected cells reveals that all of the antibodies recognize both isoforms of the PCTAIRE 3 kinase. Epitope mapping reveals three distinct classes of PCTAIRE 3 antibodies that recognize different sites within an immunodominant region of the N-terminal domain of the protein. The creation of highly specific PCTAIRE 3 monoclonal reagents will be useful for assessing the native distribution and function of this protein.

The N-Terminal Domain of MYO18A Has an ATP-Insensitive Actin-Binding Site

Myosin XVIII is the recently identified 18th class of myosins, and its members are composed of a unique N-terminal domain, a motor domain with an unusual sequence around the ATPase site, one IQ motif, a segmented coiled-coil region for dimerization, and a C-terminal globular tail. To gain insight into the functions of this unique myosin, we characterized its human homologue, MYO18A, focusing on the functional roles of the characteristic N-terminal domain that contains a PDZ module known to mediate protein-protein interaction. GFP-tagged full-length and C-terminally truncated MYO18A molecules that were expressed in HeLa cells exhibited colocalization with actin filaments. Chemical cross-linking of these molecules showed that they form stable dimers as expected from their putative coiled-coil tails. Cosedimentation of the various types of truncated MYO18A constructs with actin filaments indicated the presence of an ATP-insensitive actin-binding site in the N-terminal domain. Further studies on truncated constructs of the N-terminal domain indicated that this actin-binding site is located outside the PDZ module, but within the middle region of this domain, which does not show any homology with the known actin-binding motifs. These results imply that this dimeric myosin might stably cross-link actin filaments by two ATP-insensitive actin-binding sites at the N-terminal domains for higher-order organization of the actin cytoskeleton.

The Functionally Conserved Nucleoporins Nup124p from Fission Yeast and the Human Nup153 Mediate Nuclear Import and Activity of the Tf1 Retrotransposon and HIV-1 Vpr

We report that the fission yeast nucleoporin Nup124p is required for the nuclear import of both, retrotransposon Tf1-Gag as well as the retroviral HIV-1 Vpr. Failure to import Tf1-Gag into the nucleus in a nup124 null mutant resulted in complete loss of Tf1 transposition. Similarly, nuclear import of HIV-1 Vpr was impaired in nup124 null mutant strains and cells became resistant to Vpr's cell-killing activity. On the basis of protein domain similarity, the human nucleoporin Nup153 was identified as a putative homolog of Nup124p. We demonstrate that in vitro-translated Nup124p and Nup153 coimmunoprecipitate Tf1-Gag or HIV-1 Vpr. Though full-length Nup153 was unable to complement the Tf1 transposition defect in a nup124 null mutant, we provide evidence that both nucleoporins share a unique N-terminal domain, Nup124p(AA264-454) and Nup153(AA448-634) that is absolutely essential for Tf1 transposition. Epigenetic overexpression of this domain in a wild-type (nup124(+)) background blocked Tf1 activity implying that sequences from Nup124p and the human Nup153 challenged the same pathway affecting Tf1 transposition. Our results establish a unique relationship between two analogous nucleoporins Nup124p and Nup153 wherein the function of a common domain in retrotransposition is conserved.

Annexin A1 Interaction with a Zwitterionic Phospholipid Monolayer: A Fluorescence Microscopy Study

We present the results of a fluorescence microscopy study of the interaction of annexin A1 with dipalmitoylphosphatidylcholine (DPPC) monolayers as a function of the lipid monolayer phase and the pH of the aqueous subphase. We show that annexin A1-DPPC interaction depends strongly on the domain structure of the DPPC monolayer and only weakly on the subphase pH. Annexin A1 is found to be line active, with preferential adsorption at phase boundaries. Also, annexin A1 is found to form networks in the presence of a domain structure in the monolayer. Our results point toward an important contribution of the unique N-terminal domain to the organization of the protein at the interface.