Faculty Opinions recommendation of A New Method, "Reverse Yeast Two-Hybrid Array" (RYTHA), Identifies Mutants that Dissociate the Physical Interaction Between Elg1 and Slx5.

Autophagy is a highly conserved degrading process and is crucial for cell growth and development in eukaryotes, especially when they face starvation and stressful conditions. To evaluate the functions of Atg4 and Atg8 in mycelial growth, asexual and sexual development, and virulence in Cochliobolus heterostrophus, ΔChatg4 and ΔChatg8 mutants were generated by gene replacement. Strains deleted for ChATG4 and ChATG8 genes showed significant changes in vegetative growth and in development of conidia and ascospores compared with the wild-type strain. The autophagy process was blocked and the virulence was reduced dramatically in ΔChatg4 and ΔChatg8 mutants. In addition, deletion of ChATG4 and ChATG8 disordered Cdc10 subcellular localization and formation of septin rings. The direct physical interaction between ChAtg4 and ChAtg8 was detected by Yeast-two-hybrid, and ChAtg4-GFP was dispersed throughout the cytoplasm, although GFP-ChAtg8 appeared as punctate structures. All phenotypes were restored in complemented strains. Taken together, these findings indicated that ChATG4 and ChATG8 were crucial for autophagy to regulate fungal growth, development, virulence, and localization of septin in C. heterostrophus.

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Interaction between NifL and NifA in the nitrogen-fixing Pseudomonas stutzeri A1501

Microbiology ◽

10.1099/mic.0.29171-0 ◽

2006 ◽

Vol 152 (12) ◽

pp. 3535-3542 ◽

Cited By ~ 17

Author(s):

Zhihong Xie ◽

Yuetang Dou ◽

Shuzheng Ping ◽

Ming Chen ◽

Guoying Wang ◽

...

Keyword(s):

Nitrogenase Activity ◽

Pseudomonas Stutzeri ◽

Physical Interaction ◽

Wild Type ◽

Yeast Two Hybrid ◽

Free Living ◽

Yeast Two Hybrid System ◽

Living State ◽

Microaerobic Conditions ◽

Two Hybrid

Pseudomonas stutzeri strain A1501 isolated from rice fixes nitrogen under microaerobic conditions in the free-living state. This paper describes the properties of nifL and nifA mutants as well as the physical interaction between NifL and NifA proteins. A nifL mutant strain that carried a mutation non-polar on nifA expression retained nitrogenase activity. Complementation with a plasmid containing only nifL led to a decrease in nitrogenase activity in both the wild-type and the nifL mutant, suggesting that NifL acts as an antiactivator of NifA activity. Using the yeast two-hybrid system and purified protein domains of NifA and NifL, an interaction was shown between the C-terminal domain of NifL and the central domain of NifA, suggesting that NifL antiactivator activity is mediated by direct protein interaction with NifA.

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Heterodimeric interaction and interfaces of S100A1 and S100P

Biochemical Journal ◽

10.1042/bj20040142 ◽

2004 ◽

Vol 382 (1) ◽

pp. 375-383 ◽

Cited By ~ 18

Author(s):

Guozheng WANG ◽

Shu ZHANG ◽

David G. FERNIG ◽

David SPILLER ◽

Marisa MARTIN-FERNANDEZ ◽

...

Keyword(s):

Mammalian Cells ◽

Hydrophobic Interactions ◽

Homology Modelling ◽

S100 Protein ◽

Biological Significance ◽

Resonance Energy ◽

Optical Biosensor ◽

Physical Interaction ◽

Yeast Two Hybrid ◽

Two Hybrid

With the widespread use of yeast two-hybrid systems, many heterodimeric forms of S100 proteins have been found, although their biological significance is unknown. In the present study, S100A1 was found to interact with another S100 protein, S100P, by using the yeast two-hybrid system. The binding parameters of the interaction were obtained using an optical biosensor and show that S100P has a slightly higher affinity for S100A1 (Kd=10–20 nM) when compared with that for self-association (Kd=40–120 nM). The physical interaction of S100A1 and S100P was also demonstrated in living mammalian cells using a fluorescence resonance energy transfer technique. Preincubation of recombinant S100P with S100A1, before the biosensor assay, reduced by up to 50% the binding of S100P to a recombinant C-terminal fragment of non-muscle myosin A, one of its target molecules. Site-specific mutations of S100P and S100A1, combined with homology modelling of an S100P/S100A1 heterodimer using known S100P and S100A1 structures, allowed the hydrophobic interactions at the dimeric interface of the heterodimer to be defined and provide an explanation for the heterodimerization of S100P and S100A1 at the molecular level. These results have revealed the similarities and the differences between the S100P homodimer and the S100A1/S100P heterodimer.

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Dengue virus NS4B interacts with NS3 and dissociates it from single-stranded RNA

Journal of General Virology ◽

10.1099/vir.0.81844-0 ◽

2006 ◽

Vol 87 (9) ◽

pp. 2605-2614 ◽

Cited By ~ 111

Author(s):

Indira Umareddy ◽

Alex Chao ◽

Aruna Sampath ◽

Feng Gu ◽

Subhash G. Vasudevan

Keyword(s):

Dengue Virus ◽

Virus Replication ◽

Physical Interaction ◽

Helicase Domain ◽

Virus Infected Cell ◽

Yeast Two Hybrid ◽

The Family ◽

Positive Strand Rna ◽

Two Hybrid

Dengue virus, a member of the family Flaviviridae of positive-strand RNA viruses, has seven non-structural proteins: NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5. Except for enzymic activities contained within NS3 and NS5, the roles of the other proteins in virus replication and pathogenesis are not well defined. In this study, a physical interaction between NS4B and the helicase domain of NS3 was identified by using a yeast two-hybrid assay. This interaction was further confirmed by biochemical pull-down and immunoprecipitation assays, both with purified proteins and with dengue virus-infected cell lysates. NS4B co-localized with NS3 in the perinuclear region of infected human cells. Furthermore, NS4B dissociated NS3 from single-stranded RNA and consequently enhanced the helicase activity of NS3 in an in vitro unwinding assay. These results suggest that NS4B modulates dengue virus replication via its interaction with NS3.

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Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein

Journal of Virology ◽

10.1128/jvi.01459-18 ◽

2018 ◽

Vol 93 (6) ◽

Cited By ~ 3

Author(s):

Nadjet Lebsir ◽

Lucie Goueslain ◽

Rayan Farhat ◽

Nathalie Callens ◽

Jean Dubuisson ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Viruses ◽

Nonstructural Protein ◽

Intracellular Localization ◽

Physical Interaction ◽

Genome Replication ◽

Yeast Two Hybrid ◽

Critical Function ◽

Two Hybrid

ABSTRACTGBF1 has emerged as a host factor required for the genome replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of genome replication but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, coimmunoprecipitation, and proximity ligation assays and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A reverse yeast two-hybrid screen to identify mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is nonreplicative despite conserved protease activity and does not interact with GBF1. The mutated residue is exposed at the surface of NS3, suggesting it is part of the domain of NS3 that interacts with GBF1. The corresponding mutation in strain JFH-1 (S77D) produces a similar phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV genome replication.IMPORTANCESingle-stranded, positive-sense RNA viruses rely to a significant extent on host factors to achieve the replication of their genome. GBF1 is such a cellular protein that is required for the replication of several RNA viruses, but its mechanism of action during viral infections is not yet defined. In this study, we investigated potential interactions that GBF1 might engage in with proteins of HCV, a GBF1-dependent virus. We found that GBF1 interacts with NS3, a nonstructural protein involved in HCV genome replication, and our results suggest that this interaction is important for GBF1 function during HCV replication. Interestingly, GBF1 interaction with HCV appears different from its interaction with enteroviruses, another group of GBF1-dependent RNA viruses, in keeping with the fact that HCV and enteroviruses use different functions of GBF1.

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