scholarly journals Interplay Between the N-Terminal Domains of Arabidopsis Starch Synthase 3 Determines the Interaction of the Enzyme With the Starch Granule

2021 ◽  
Vol 12 ◽  
Author(s):  
Francisco M. Gámez-Arjona ◽  
Ángel Mérida
Keyword(s):  
Starch Granule ◽  
Coiled Coil ◽  
Terminal Region ◽  
Starch Synthase ◽  
Full Length ◽  
Localization Pattern ◽  
Binding Domains ◽  
Starch Synthases ◽  
Linear Chains

The elongation of the linear chains of starch is undertaken by starch synthases. class 3 of starch synthase (SS3) has a specific feature: a long N-terminal region containing starch binding domains (SBDs). In this work, we analyze in vivo the contribution of these domains to the localization pattern of the enzyme. For this purpose, we divided the N-terminal region of Arabidopsis SS3 in three domains: D1, D2, and D3 (each of which contains an SBD and a coiled-coil site). Our analyses indicate that the N-terminal region is sufficient to determine the same localization pattern observed with the full-length protein. D2 binds tightly the polypeptide to the polymer and it is necessary the contribution of D1 and D3 to avoid the polypeptide to be trapped in the growing polymer. The localization pattern of Arabidopsis SS3 appears to be the result of the counterbalanced action of the different domains present in its N-terminal region.

scholarly journals The KRAB-associated co-repressor KAP-1 is a coiled-coil binding partner, substrate and activator of the c-Fes protein tyrosine kinase

2006 ◽  
Vol 399 (1) ◽  
pp. 141-150 ◽  
Author(s):  
Frank J. Delfino ◽  
Jonathan M. Shaffer ◽  
Thomas E. Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Protein Tyrosine Kinase ◽  
Coiled Coil ◽  
Cell Types ◽  
Terminal Region ◽  
Full Length ◽  
Protein Tyrosine ◽  
Downstream Effector ◽  
Src Homology

The c-Fes protein tyrosine kinase is implicated in the differentiation of a number of cell types including neuronal, endothelial and myeloid cells. Structurally, Fes consists of a unique N-terminal region, followed by SH2 (Src homology domain 2) and kinase domains. Two coiled-coil (CC) domains (CC1 and CC2) located within the unique N-terminal region are critical regulators of Fes activity in vivo and may function to recruit Fes activators and/or substrates. A yeast two-hybrid screen, utilizing a K-562 cell cDNA library and the Fes CC2 domain as bait, identified an interacting clone encoding the CC domain and B-box motifs (residues 114–357) of the transcriptional co-repressor KRAB-associated protein (KAP)-1. KAP-1114–357 interacted with full-length Fes in yeast, and the KAP-1 CC domain was sufficient to bind the Fes N-terminal region in Sf-9 cells. Co-expression of Fes with full-length KAP-1 in human 293T cells stimulated Fes autophosphorylation and led to KAP-1 tyrosine phosphorylation. Association of endogenous Fes and KAP-1 was also observed in HL-60 myeloid leukaemia cells. Together, these data identify a novel Fes–KAP-1 interaction, and suggest a dual role for KAP-1 as both a Fes activator and downstream effector.

scholarly journals Microtubule Actin Cross-Linking Factor (Macf)

1999 ◽  
Vol 147 (6) ◽  
pp. 1275-1286 ◽  
Author(s):  
Conrad L. Leung ◽  
Dongming Sun ◽  
Min Zheng ◽  
David R. Knowles ◽  
Ronald K.H. Liem
Keyword(s):  
Calcium Binding ◽  
Coiled Coil ◽  
Specific Protein ◽  
Binding Domain ◽  
Full Length ◽  
Actin Binding ◽  
Cross Linking ◽  
Actin Binding Domain

We cloned and characterized a full-length cDNA of mouse actin cross-linking family 7 (mACF7) by sequential rapid amplification of cDNA ends–PCR. The completed mACF7 cDNA is 17 kb and codes for a 608-kD protein. The closest relative of mACF7 is the Drosophila protein Kakapo, which shares similar architecture with mACF7. mACF7 contains a putative actin-binding domain and a plakin-like domain that are highly homologous to dystonin (BPAG1-n) at its NH2 terminus. However, unlike dystonin, mACF7 does not contain a coiled–coil rod domain; instead, the rod domain of mACF7 is made up of 23 dystrophin-like spectrin repeats. At its COOH terminus, mACF7 contains two putative EF-hand calcium-binding motifs and a segment homologous to the growth arrest–specific protein, Gas2. In this paper, we demonstrate that the NH2-terminal actin-binding domain of mACF7 is functional both in vivo and in vitro. More importantly, we found that the COOH-terminal domain of mACF7 interacts with and stabilizes microtubules. In transfected cells full-length mACF7 can associate not only with actin but also with microtubules. Hence, we suggest a modified name: MACF (microtubule actin cross-linking factor). The properties of MACF are consistent with the observation that mutations in kakapo cause disorganization of microtubules in epidermal muscle attachment cells and some sensory neurons.

scholarly journals Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis.

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000 ◽  
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals The Two Variants of the Streptococcus pneumoniae Pilus 1 RrgA Adhesin Retain the Same Function and Elicit Cross-Protection In Vivo

2010 ◽  
Vol 78 (12) ◽  
pp. 5033-5042 ◽  
Author(s):  
Monica Moschioni ◽  
Carla Emolo ◽  
Massimiliano Biagini ◽  
Silvia Maccari ◽  
Werner Pansegrau ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Passive Immunization ◽  
Full Length ◽  
Lung Epithelial Cells ◽  
Binding Domains ◽  
Lung Epithelial ◽  
Islet 1 ◽  
Recombinant Fragments

ABSTRACT Thirty percent of Streptococcus pneumoniae isolates contain pilus islet 1, coding for a pilus composed of the backbone subunit RrgB and two ancillary proteins, RrgA and RrgC. RrgA is the major determinant of in vitro adhesion associated with pilus 1, is protective in vivo in mouse models, and exists in two variants (clades I and II). Mapping of the sequence variability onto the RrgA structure predicted from X-ray data showed that the diversity was restricted to the “head” of the protein, which contains the putative binding domains, whereas the elongated “stalk” was mostly conserved. To investigate whether this variability could influence the adhesive capacity of RrgA and to map the regions important for binding, two full-length protein variants and three recombinant RrgA portions were tested for adhesion to lung epithelial cells and to purified extracellular matrix (ECM) components. The two RrgA variants displayed similar binding abilities, whereas none of the recombinant fragments adhered at levels comparable to those of the full-length protein, suggesting that proper folding and structural arrangement are crucial to retain protein functionality. Furthermore, the two RrgA variants were shown to be cross-reactive in vitro and cross-protective in vivo in a murine model of passive immunization. Taken together, these data indicate that the region implicated in adhesion and the functional epitopes responsible for the protective ability of RrgA may be conserved and that the considerable level of variation found within the “head” domain of RrgA may have been generated by immunologic pressure without impairing the functional integrity of the pilus.

scholarly journals Cingulin Contains Globular and Coiled-Coil Domains and Interacts with Zo-1, Zo-2, Zo-3, and Myosin

1999 ◽  
Vol 147 (7) ◽  
pp. 1569-1582 ◽  
Author(s):  
Michelangelo Cordenonsi ◽  
Fabio D'Atri ◽  
Eva Hammar ◽  
David A.D. Parry ◽  
John Kendrick-Jones ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Mdck Cells ◽  
Coiled Coil ◽  
Full Length ◽  
Cytoplasmic Surface ◽  
Terminal Fragment ◽  
Reticulocyte Lysates ◽  
Actomyosin Cytoskeleton

We characterized the sequence and protein interactions of cingulin, an Mr 140–160-kD phosphoprotein localized on the cytoplasmic surface of epithelial tight junctions (TJ). The derived amino acid sequence of a full-length Xenopus laevis cingulin cDNA shows globular head (residues 1–439) and tail (1,326–1,368) domains and a central α-helical rod domain (440–1,325). Sequence analysis, electron microscopy, and pull-down assays indicate that the cingulin rod is responsible for the formation of coiled-coil parallel dimers, which can further aggregate through intermolecular interactions. Pull-down assays from epithelial, insect cell, and reticulocyte lysates show that an NH2-terminal fragment of cingulin (1–378) interacts in vitro with ZO-1 (Kd ∼5 nM), ZO-2, ZO-3, myosin, and AF-6, but not with symplekin, and a COOH-terminal fragment (377–1,368) interacts with myosin and ZO-3. ZO-1 and ZO-2 immunoprecipitates contain cingulin, suggesting in vivo interactions. Full-length cingulin, but not NH2-terminal and COOH-terminal fragments, colocalizes with endogenous cingulin in transfected MDCK cells, indicating that sequences within both head and rod domains are required for TJ localization. We propose that cingulin is a functionally important component of TJ, linking the submembrane plaque domain of TJ to the actomyosin cytoskeleton.

scholarly journals Calpain A modulates Toll responses by limited Cactus/IκB proteolysis

2013 ◽  
Vol 24 (18) ◽  
pp. 2966-2980 ◽  
Author(s):  
Marcio Fontenele ◽  
Bomyi Lim ◽  
Danielle Oliveira ◽  
Márcio Buffolo ◽  
David H. Perlman ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Immune System ◽  
Cysteine Proteases ◽  
Proteolytic Cleavage ◽  
Terminal Region ◽  
Full Length ◽  
Calcium Dependent ◽  
Protein Functions ◽  
Novel Protein

Calcium-dependent cysteine proteases of the calpain family are modulatory proteases that cleave their substrates in a limited manner. Among their substrates, calpains target vertebrate and invertebrate IκB proteins. Because proteolysis by calpains potentially generates novel protein functions, it is important to understand how this affects NFκB activity. We investigate the action of Calpain A (CalpA) on the Drosophila melanogaster IκB homologue Cactus in vivo. CalpA alters the absolute amounts of Cactus protein. Our data indicate, however, that CalpA uses additional mechanisms to regulate NFκB function. We provide evidence that CalpA interacts physically with Cactus, recognizing a Cactus pool that is not bound to Dorsal, a fly NFκB/Rel homologue. We show that proteolytic cleavage by CalpA generates Cactus fragments lacking an N-terminal region required for Toll responsiveness. These fragments are generated in vivo and display properties distinct from those of full-length Cactus. We propose that CalpA targets free Cactus, which is incorporated into and modulates Toll-responsive complexes in the embryo and immune system.

scholarly journals The RhoGEF Cysts couples apical polarity proteins to Rho and myosin activity at adherens junctions

2019 ◽  
Author(s):  
Jordan T. Silver ◽  
Frederik Wirtz-Peitz ◽  
Sérgio Simões ◽  
Milena Pellikka ◽  
Dong Yan ◽  
...  
Keyword(s):  
Rho Gtpases ◽  
Rho Gtpase ◽  
Adherens Junctions ◽  
Coiled Coil ◽  
Terminal Region ◽  
Nucleotide Exchange ◽  
Apical Polarity ◽  
Polarity Proteins ◽  
Spatio Temporal

AbstractThe spatio-temporal regulation of small Rho GTPases is crucial for the dynamic stability of epithelial tissues. However, how RhoGTPase activity is controlled during development remains largely unknown. To explore the regulation of Rho GTPases in vivo we analyzed the Rho GTPase guanine nucleotide exchange factor (RhoGEF) Cysts, the Drosophila orthologue of mammalian p114RhoGEF, GEF-H1, p190RhoGEF, and AKAP-13. Loss of Cysts causes a phenotype that closely resemble the mutant phenotype of the apical polarity regulator Crumbs. This phenotype can be suppressed by the loss of basolateral polarity proteins suggesting that Cysts in an integral component of the apical polarity protein network. Cysts activates Rho at adherens junctions to promote junctional enrichment of myosin II, which requires the RhoGEF domain and the coiled-coil domain containing C-terminal region of Cysts. Cysts recruitment to the apico-lateral cortex depends on Crumbs and Bazooka/Par3 and requires multiple domains within Cysts including the C-terminal region. Together, our findings indicate that Cysts links apical polarity proteins to Rho1 and myosin activation at adherens junctions to support junctional and epithelial integrity in the Drosophila ectoderm.

Organizational analysis of elav gene and functional analysis of ELAV protein of Drosophila melanogaster and Drosophila virilis

1991 ◽  
Vol 11 (6) ◽  
pp. 2994-3000
Author(s):  
K M Yao ◽  
K White
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rna Binding ◽  
Terminal Region ◽  
Drosophila Virilis ◽  
Functional Domain ◽  
Amino Terminal ◽  
Binding Domains ◽  
Rna Binding Domains

Drosophila virilis genomic DNA corresponding to the D. melanogaster embryonic lethal abnormal visual system (elav) locus was cloned. DNA sequence analysis of a 3.8-kb genomic piece allowed identification of (i) an open reading frame (ORF) with striking homology to the previously identified D. melanogaster ORF and (ii) conserved sequence elements of possible regulatory relevance within and flanking the second intron. Conceptual translation of the D. virilis ORF predicts a 519-amino-acid-long ribonucleoprotein consensus sequence-type protein. Similar to D. melanogaster ELAV protein, it contains three tandem RNA-binding domains and an alanine/glutamine-rich amino-terminal region. The sequence throughout the RNA-binding domains, comprising the carboxy-terminal 346 amino acids, shows an extraordinary 100% identity at the amino acid level, indicating a strong structural constraint for this functional domain. The amino-terminal region is 36 amino acids longer in D. virilis, and the conservation is 66%. In in vivo functional tests, the D. virilis ORF was indistinguishable from the D. melanogaster ORF. Furthermore, a D. melanogaster ORF encoding an ELAV protein with a 40-amino-acid deletion within the alanine/glutamine-rich region was also able to supply elav function in vivo. Thus, the divergence of the amino-terminal region of the ELAV protein reflects lowered functional constraint rather than species-specific functional specification.

scholarly journals Homotypic dimerization of the actin-binding protein p57/coronin-1 mediated by a leucine zipper motif in the C-terminal region

2005 ◽  
Vol 387 (2) ◽  
pp. 325-331 ◽  
Author(s):  
Teruaki OKU ◽  
Saotomo ITOH ◽  
Rie ISHII ◽  
Kensuke SUZUKI ◽  
William M. NAUSEEF ◽  
...  
Keyword(s):  
Leucine Zipper ◽  
Fluorescent Protein ◽  
Binding Protein ◽  
Coiled Coil ◽  
Terminal Region ◽  
Full Length ◽  
Actin Binding ◽  
Leucine Zipper Motif ◽  
Actin Binding Protein ◽  
Coiled Coil Domain

The actin-binding protein p57/coronin-1, a member of the coronin protein family, is selectively expressed in immune cells, and has been implicated in leucocyte migration and phagocytosis by virtue of its interaction with F-actin (filamentous actin). We previously identified two sites in the N-terminal region of p57/coronin-1 by which it binds actin, and in the present study we examine the role of the leucine zipper motif located in the C-terminal coiled-coil domain in mediating the homotypic association of p57/coronin-1. Recombinant p57/coronin-1 protein in solution formed a homodimer, as analysed by Superose 12 column chromatography and by sucrose density gradient centrifugation. In vivo, a truncated form consisting of the C-terminal coiled-coil domain co-precipitated with full-length p57/coronin-1 when both were co-expressed in COS-1 cells. A chimaeric construct composed of the C-terminal domain of p57/coronin-1 (which lacks the actin-binding sites) fused with green fluorescent protein co-localized with cortical F-actin-rich regions in COS-1 cells only when full-length p57/coronin-1 was expressed simultaneously in the cells, suggesting that the C-terminal region is required for the homotypic association of p57/coronin-1. Furthermore, p57LZ, a polypeptide consisting of the C-terminal 90 amino acid residues of p57/coronin-1, was sufficient for dimerization. When two leucine residues out of the four that constitute the leucine zipper structure in p57LZ or full-length p57 were replaced with alanine residues, the mutants failed to form homodimers. Taken together, these results demonstrate that p57/coronin-1 forms homodimers, that the association is mediated by the leucine zipper structure in the C-terminal region, and that it plays a role in the cross-linking of F-actin in the cell.

scholarly journals Centrosomal Proteins CG-NAP and Kendrin Provide Microtubule Nucleation Sites by Anchoring γ-Tubulin Ring Complex

2002 ◽  
Vol 13 (9) ◽  
pp. 3235-3245 ◽  
Author(s):  
Mikiko Takahashi ◽  
Akiko Yamagiwa ◽  
Tamako Nishimura ◽  
Hideyuki Mukai ◽  
Yoshitaka Ono
Keyword(s):  
Spindle Pole Body ◽  
Coiled Coil ◽  
Spindle Pole ◽  
Terminal Region ◽  
Microtubule Assembly ◽  
Microtubule Nucleation ◽  
Amino Terminal ◽  
Ring Complex ◽  
Carboxyl Terminal

Microtubule assembly is initiated by the γ-tubulin ring complex (γ-TuRC). In yeast, the microtubule is nucleated from γ-TuRC anchored to the amino-terminus of the spindle pole body component Spc110p, which interacts with calmodulin (Cmd1p) at the carboxy-terminus. However, mammalian protein that anchors γ-TuRC remains to be elucidated. A giant coiled-coil protein, CG-NAP (centrosome and Golgi localized PKN-associated protein), was localized to the centrosome via the carboxyl-terminal region. This region was found to interact with calmodulin by yeast two-hybrid screening, and it shares high homology with the carboxyl-terminal region of another centrosomal coiled-coil protein, kendrin. The amino-terminal region of either CG-NAP or kendrin indirectly associated with γ-tubulin through binding with γ-tubulin complex protein 2 (GCP2) and/or GCP3. Furthermore, endogenous CG-NAP and kendrin were coimmunoprecipitated with each other and with endogenous GCP2 and γ-tubulin, suggesting that CG-NAP and kendrin form complexes and interact with γ-TuRC in vivo. These proteins were localized to the center of microtubule asters nucleated from isolated centrosomes. Pretreatment of the centrosomes by antibody to CG-NAP or kendrin moderately inhibited the microtubule nucleation; moreover, the combination of these antibodies resulted in stronger inhibition. These results imply that CG-NAP and kendrin provide sites for microtubule nucleation in the mammalian centrosome by anchoring γ-TuRC.

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