scholarly journals ODA16 aids axonemal outer row dynein assembly through an interaction with the intraflagellar transport machinery

2008 ◽  
Vol 183 (2) ◽  
pp. 313-322 ◽  
Author(s):  
Noveera T. Ahmed ◽  
Chunlei Gao ◽  
Ben F. Lucker ◽  
Douglas G. Cole ◽  
David R. Mitchell
Keyword(s):  
Binding Site ◽  
Intraflagellar Transport ◽  
Site Formation ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
B Subunit ◽  
Dynein Arms ◽  
Two Hybrid

Formation of flagellar outer dynein arms in Chlamydomonas reinhardtii requires the ODA16 protein at a previously uncharacterized assembly step. Here, we show that dynein extracted from wild-type axonemes can rebind to oda16 axonemes in vitro, and dynein in oda16 cytoplasmic extracts can bind to docking sites on pf28 (oda) axonemes, which is consistent with a role for ODA16 in dynein transport, rather than subunit preassembly or binding site formation. ODA16 localization resembles that seen for intraflagellar transport (IFT) proteins, and flagellar abundance of ODA16 depends on IFT. Yeast two-hybrid analysis with mammalian homologues identified an IFT complex B subunit, IFT46, as a directly interacting partner of ODA16. Interaction between Chlamydomonas ODA16 and IFT46 was confirmed through in vitro pull-down assays and coimmunoprecipitation from flagellar extracts. ODA16 appears to function as a cargo-specific adaptor between IFT particles and outer row dynein needed for efficient dynein transport into the flagellar compartment.

scholarly journals Inhibition of Ubiquitination and Stabilization of Human Ubiquitin E3 Ligase PIRH2 by Measles Virus Phosphoprotein

2005 ◽  
Vol 79 (18) ◽  
pp. 11824-11836 ◽  
Author(s):  
Mingzhou Chen ◽  
Jean-Claude Cortay ◽  
Ian R. Logan ◽  
Vasileia Sapountzi ◽  
Craig N. Robson ◽  
...  
Keyword(s):  
Binding Site ◽  
Measles Virus ◽  
Fluorescent Protein ◽  
E3 Ligase ◽  
Yeast Two Hybrid ◽  
Ubiquitin E3 Ligase ◽  
P Protein ◽  
Two Hybrid

ABSTRACT Using a C-terminal domain (PCT) of the measles virus (MV) phosphoprotein (P protein) as bait in a yeast two-hybrid screen, a cDNA identical to the recently described human p53-induced-RING-H2 (hPIRH2) cDNA was isolated. A glutathione S-transferase-hPIRH2 fusion protein expressed in bacteria was able to pull down P protein when mixed with an extract from P-expressing HeLa cells in vitro, and myc-tagged hPIRH2 could be reciprocally coimmunoprecipitated with MV P protein from human cells. Additionally, immunoprecipitation experiments demonstrated that hPIRH2-myc, MV P, and nucleocapsid (N) proteins form a ternary complex. The hPIRH2 binding site was mapped to the C-terminal X domain region of the P protein by using a yeast two-hybrid assay. The PCT binding site was mapped on hPIRH2 by using a novel yeast two-hybrid tagged PCR approach and by coimmunoprecipitation of hPIRH2 cysteine mutants and mouse/human PIRH2 chimeras. The hPIRH2 C terminus could mediate the interaction with MV P which was favored by the RING-H2 motif. When coexpressed with an enhanced green fluorescent protein-tagged hPIRH2 protein, MV P alone or in a complex with MV N was able to redistribute hPIRH2 to outside the nucleus, within intracellular aggregates. Finally, MV P efficiently stabilized hPIRH2-myc expression and prevented its ubiquitination in vivo but had no effect on the stability or ubiquitination of an alternative ubiquitin E3 ligase, Mdm2. Thus, MV P protein is the first protein from a pathogen that is able to specifically interact with and stabilize the ubiquitin E3 ligase hPIRH2 by preventing its ubiquitination.

scholarly journals Genetic Analysis of theEscherichia coliFtsZ·ZipA Interaction in the Yeast Two-hybrid System

2001 ◽  
Vol 276 (15) ◽  
pp. 11980-11987 ◽  
Author(s):  
Steven A. Haney ◽  
Elizabeth Glasfeld ◽  
Cynthia Hale ◽  
David Keeney ◽  
Zhizhen He ◽  
...  
Keyword(s):  
Hybrid System ◽  
Enzyme Linked Immunosorbent Assay ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Sequence ◽  
Yeast Two Hybrid System ◽  
C Terminus ◽  
Two Hybrid

The recruitment of ZipA to the septum by FtsZ is an early, essential step in cell division inEscherichia coli. We have used polymerase chain reaction-mediated random mutagenesis in the yeast two-hybrid system to analyze this interaction and have identified residues within a highly conserved sequence at the C terminus of FtsZ as the ZipA binding site. A search for suppressors of a mutation that causes a loss of interaction (ftsZD373G) identified eight different changes at two residues within this sequence.In vitro, wild type FtsZ interacted with ZipA with a high affinity in an enzyme-linked immunosorbent assay, whereas FtsZD373Gfailed to interact. Two mutant proteins examined restored this interaction significantly.In vivo, the alleles tested are significantly more toxic than the wild typeftsZand cannot complement a deletion. We have shown that a fusion, which encodes the last 70 residues of FtsZ in the two-hybrid system, is sufficient for the interaction with FtsA and ZipA. However, when the wild type sequence is compared with one that encodes FtsZD373G, no interaction was seen with either protein. Mutations surrounding Asp-373 differentially affected the interactions of FtsZ with ZipA and FtsA, indicating that these proteins bind the C terminus of FtsZ differently.

scholarly journals Identification of SH2-Bbeta as a substrate of the tyrosine kinase JAK2 involved in growth hormone signaling.

1997 ◽  
Vol 17 (11) ◽  
pp. 6633-6644 ◽  
Author(s):  
L Rui ◽  
L S Mathews ◽  
K Hotta ◽  
T A Gustafson ◽  
C Carter-Su
Keyword(s):  
Growth Hormone ◽  
Tyrosine Kinase ◽  
Signaling Molecules ◽  
Sh2 Domain ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Cos Cells ◽  
Two Hybrid

Activation of the tyrosine kinase JAK2 is an essential step in cellular signaling by growth hormone (GH) and multiple other hormones and cytokines. Murine JAK2 has a total of 49 tyrosines which, if phosphorylated, could serve as docking sites for Src homology 2 (SH2) or phosphotyrosine binding domain-containing signaling molecules. Using a yeast two-hybrid screen of a rat adipocyte cDNA library, we identified a splicing variant of the SH2 domain-containing protein SH2-B, designated SH2-Bbeta, as a JAK2-interacting protein. The carboxyl terminus of SH2-Bbeta (SH2-Bbetac), which contains the SH2 domain, specifically interacts with kinase-active, tyrosyl-phosphorylated JAK2 but not kinase-inactive, unphosphorylated JAK2 in the yeast two-hybrid system. In COS cells coexpressing SH2-Bbeta or SH2-Bbetac and murine JAK2, both SH2-Bbetac and SH2-Bbeta coimmunoprecipitate to a significantly greater extent with wild-type, tyrosyl-phosphorylated JAK2 than with kinase-inactive, unphosphorylated JAK2. SH2-Bbetac also binds to immunoprecipitated wild-type but not kinase-inactive JAK2 in a far Western blot. In 3T3-F442A cells, GH stimulates the interaction of SH2-Bbeta with tyrosyl-phosphorylated JAK2 both in vitro, as assessed by binding of JAK2 in cell lysates to glutathione S-transferase (GST)-SH2-Bbetac or GST-SH2-Bbeta fusion proteins, and in vivo, as assessed by coimmunoprecipitation of JAK2 with SH2-Bbeta. GH promoted a transient and dose-dependent tyrosyl phosphorylation of SH2-Bbeta in 3T3-F442A cells, further suggesting the involvement of SH2-Bbeta in GH signaling. Consistent with SH2-Bbeta being a substrate of JAK2, SH2-Bbetac is tyrosyl phosphorylated when coexpressed with wild-type but not kinase-inactive JAK2 in both yeast and COS cells. SH2-Bbeta was also tyrosyl phosphorylated in response to gamma interferon, a cytokine that activates JAK2 and JAK1. These data suggest that GH-induced activation and phosphorylation of JAK2 recruits SH2-Bbeta and its associated signaling molecules into a GHR-JAK2 complex, thereby initiating some as yet unidentified signal transduction pathways. These pathways are likely to be shared by other cytokines that activate JAK2.

scholarly journals Conserved Residues in the C-Terminal Domain Affect the Structure and Function of CYP38 in Arabidopsis

2021 ◽  
Vol 12 ◽  
Author(s):  
Lujing Shi ◽  
Lele Du ◽  
Jingru Wen ◽  
Xiumei Zong ◽  
Wene Zhao ◽  
...  
Keyword(s):  
Thylakoid Membrane ◽  
Structure And Function ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Conserved Residues ◽  
Terminal Domain ◽  
Target Binding ◽  
And Function ◽  
Two Hybrid

Arabidopsis cyclophilin38 (CYP38) is a thylakoid lumen protein critial for PSII assembly and maintenance, and its C-terminal region serves as the target binding domain. We hypothesized that four conserved residues (R290, F294, Q372, and F374) in the C-terminal domain are critical for the structure and function of CYP38. In yeast two-hybrid and protein pull-down assays, CYP38s with single-sited mutations (R290A, F294A, Q372A, or F374A) did not interact with the CP47 E-loop as the wild-type CYP38. In contrast, CYP38 with the R290A/F294A/Q372A/F374A quadruple mutation could bind the CP47 E-loop. Gene transformation analysis showed that the quadruple mutation prevented CYP38 to efficiently complement the mutant phenotype of cyp38. The C-terminal domain half protein with the quadruple mutation, like the wild-type one, could interact with the N-terminal domain or the CP47 E-loop in vitro. The cyp38 plants expressing CYP38 with the quadruple mutation showed a similar BN-PAGE profile as cyp38, but distinct from the wild type. The CYP38 protein with the quadruple mutation associated with the thylakoid membrane less efficiently than the wild-type CYP38. We concluded that these four conserved residues are indispensable as changes of all these residues together resulted in a subtle conformational change of CYP38 and reduced its intramolecular N-C interaction and the ability to associate with the thylakoid membrane, thus impairing its function in chloroplast.

scholarly journals Functional Analyses of the Pto Resistance Gene Family in Tomato and the Identification of a Minor Resistance Determinant in a Susceptible Haplotype

2002 ◽  
Vol 15 (3) ◽  
pp. 281-291 ◽  
Author(s):  
Jeff H. Chang ◽  
Yin-Shan Tai ◽  
Adriana J. Bernal ◽  
Daniel T. Lavelle ◽  
Brian J. Staskawicz ◽  
...  
Keyword(s):  
Cell Death ◽  
Amino Acid ◽  
Hybrid System ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Yeast Two Hybrid System ◽  
A Minor ◽  
Two Hybrid

Pto is a member of a multigene family and encodes a serine/threonine kinase that mediates gene-for-gene resistance to strains of Pseudomonas syringae pv. tomato expressing avrPto. The inferred amino acid sequence of the Pto homologs from both resistant (LpimPth2 to LpimPth4,) and susceptible (LescFen, LescPth2 to LescPth5) haplotypes suggested that most could encode functional serine/threonine kinases. In addition, the activation segments of the homologs are similar in sequence to that of Pto, and some have residues previously identified as required for binding of AvrPto by Pto in the yeast two-hybrid system. The Pto homologs were therefore characterized for transcription, for the ability of their products to interact with AvrPto in the yeast two-hybrid system, for their autophos-phorylation activity, and for their potential to elicit cell death in the presence of and absence of a ligand, as well as their dependence on Prf. LpimPth5, LpimPth4, and LescPth4 were not transcribed at levels detectable by reverse transcription-polymerase chain reaction. The interaction with AvrPto was unique to Pto in the yeast two-hybrid system. LescPth2 autophosphorylated in vitro as a fusion protein. LpimPth2, LpimPth3, LpimPth4, LescPth3, and LescPth4 did not autophosphorylate in vitro. Transient expression of wild-type Fen and wild-type LpimPth3, as well as LescFen, LescPth3, and LescPth5 with perturbations in their P+1 loop caused cell death in Nicotiana benthamiana. LpimPth3 and LescPth3 with amino acid substitutions in the P+1 loop also elicited cell death in tomato; this was dependent on the presence of wild-type Prf. Consequently, some homologs could potentially encode functional resistance proteins. LescPth5 induced cell death specifically in response to expression of AvrPto in tobacco in a Prf-dependent manner; this is consistent with a homolog from a ‘susceptible’ haplotype encoding a minor recognition determinant.

scholarly journals Binding of Integrin α6β4 to Plectin Prevents Plectin Association with F-Actin but Does Not Interfere with Intermediate Filament Binding

1999 ◽  
Vol 147 (2) ◽  
pp. 417-434 ◽  
Author(s):  
Dirk Geerts ◽  
Lionel Fontao ◽  
Mirjam G. Nievers ◽  
Roel Q.J. Schaapveld ◽  
Patricia E. Purkis ◽  
...  
Keyword(s):  
Binding Site ◽  
Intermediate Filament ◽  
Binding Sites ◽  
Cytoplasmic Domain ◽  
Actin Binding ◽  
Yeast Two Hybrid ◽  
Dot Blot ◽  
Intermediate Filament Proteins ◽  
Two Hybrid

Hemidesmosomes are stable adhesion complexes in basal epithelial cells that provide a link between the intermediate filament network and the extracellular matrix. We have investigated the recruitment of plectin into hemidesmosomes by the α6β4 integrin and have shown that the cytoplasmic domain of the β4 subunit associates with an NH2-terminal fragment of plectin that contains the actin-binding domain (ABD). When expressed in immortalized plectin-deficient keratinocytes from human patients with epidermol- ysis bullosa (EB) simplex with muscular dystrophy (MD-EBS), this fragment is colocalized with α6β4 in basal hemidesmosome-like clusters or associated with F-actin in stress fibers or focal contacts. We used a yeast two-hybrid binding assay in combination with an in vitro dot blot overlay assay to demonstrate that β4 interacts directly with plectin, and identified a major plectin-binding site on the second fibronectin type III repeat of the β4 cytoplasmic domain. Mapping of the β4 and actin-binding sites on plectin showed that the binding sites overlap and are both located in the plectin ABD. Using an in vitro competition assay, we could show that β4 can compete out the plectin ABD fragment from its association with F-actin. The ability of β4 to prevent binding of F-actin to plectin explains why F-actin has never been found in association with hemidesmosomes, and provides a molecular mechanism for a switch in plectin localization from actin filaments to basal intermediate filament–anchoring hemidesmosomes when β4 is expressed. Finally, by mapping of the COOH-terminally located binding site for several different intermediate filament proteins on plectin using yeast two-hybrid assays and cell transfection experiments with MD-EBS keratinocytes, we confirm that plectin interacts with different cytoskeletal networks.

scholarly journals p21-Activated Kinase 1 Regulates Microtubule Dynamics by Phosphorylating Tubulin Cofactor B

2005 ◽  
Vol 25 (9) ◽  
pp. 3726-3736 ◽  
Author(s):  
Ratna K. Vadlamudi ◽  
Christopher J. Barnes ◽  
Suresh Rayala ◽  
Feng Li ◽  
Seetharaman Balasenthil ◽  
...  
Keyword(s):  
Microtubule Dynamics ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Microtubule Polymerization ◽  
Cytoskeleton Reorganization ◽  
P21 Activated Kinase ◽  
Gtpase Binding Domain ◽  
Two Hybrid

ABSTRACT p21-activated kinase 1 (Pak1) induces cytoskeleton reorganization in part by regulating microtubule dynamics through an elusive mechanism. Using a yeast two-hybrid screen, we identified tubulin cofactor B (TCoB) (a cofactor in the assembly of the α/β-tubulin heterodimers) as an interacting substrate of Pak1. Pak1 directly phosphorylated TCoB in vitro and in vivo on serines 65 and 128 and colocalized with TCoB on newly polymerized microtubules and on centrosomes. TCoB interacted with the GTPase-binding domain of Pak1 and activated Pak1 in vitro and in vivo. In contrast to wild-type TCoB, an S65A, S128A double mutant and knock-down of the endogenous TCoB or Pak1 reduced microtubule polymerization, suggesting that Pak1 phosphorylation is necessary for normal TCoB function. Overexpression of TCoB dramatically increased the number of γ-tubulin-containing microtubule-organizing centers, a phenotype reminiscent of cells overexpressing Pak1. TCoB was overexpressed and phosphorylated in breast tumors. These findings reveal a novel role for TCoB and Pak1 in regulating microtubule dynamics.

scholarly journals A Map of Interactions between the Proteins of a Retrotransposon

1998 ◽  
Vol 72 (11) ◽  
pp. 9318-9322 ◽  
Author(s):  
Scott J. S. Steele ◽  
Henry L. Levin
Keyword(s):  
Rnase H ◽  
Binding Assays ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Core Domain ◽  
Yeast Two Hybrid System ◽  
Vitro Binding ◽  
Potential Interactions ◽  
Two Hybrid

ABSTRACT The yeast two-hybrid system and in vitro binding assays were used to characterize 54 potential interactions between the proteins of Tf1, an LTR-retrotransposon found in Schizosaccharomyces pombe. The Tf1 integrase (IN) protein was found to interact strongly with itself and not with other control proteins. In addition, the IN core domain interacted strongly with itself and full-length IN. Interestingly, the two-hybrid analysis detected an interaction between the RNase H domain of reverse transcriptase and IN. The biological implications of these interactions are discussed.

scholarly journals Mutations That Affect Dimer Formation and Helicase Activity of the Hepatitis C Virus Helicase

2001 ◽  
Vol 75 (1) ◽  
pp. 205-214 ◽  
Author(s):  
Yee-Ling Khu ◽  
Esther Koh ◽  
Siew Pheng Lim ◽  
Yin Hwee Tan ◽  
Sydney Brenner ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Gel Filtration ◽  
Helicase Activity ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Particle Assembly ◽  
Two Hybrid ◽  
Minimal Region

ABSTRACT Interaction between viral proteins is necessary for viral replication and viral particle assembly. We used the yeast two-hybrid assay to identify interactions among all the mature proteins of the hepatitis C virus. The interaction between NS3 and NS3 was one of the strongest viral protein-protein interactions detected. The minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region. Random mutations in the minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using the yeast two-hybrid assay as a screen. Three of these mutations resulted in a reduction or a loss of interaction between helicases. Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutants remain mostly monomeric. All three mutants were partially or almost inactive when assayed for helicase activity in vitro. Mixing a mutant helicase (Y267S) with wild-type helicase did not dramatically affect helicase activity. These data indicate that dimerization of the helicase is important for helicase activity. The mutations that reduce self-association of the helicase may define the key residues involved in NS3-NS3 dimerization.

scholarly journals The Tomato Metallocarboxypeptidase Inhibitor I, which Interacts with a Heavy Metal-Associated Isoprenylated Protein, Is Implicated in Plant Response to Cadmium

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 700 ◽  
Author(s):  
Anna Manara ◽  
Elisa Fasani ◽  
Barbara Molesini ◽  
Giovanni DalCorso ◽  
Federica Pennisi ◽  
...  
Keyword(s):  
Heavy Metal ◽  
Active Site ◽  
Abiotic Stresses ◽  
Lower Amount ◽  
Interacting Proteins ◽  
Wild Type ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Model Species ◽  
Two Hybrid

Metallocarboxypeptidases are metal-dependent enzymes, whose biological activity is regulated by inhibitors directed on the metal-containing active site. Some metallocarboxypeptidase inhibitors are induced under stress conditions and have a role in defense against pests. This paper is aimed at investigating the response of the tomato metallocarboxypeptidase inhibitor (TCMP)-1 to Cd and other abiotic stresses. To this aim, the tomato TCMP-1 was ectopically expressed in the model species Arabidopsis thaliana, and a yeast two-hybrid analysis was performed to identify interacting proteins. We demonstrate that TCMP-1 is responsive to Cd, NaCl, and abscisic acid (ABA) and interacts with the tomato heavy metal-associated isoprenylated plant protein (HIPP)26. A. thaliana plants overexpressing TCMP-1 accumulate lower amount of Cd in shoots, display an increased expression of AtHIPP26 in comparison with wild-type plants, and are characterized by a modulation in the expression of antioxidant enzymes. Overall, these results suggest a possible role for the TCMP-1/HIPP26 complex in Cd response and compartmentalization.

Sign in / Sign up

Export Citation Format

Share Document