Yeast Two-Hybrid Analysis of Jasmonate Signaling Proteins

2013 ◽  
pp. 173-185 ◽  
Author(s):  
Amparo Pérez Cuéllar ◽  
Laurens Pauwels ◽  
Rebecca De Clercq ◽  
Alain Goossens
Keyword(s):  
Signaling Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Jasmonate Signaling ◽  
Two Hybrid

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 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.

Identification of a possible MAP kinase cascade in Arabidopsis thaliana based on pairwise yeast two-hybrid analysis and functional complementation tests of yeast mutants

FEBS Letters ◽  
1998 ◽  
Vol 437 (1-2) ◽  
pp. 56-60 ◽  
Author(s):  
Tsuyoshi Mizoguchi ◽  
Kazuya Ichimura ◽  
Kenji Irie ◽  
Peter Morris ◽  
Jérôme Giraudat ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Map Kinase ◽  
Functional Complementation ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Map Kinase Cascade ◽  
Kinase Cascade ◽  
Yeast Mutants ◽  
Two Hybrid

Type III secretion chaperones ShcS1 and ShcO1 from Pseudomonas syringae pv. tomato DC3000 bind more than one effector

Microbiology ◽  
2005 ◽  
Vol 151 (1) ◽  
pp. 269-280 ◽  
Author(s):  
Ute Kabisch ◽  
Angelika Landgraf ◽  
Jana Krause ◽  
Ulla Bonas ◽  
Jens Boch
Keyword(s):  
Pseudomonas Syringae ◽  
Type Iii Secretion ◽  
Gel Filtration ◽  
Effector Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Tomato Dc3000 ◽  
Type Iii ◽  
Effector Genes ◽  
Two Hybrid

The hrp-type III secretion (TTS) system is a key pathogenicity factor of the plant pathogen Pseudomonas syringae pv. tomato DC3000 that translocates effector proteins into the cytosol of the eukaryotic host cell. The translocation of a subset of effectors is dependent on specific chaperones. In this study an operon encoding a TTS chaperone (ShcS1) and the truncated effector HopS1′ was characterized. Yeast two-hybrid analysis and pull-down assays demonstrated that these proteins interact. Using protein fusions to AvrRpt2 it was shown that ShcS1 facilitates the translocation of HopS1′, suggesting that ShcS1 is a TTS chaperone for HopS1′ and that amino acids 1 to 118 of HopS1′ are required for translocation. P. syringae pv. tomato DC3000 carries two shcS1 homologues, shcO1 and shcS2, which are located in different operons, and both operons include additional putative effector genes. Transcomplementation experiments showed that ShcS1 and ShcO1, but not ShcS2, can facilitate the translocation of HopS1′ : : AvrRpt2. To characterize the specificities of the putative chaperones, yeast two-hybrid interaction studies were performed between the three chaperones and putative target effectors. These experiments showed that both ShcS1 and ShcO1 bind to two different effectors, HopS1′ and HopO1-1, that share only 16 % amino acid sequence identity. Using gel filtration it was shown that ShcS1 forms homodimers, and this was confirmed by yeast two-hybrid experiments. In addition, ShcS1 is also able to form heterodimers with ShcO1. These data demonstrate that ShcS1 and ShcO1 are exceptional class IA TTS chaperones because they can bind more than one target effector.

scholarly journals A multipleATGgene knockout strain for yeast two-hybrid analysis

Autophagy ◽  
2009 ◽  
Vol 5 (5) ◽  
pp. 699-705 ◽  
Author(s):  
Yang Cao ◽  
Usha Nair ◽  
Kyoko Yasumura-Yorimitsu ◽  
Daniel J. Klionsky
Keyword(s):  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Knockout Strain ◽  
Two Hybrid

scholarly journals Rac3-induced Neuritogenesis Requires Binding to Neurabin I

2006 ◽  
Vol 17 (5) ◽  
pp. 2391-2400 ◽  
Author(s):  
Donata Orioli ◽  
Ivan N. Colaluca ◽  
Miria Stefanini ◽  
Silvano Riva ◽  
Carlos G. Dotti ◽  
...  
Keyword(s):  
Antisense Oligonucleotides ◽  
Binding Protein ◽  
Growth Cones ◽  
Actin Binding ◽  
Primary Neurons ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Gtp Binding ◽  
Rho Family ◽  
Two Hybrid

Rac3, a neuronal GTP-binding protein of the Rho family, induces neuritogenesis in primary neurons. Using yeast two-hybrid analysis, we show that Neurabin I, the neuronal F-actin binding protein, is a direct Rac3-interacting molecule. Biochemical and light microscopy studies indicate that Neurabin I copartitions and colocalizes with Rac3 at the growth cones of neurites, inducing Neurabin I association to the cytoskeleton. Moreover, Neurabin I antisense oligonucleotides abolish Rac3-induced neuritogenesis, which in turn is rescued by exogenous Neurabin I but not by Neurabin I mutant lacking the Rac3-binding domain. These results show that Neurabin I mediates Rac3-induced neuritogenesis, possibly by anchoring Rac3 to growth cone F-actin.

Interactive cellular proteins related to classical swine fever virus non-structure protein 2 by yeast two-hybrid analysis

2012 ◽  
Vol 39 (12) ◽  
pp. 10515-10524 ◽  
Author(s):  
Kai Kang ◽  
Kangkang Guo ◽  
Qinhai Tang ◽  
Yanming Zhang ◽  
Jiang Wu ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Cellular Proteins ◽  
Yeast Two Hybrid ◽  
Hybrid Analysis ◽  
Two Hybrid
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