scholarly journals The Complex Interplay between the Neck and Hinge Domains in Kinesin-1 Dimerization and Motor Activity

2005 ◽  
Vol 16 (8) ◽  
pp. 3529-3537 ◽  
Author(s):  
Friederike Bathe ◽  
Katrin Hahlen ◽  
Renate Dombi ◽  
Lucia Driller ◽  
Manfred Schliwa ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
Coiled Coil ◽  
Proximal Part ◽  
Tail Domain ◽  
Yeast Two Hybrid ◽  
Complex Interplay ◽  
Hybrid Data ◽  
Neck Domain ◽  
Two Hybrid ◽  
Complex Manner

Kinesin-1 dimerizes via the coiled-coil neck domain. In contrast to animal kinesins, neck dimerization of the fungal kinesin-1 NcKin requires additional residues from the hinge. Using chimeric constructs containing or lacking fungal-specific elements, the proximal part of the hinge was shown to stabilize the neck coiled-coil conformation in a complex manner. The conserved fungal kinesin hinge residue W384 caused neck coiled-coil formation in a chimeric NcKin construct, including parts of the human kinesin-1 stalk. The stabilizing effect was retained in a NcKinW384F mutant, suggesting important π -stacking interactions. Without the stalk, W384 was not sufficient to induce coiled-coil formation, indicating that W384 is part of a cluster of several residues required for neck coiled-coil folding. A W384-less chimera of NcKin and human kinesin possessed a non–coiled-coil neck conformation and showed inhibited activity that could be reactivated when artificial interstrand disulfide bonds were used to stabilize the neck coiled-coil conformation. On the basis of yeast two-hybrid data, we propose that the proximal hinge can bind kinesin's cargo-free tail domain and causes inactivation of kinesin by disrupting the neck coiled-coil conformation.

The SPI2-encoded SseA chaperone has discrete domains required for SseB stabilization and export, and binds within the C-terminus of SseB and SseD

Microbiology ◽  
2004 ◽  
Vol 150 (7) ◽  
pp. 2055-2068 ◽  
Author(s):  
Daniel V. Zurawski ◽  
Murry A. Stein
Keyword(s):  
Type Iii Secretion ◽  
Coiled Coil ◽  
Amphipathic Helix ◽  
Yeast Two Hybrid ◽  
C Terminus ◽  
N Terminus ◽  
Domain Mapping ◽  
Self Association ◽  
Discrete Domains ◽  
Two Hybrid

SseA, a key Salmonella virulence determinant, is a small, basic pI protein encoded within the Salmonella pathogenicity island 2 and serves as a type III secretion system chaperone for SseB and SseD. Both SseA partners are subunits of the surface-localized translocon module that delivers effectors into the host cell; SseB is predicted to compose the translocon sheath and SseD is a putative translocon pore subunit. In this study, SseA molecular interactions with its partners were characterized further. Yeast two-hybrid screens indicate that SseA binding requires a C-terminal domain within both partners. An additional central domain within SseD was found to influence binding. The SseA-binding region within SseB was found to encompass a predicted amphipathic helix of a type participating in coiled-coil interactions that are implicated in the assembly of translocon sheaths. Deletions that impinge upon this putative coiled-coiled domain prevent SseA binding, suggesting that SseA occupies a portion of the coiled-coil. SseA occupancy of this motif is envisioned to be sufficient to prevent premature SseB self-association inside bacteria. Domain mapping on the chaperone was also performed. A deletion of the SseA N-terminus, or site-directed mutations within this region, allowed stabilization of SseB, but its export was disrupted. Therefore, the N-terminus of SseA provides a function that is essential for SseB export, but dispensable for partner binding and stabilization.

HZwint-1, a novel human kinetochore component that interacts with HZW10

2000 ◽  
Vol 113 (11) ◽  
pp. 1939-1950 ◽  
Author(s):  
D.A. Starr ◽  
R. Saffery ◽  
Z. Li ◽  
A.E. Simpson ◽  
K.H. Choo ◽  
...  
Keyword(s):  
Hela Cells ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Interacting Protein ◽  
Dicentric Chromosomes ◽  
Centromere Function ◽  
Coiled Coil Domain ◽  
Gfp Fluorescence ◽  
Two Hybrid ◽  
Active Centromere

HZwint-1 (Human ZW10 interacting protein-1) was identified in a yeast two hybrid screen for proteins that interact with HZW10. HZwint-1 cDNA encodes a 43 kDa protein predicted to contain an extended coiled-coil domain. Immunofluorescence studies with sera raised against HZwint-1 protein revealed strong kinetochore staining in nocodazole-arrested chromosome spreads. This signal co-localizes at the kinetochore with HZW10, at a position slightly outside of the central part of the centromere as revealed by staining with a CREST serum. The kinetochore localization of HZwint-1 has been confirmed by following GFP fluorescence in HeLa cells transiently transfected with a plasmid encoding a GFP/HZwint-1 fusion protein. In cycling HeLa cells, HZwint-1 localizes to the kinetochore of prophase HeLa cells prior to HZW10 localization, and remains at the kinetochore until late in anaphase. This localization pattern, combined with the two-hybrid results, suggests that HZwint-1 may play a role in targeting HZW10 to the kinetochore at prometaphase. HZwint-1 was also found to localize to neocentromeres and to the active centromere of dicentric chromosomes. HZwint-1 thus appears to associate with all active centromeres, implying that it plays an important role in correct centromere function.

scholarly journals IE1 and hr facilitate the localization of Bombyx mori nucleopolyhedrovirus ORF8 to specific nuclear sites

2005 ◽  
Vol 86 (11) ◽  
pp. 3031-3038 ◽  
Author(s):  
WonKyung Kang ◽  
Noriko Imai ◽  
Yu Kawasaki ◽  
Toshihiro Nagamine ◽  
Shogo Matsumoto
Keyword(s):  
Bombyx Mori ◽  
Fluorescent Protein ◽  
Coiled Coil ◽  
Terminal Region ◽  
Mutant Protein ◽  
Yeast Two Hybrid ◽  
Focus Formation ◽  
Coiled Coil Domain ◽  
Nuclear Foci ◽  
Two Hybrid

The Bombyx mori nucleopolyhedrovirus (BmNPV) ORF8 protein has previously been reported to colocalize with IE1 to specific nuclear sites during infection. Transient expression of green fluorescent protein (GFP)-fused ORF8 showed the protein to have cytoplasmic localization, but following BmNPV infection the protein formed foci, suggesting that ORF8 requires some other viral factor(s) for this. Therefore, interacting factors were looked for using the yeast two-hybrid system and IE1 was identified. We mapped the interacting region of ORF8 using a yeast two-hybrid assay. An N-terminal region (residues 1–110) containing a predicted coiled-coil domain interacted with IE1, while a truncated N-terminal region (residues 1–78) that lacks this domain did not. In addition, a protein with a complete deletion of the N-terminal region failed to interact with IE1. These results suggest that the ORF8 N-terminal region containing the coiled-coil domain is required for the interaction with IE1. Next, whether IE1 plays a role in ORF8 localization was investigated. In the presence of IE1, GFP-ORF8 localized to the nucleus. In addition, cotransfection with a plasmid expressing IE1 and a plasmid containing the hr3 element resulted in nuclear foci formation. A GFP-fused ORF8 mutant protein containing the coiled-coil domain, previously shown to interact with IE1, also formed nuclear foci in the presence of IE1 and hr3. However, ORF8 mutant proteins that did not interact with IE1 failed to form nuclear foci. In contrast to wild-type IE1, focus formation was not observed for an IE1 mutant protein that was deficient in hr binding. These results suggest that IE1 and hr facilitate the localization of BmNPV ORF8 to specific nuclear sites.

scholarly journals PDZD8 Is a Novel Gag-Interacting Factor That Promotes Retroviral Infection

2010 ◽  
Vol 84 (17) ◽  
pp. 8990-8995 ◽  
Author(s):  
Matthew S. Henning ◽  
Scott G. Morham ◽  
Stephen P. Goff ◽  
Mojgan H. Naghavi
Keyword(s):  
Viral Entry ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Retroviral Infection ◽  
Gag Protein ◽  
Cellular Factors ◽  
Immunodeficiency Virus ◽  
Two Hybrid ◽  
Hiv 1

ABSTRACT In a yeast two-hybrid screen for cellular factors that could interact with human immunodeficiency virus type 1 (HIV-1) Gag protein, we identified PDZD8 and confirmed the interaction by coimmunoprecipitation (co-IP). PDZD8 overexpression promoted the initiation of reverse transcription and increased infection by pseudotyped retroviruses independent of the route of viral entry, while transient knockdown of endogenous levels decreased HIV-1 infection. A mutant of PDZD8 lacking a predicted coiled-coil domain in its Gag-interacting region failed to bind Gag and promote HIV-1 infection, identifying the domain of PDZD8 required for mediating these effects. As such, we identify PDZD8 as a novel positive mediator of retroviral infection.

scholarly journals Requirement for Bbp1p in the Proper Mitotic Functions of Cdc5p in Saccharomyces cerevisiae

2004 ◽  
Vol 15 (4) ◽  
pp. 1711-1723 ◽  
Author(s):  
Chong J. Park ◽  
Sukgil Song ◽  
Thomas H. Giddings ◽  
Hyeon-Su Ro ◽  
Krisada Sakchaisri ◽  
...  
Keyword(s):  
Coiled Coil ◽  
Spindle Pole ◽  
Stable Complex ◽  
Yeast Two Hybrid ◽  
Binding Studies ◽  
Interacting Protein ◽  
Spindle Pole Bodies ◽  
Vitro Binding ◽  
Two Hybrid

The polo-box domain of the budding yeast polo kinase Cdc5p plays an essential role for targeting the catalytic activity of Cdc5p to spindle pole bodies (SPBs) and cytokinetic neck-filaments. Here, we report the isolation of Bbp1p as a polo-box interacting protein by a yeast two-hybrid screen. Bbp1p localizes to the periphery of the central plaque of the SPB and plays an important role in SPB duplication. Similarly, Cdc5p localized to the cytoplasmic periphery of the SPB. In vitro binding studies showed that Cdc5p interacted with the N-terminal domain of Bbp1p (Bbp1pΔC), but apparently not with Mps2p, a component shown to form a stable complex with Bbp1p. In addition, Bbp1p, but likely not Mps2p, was required for proper localization of Cdc5p to the SPB. The C-terminal coiled-coil domain of Bbp1p (Bbp1p243–385), which is crucial for both the homodimerization and the SPB localization, could target the localization-defective Cdc5pΔC to the SPB and induce the release of Cdc14p from the nucleolus. Consistent with this observation, expression of CDC5ΔC-BBP1243–385 under CDC5 promoter control partially complemented the cdc5Δ defect. These data suggest that Bbp1pΔC interacts with the polo-box domain of Cdc5p, and this interaction is critical for the subcellular localization and mitotic functions of Cdc5p.

scholarly journals The nonhelical tail domain of keratin 14 promotes filament bundling and enhances the mechanical properties of keratin intermediate filaments in vitro

2001 ◽  
Vol 155 (5) ◽  
pp. 747-754 ◽  
Author(s):  
Olivier Bousquet ◽  
Linglei Ma ◽  
Soichiro Yamada ◽  
Changhong Gu ◽  
Toshihiro Idei ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Mechanical Strain ◽  
Type I ◽  
Basal Cells ◽  
Tail Domain ◽  
Yeast Two Hybrid ◽  
Keratin Filaments ◽  
Keratin Intermediate Filaments ◽  
Two Hybrid

Keratin filaments arise from the copolymerization of type I and II sequences, and form a pancytoplasmic network that provides vital mechanical support to epithelial cells. Keratins 5 and 14 are expressed as a pair in basal cells of stratified epithelia, where they occur as bundled arrays of filaments. In vitro, bundles of K5–K14 filaments can be induced in the absence of cross-linkers, and exhibit enhanced resistance to mechanical strain. This property is not exhibited by copolymers of K5 and tailless K14, in which the nonhelical tail domain has been removed, or copolymers of K5 and K19, a type I keratin featuring a short tail domain. The purified K14 tail domain binds keratin filaments in vitro with specificity (kD ∼2 μM). When transiently expressed in cultured cells, the K14 tail domain associates with endogenous keratin filaments. Utilization of the K14 tail domain as a bait in a yeast two-hybrid screen pulls out type I keratin sequences from a skin cDNA library. These data suggest that the tail domain of K14 contributes to the ability of K5–K14 filaments to self-organize into large bundles showing enhanced mechanical resilience in vitro.

scholarly journals Ang2/Fat-Free Is a Conserved Subunit of the Golgi-associated Retrograde Protein Complex

2010 ◽  
Vol 21 (19) ◽  
pp. 3386-3395 ◽  
Author(s):  
F. Javier Pérez-Victoria ◽  
Christina Schindler ◽  
Javier G. Magadán ◽  
Gonzalo A. Mardones ◽  
Cédric Delevoye ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Yeast Saccharomyces Cerevisiae ◽  
Coiled Coil Region ◽  
Significant Homology ◽  
Biochemical Analyses ◽  
Golgi Network ◽  
Two Hybrid ◽  
Garp Complex

The Golgi-associated retrograde protein (GARP) complex mediates tethering and fusion of endosome-derived transport carriers to the trans-Golgi network (TGN). In the yeast Saccharomyces cerevisiae, GARP comprises four subunits named Vps51p, Vps52p, Vps53p, and Vps54p. Orthologues of the GARP subunits, except for Vps51p, have been identified in all other eukaryotes. A yeast two-hybrid screen of a human cDNA library yielded a phylogenetically conserved protein, Ang2/Fat-free, which interacts with human Vps52, Vps53 and Vps54. Human Ang2 is larger than yeast Vps51p, but exhibits significant homology in an N-terminal coiled-coil region that mediates assembly with other GARP subunits. Biochemical analyses show that human Ang2, Vps52, Vps53 and Vps54 form an obligatory 1:1:1:1 complex that strongly interacts with the regulatory Habc domain of the TGN SNARE, Syntaxin 6. Depletion of Ang2 or the GARP subunits similarly impairs protein retrieval to the TGN, lysosomal enzyme sorting, endosomal cholesterol traffic¤ and autophagy. These findings indicate that Ang2 is the missing component of the GARP complex in most eukaryotes.

scholarly journals Ariadne-1: A Vital Drosophila Gene Is Required in Development and Defines a New Conserved Family of RING-Finger Proteins

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1231-1244
Author(s):  
Miguel Aguilera ◽  
Mariano Oliveros ◽  
Manuel Martínez-Padrón ◽  
Julio A Barbas ◽  
Alberto Ferrús
Keyword(s):  
Functional Characterization ◽  
Coiled Coil ◽  
Ring Finger ◽  
Cell Types ◽  
Null Alleles ◽  
Yeast Two Hybrid ◽  
Motor Impairments ◽  
Drosophila Gene ◽  
Human Pathology ◽  
Two Hybrid

Abstract We report the identification and functional characterization of ariadne-1 (ari-1), a novel and vital Drosophila gene required for the correct differentiation of most cell types in the adult organism. Also, we identify a sequence-related gene, ari-2, and the corresponding mouse and human homologues of both genes. All these sequences define a new protein family by the Acid-rich, RING finger, B-box, RING finger, coiled-coil (ARBRCC) motif string. In Drosophila, ari-1 is expressed throughout development in all tissues. The mutant phenotypes are most noticeable in cells that undergo a large and rapid membrane deposition, such as rewiring neurons during metamorphosis, large tubular muscles during adult myogenesis, and photoreceptors. Occasional survivors of null alleles exhibit reduced life span, motor impairments, and short and thin bristles. Single substitutions at key cysteines in each RING finger cause lethality with no survivors and a drastic reduction of rough endoplasmic reticulum that can be observed in the photoreceptors of mosaic eyes. In yeast two-hybrid assays, the protein ARI-1 interacts with a novel ubiquitin-conjugating enzyme, UbcD10, whose sequence is also reported here. The N-terminal RING-finger motif is necessary and sufficient to mediate this interaction. Mouse and fly homologues of both ARI proteins and the Ubc can substitute for each other in the yeast two-hybrid assay, indicating that ARI represents a conserved novel mechanism in development. In addition to ARI homologues, the RBR signature is also found in the Parkinson-disease-related protein Parkin adjacent to an ubiquitin-like domain, suggesting that the study of this mechanism could be relevant for human pathology.

Ptr ToxA Interacts with a Chloroplast-Localized Protein

2007 ◽  
Vol 20 (2) ◽  
pp. 168-177 ◽  
Author(s):  
Viola A. Manning ◽  
Linda K. Hardison ◽  
Lynda M. Ciuffetti
Keyword(s):  
Coiled Coil ◽  
Tan Spot ◽  
Yeast Two Hybrid ◽  
Mesophyll Cells ◽  
Pyrenophora Tritici Repentis ◽  
Ptr Toxa ◽  
Coiled Coil Domain ◽  
Multimeric Complex ◽  
Chloroplast Stroma ◽  
Two Hybrid

Pyrenophora tritici-repentis, causal agent of tan spot of wheat, produces host-selective toxins that are determinants of pathogenicity or virulence. Ptr ToxA (ToxA), a proteina-ceous toxin produced by P. tritici-repentis, is a necrotizing toxin produced by the most common races isolated from infected wheat. Recent studies have shown that ToxA is internalized into the mesophyll cells and localizes to chloroplasts of sensitive wheat cultivars only. We employed a yeast two-hybrid screen in an effort to determine plant proteins that interact with ToxA and found that ToxA interacts with a chloroplast protein, designated ToxA binding protein 1 (ToxABP1). ToxABP1 contains a lysine-rich region within a coiled-coil domain that is similar to phosphotidyl-inositol binding sites present in animal proteins involved in endocytosis. In both ToxA-sensitive and -insensitive cultivars, ToxABP1 is expressed at similar levels and encodes an identical protein. ToxABP1 protein is present in both chloroplast membranes and chloroplast stroma. ToxA appears to interact primarily with a multimeric complex of ToxABP1 protein associated with the chloroplast membrane.

scholarly journals Mapping the MinE Site Involved in Interaction with the MinD Division Site Selection Protein of Escherichia coli

2003 ◽  
Vol 185 (16) ◽  
pp. 4948-4955 ◽  
Author(s):  
Lu-Yan Ma ◽  
Glenn King ◽  
Lawrence Rothfield
Keyword(s):  
Escherichia Coli ◽  
Coiled Coil ◽  
Yeast Two Hybrid ◽  
Terminal Domain ◽  
Division Site ◽  
Coil Structure ◽  
Extended Surface ◽  
The Mind ◽  
Two Hybrid ◽  
Helical Region

ABSTRACT Interactions between the MinD and MinE proteins are required for proper placement of the Escherichia coli division septum. The site within MinE that is required for interaction with MinD was mapped by studying the effects of site-directed minE mutations on MinD-MinE interactions in yeast two-hybrid and three-hybrid experiments. This confirmed that the MinE N-terminal domain is responsible for the interaction of MinE with MinD. Mutations that interfered with the interaction defined an extended surface on one face of the α-helical region of the MinE N-terminal domain, consistent with the idea that the MinE-MinD interaction involves formation of a coiled-coil structure by interaction with a complementary helical surface within MinD.

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