Plasmids for One- and Two-Hybrid Analysis in Mammalian Cells

1998 ◽  
Vol 256 (2) ◽  
pp. 245-247 ◽  
Author(s):  
Ivan Sadowski
Keyword(s):  
Mammalian Cells ◽  
Hybrid Analysis ◽  
Two Hybrid

scholarly journals The git5 Gβ and git11 Gγ Form an Atypical Gβγ Dimer Acting in the Fission Yeast Glucose/cAMP Pathway

Genetics ◽  
2001 ◽  
Vol 157 (3) ◽  
pp. 1159-1168 ◽  
Author(s):  
Sheila Landry ◽  
Charles S Hoffman
Keyword(s):  
Fission Yeast ◽  
Mammalian Cells ◽  
Glucose Repression ◽  
Coiled Coil ◽  
Carboxy Terminus ◽  
Amino Terminal ◽  
Camp Pathway ◽  
Coiled Coil Domain ◽  
Mutational Activation ◽  
Two Hybrid

AbstractFission yeast adenylate cyclase, like mammalian adenylate cyclases, is regulated by a heterotrimeric G protein. The gpa2 Gα and git5 Gβ are both required for glucose-triggered cAMP signaling. The git5 Gβ is a unique member of the Gβ family in that it lacks an amino-terminal coiled-coil domain shown to be essential for mammalian Gβ folding and interaction with Gγ subunits. Using a git5 bait in a two-hybrid screen, we identified the git11 Gγ gene. Co-immunoprecipitation studies confirm the composition of this Gβγ dimer. Cells deleted for git11 are defective in glucose repression of both fbp1 transcription and sexual development, resembling cells lacking either the gpa2 Gα or the git5 Gβ. Overexpression of the gpa2 Gα partially suppresses loss of either the git5 Gβ or the git11 Gγ, while mutational activation of the Gα fully suppresses loss of either Gβ or Gγ. Deletion of gpa2 (Gα), git5 (Gβ), or git11 (Gγ) confer quantitatively distinct effects on fbp1 repression, indicating that the gpa2 Gα subunit remains partially active in the absence of the Gβγ dimer and that the git5 Gβ subunit remains partially active in the absence of the git11 Gγ subunit. The addition of the CAAX box from the git11 Gγ to the carboxy-terminus of the git5 Gβ partially suppresses the loss of the Gγ. Thus the Gγ in this system is presumably required for localization of the Gβγ dimer but not for folding of the Gβ subunit. In mammalian cells, the essential roles of the Gβ amino-terminal coiled-coil domains and Gγ partners in Gβ folding may therefore reflect a mechanism used by cells that express multiple forms of both Gβ and Gγ subunits to regulate the composition and activity of its G proteins.

scholarly journals The Catenin p120ctnInteracts with Kaiso, a Novel BTB/POZ Domain Zinc Finger Transcription Factor

1999 ◽  
Vol 19 (5) ◽  
pp. 3614-3623 ◽  
Author(s):  
Juliet M. Daniel ◽  
Albert B. Reynolds
Keyword(s):  
Transcription Factor ◽  
Monoclonal Antibodies ◽  
Cell Adhesion ◽  
Mammalian Cells ◽  
Yeast Two Hybrid ◽  
Amino Terminal ◽  
Juxtamembrane Region ◽  
Carboxy Terminal ◽  
Two Hybrid ◽  
E Cadherin

ABSTRACT p120 ctn is an Armadillo repeat domain protein with structural similarity to the cell adhesion cofactors β-catenin and plakoglobin. All three proteins interact directly with the cytoplasmic domain of the transmembrane cell adhesion molecule E-cadherin; β-catenin and plakoglobin bind a carboxy-terminal region in a mutually exclusive manner, while p120 binds the juxtamembrane region. Unlike β-catenin and plakoglobin, p120 does not interact with α-catenin, the tumor suppressor adenomatous polyposis coli (APC), or the transcription factor Lef-1, suggesting that it has unique binding partners and plays a distinct role in the cadherin-catenin complex. Using p120 as bait, we conducted a yeast two-hybrid screen and identified a novel transcription factor which we named Kaiso. Kaiso’s deduced amino acid sequence revealed an amino-terminal BTB/POZ protein-protein interaction domain and three carboxy-terminal zinc fingers of the C2H2 DNA-binding type. Kaiso thus belongs to a rapidly growing family of POZ-ZF transcription factors that include the Drosophila developmental regulators Tramtrak and Bric à brac, and the human oncoproteins BCL-6 and PLZF, which are causally linked to non-Hodgkins’ lymphoma and acute promyelocytic leukemia, respectively. Monoclonal antibodies to Kaiso were generated and used to immunolocalize the protein and confirm the specificity of the p120-Kaiso interaction in mammalian cells. Kaiso specifically coprecipitated with a variety of p120-specific monoclonal antibodies but not with antibodies to α- or β-catenin, E-cadherin, or APC. Like other POZ-ZF proteins, Kaiso localized to the nucleus and was associated with specific nuclear dots. Yeast two-hybrid interaction assays mapped the binding domains to Arm repeats 1 to 7 of p120 and the carboxy-terminal 200 amino acids of Kaiso. In addition, Kaiso homodimerized via its POZ domain but it did not heterodimerize with BCL-6, which heterodimerizes with PLZF. The involvement of POZ-ZF proteins in development and cancer makes Kaiso an interesting candidate for a downstream effector of cadherin and/or p120 signaling.

scholarly journals Caenorhabditis elegans SMG-2 Selectively Marks mRNAs Containing Premature Translation Termination Codons

2007 ◽  
Vol 27 (16) ◽  
pp. 5630-5638 ◽  
Author(s):  
Lisa Johns ◽  
Andrew Grimson ◽  
Sherry L. Kuchma ◽  
Carrie Loushin Newman ◽  
Philip Anderson
Keyword(s):  
Caenorhabditis Elegans ◽  
Mammalian Cells ◽  
Translation Termination ◽  
Yeast Two Hybrid ◽  
Eukaryotic Mrnas ◽  
Nonsense Mediated Mrna Decay ◽  
Endogenous Genes ◽  
Alternatively Spliced ◽  
Two Hybrid ◽  
Premature Translation Termination

ABSTRACT Eukaryotic mRNAs containing premature translation termination codons (PTCs) are rapidly degraded by a process termed “nonsense-mediated mRNA decay” (NMD). We examined protein-protein and protein-RNA interactions among Caenorhabditis elegans proteins required for NMD. SMG-2, SMG-3, and SMG-4 are orthologs of yeast (Saccharomyces cerevisiae) and mammalian Upf1, Upf2, and Upf3, respectively. A combination of immunoprecipitation and yeast two-hybrid experiments indicated that SMG-2 interacts with SMG-3, SMG-3 interacts with SMG-4, and SMG-2 interacts indirectly with SMG-4 via shared interactions with SMG-3. Such interactions are similar to those observed in yeast and mammalian cells. SMG-2-SMG-3-SMG-4 interactions require neither SMG-2 phosphorylation, which is abolished in smg-1 mutants, nor SMG-2 dephosphorylation, which is reduced or eliminated in smg-5 mutants. SMG-2 preferentially associates with PTC-containing mRNAs. We monitored the association of SMG-2, SMG-3, and SMG-4 with mRNAs of five endogenous genes whose mRNAs are alternatively spliced to either contain or not contain PTCs. SMG-2 associates with both PTC-free and PTC-containing mRNPs, but it strongly and preferentially associates with (“marks”) those containing PTCs. SMG-2 marking of PTC-mRNPs is enhanced by SMG-3 and SMG-4, but SMG-3 and SMG-4 are not detectably associated with the same mRNPs. Neither SMG-2 phosphorylation nor dephosphorylation is required for selective association of SMG-2 with PTC-containing mRNPs, indicating that SMG-2 is phosphorylated only after premature terminations have been discriminated from normal terminations. We discuss these observations with regard to the functions of SMG-2 and its phosphorylation during NMD.

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 Mechanism of Signalling and Adaptation through the Rhodobacter sphaeroides Cytoplasmic Chemoreceptor Cluster

2019 ◽  
Vol 20 (20) ◽  
pp. 5095
Author(s):  
Beyer ◽  
Szöllössi ◽  
Byles ◽  
Fischer ◽  
Armitage
Keyword(s):  
Rhodobacter Sphaeroides ◽  
Tandem Mass ◽  
Wild Type ◽  
Hybrid Analysis ◽  
Phosphatase Domain ◽  
E Coli ◽  
Tandem Mass Spectroscopy ◽  
Membrane Spanning ◽  
State Concentration ◽  
Two Hybrid

Rhodobacter sphaeroides has two chemotaxis clusters, an Escherichia coli-like cluster with membrane-spanning chemoreceptors and a less-understood cytoplasmic cluster. The cytoplasmic CheA is split into CheA4, a kinase, and CheA3, a His-domain phosphorylated by CheA4 and a phosphatase domain, which together phosphorylate and dephosphorylate motor-stopping CheY6. In bacterial two-hybrid analysis, one major cytoplasmic chemoreceptor, TlpT, interacted with CheA4, while the other, TlpC, interacted with CheA3. Both clusters have associated adaptation proteins. Deleting their methyltransferases and methylesterases singly and together removed chemotaxis, but with opposite effects. The cytoplasmic cluster signal overrode the membrane cluster signal. Methylation and demethylation of specific chemoreceptor glutamates controls adaptation. Tandem mass spectroscopy and bioinformatics identified four putative sites on TlpT, three glutamates and a glutamine. Mutating each glutamate to alanine resulted in smooth swimming and loss of chemotaxis, unlike similar mutations in E. coli chemoreceptors. Cells with two mutated glutamates were more stoppy than wild-type and responded and adapted to attractant addition, not removal. Mutating all four sites amplified the effect. Cells were non-motile, began smooth swimming on attractant addition, and rapidly adapted back to non-motile before attractant removal. We propose that TlpT responds and adapts to the cell’s metabolic state, generating the steady-state concentration of motor-stopping CheY6~P. Membrane-cluster signalling produces a pulse of CheY3/CheY4~P that displaces CheY6~P and allows flagellar rotation and smooth swimming before both clusters adapt.

scholarly journals Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

mBio ◽  
2016 ◽  
Vol 7 (3) ◽  
Author(s):  
Anna Beier ◽  
Ines Teichert ◽  
Christoph Krisp ◽  
Dirk A. Wolters ◽  
Ulrich Kück
Keyword(s):  
Sexual Development ◽  
Protein Phosphatase ◽  
Affinity Purification ◽  
Three Dimensional ◽  
Catalytic Subunit ◽  
Fruiting Bodies ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
A Subunit ◽  
Two Hybrid

ABSTRACT The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora . Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δ pp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. IMPORTANCE The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood. The first fungal STRIPAK was described in Sordaria macrospora , which is a well-established model organism used to study the formation of fungal fruiting bodies, three-dimensional organ-like structures. We analyzed STRIPAK subunit PP2Ac1, catalytic subunit 1 of protein phosphatase PP2A, to study the importance of the catalytic activity of this protein during sexual development. The results of our yeast two-hybrid analysis and tandem affinity purification, followed by mass spectrometry, indicate that PP2Ac1 activity connects STRIPAK with other signaling pathways and thus forms a large interconnected signaling network.

MVP interacts with YPEL4 and inhibits YPEL4-mediated activities of the ERK signal pathway

2010 ◽  
Vol 88 (3) ◽  
pp. 445-450 ◽  
Author(s):  
Pei Liang ◽  
Yongqi Wan ◽  
Yan Yan ◽  
Yuequn Wang ◽  
Na Luo ◽  
...  
Keyword(s):  
Metal Binding ◽  
Mammalian Cells ◽  
Mapk Signaling ◽  
Reporter System ◽  
Biochemical Assays ◽  
Metal Binding Domain ◽  
Erk Signal Pathway ◽  
Gene Maps ◽  
Putative Zinc Finger ◽  
Two Hybrid

Human YPEL4 is a member of YPEL family. It contains a Yippee domain, which is a putative zinc-finger-like, metal-binding domain. The human YPEL4 gene maps to chromosome 11q12.1, is ubiquitously expressed in adult tissues, and encodes a nuclear protein of 127 amino acids, the function of which remains unknown. To gain insights into the cellular function of this protein, we searched for YPEL4-interacting proteins using a yeast two-hybrid screen. The major vault protein (MVP), a lung resistance associated protein, was identified as a binding partner of YPEL4. The interaction between YPEL4 and MVP in mammalian cells was further demonstrated by a series of biochemical assays including the mammalian two-hybrid assay, GST pull-down assay, co-immunoprecipitation assay, and immunocytochemistry. Using a reporter system, we found that MVP can inhibit YPEL4’s ability to activate Elk-1 in the MAPK signaling pathway. This study provides new clues for understanding the molecular mechanism of YPEL4 in cell division and signal transduction pathways and should be helpful for understanding molecular functions of the YPEL family.

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