Protein‐protein interaction screening with the Ras‐recruitment system

Kruse, Claudia; Hanke, Sabine; Vasiliev, Sergey; Hennemann, Hanjo

doi:10.1002/sita.200600089

Cited by 4 publications

(6 citation statements)

References 59 publications

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“…Among them, split-ubiquitin system is a well-established, useful technique to screen the candidate proteins with the PPIs for membrane target proteins 28 29 . As in other yeast systems, small G-protein-based methods, including the Sos recruitment system and the Ras recruitment system, are occasionally used to study the PPIs of membrane proteins 23 30 31 . These methods remain useful alternatives to the original two-hybrid system; however, they suffer from technical complexities, such as the different temperatures required for growth and screening (25 °C and 36 °C), slow growth at suboptimal temperatures, obligatory replica-plating steps (glucose to galactose medium), and the total time required for the procedure (~7 days including precultivation) 32 33 34 .…”

mentioning

confidence: 99%

Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets

Kaishima

Ishii

Fukuda

et al. 2015

Sci Rep

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Protein-protein interactions (PPIs) are crucial for the vast majority of biological processes. We previously constructed a Gγ recruitment system to screen PPI candidate proteins and desirable affinity-altered (affinity-enhanced and affinity-attenuated) protein variants. The methods utilized a target protein fused to a mutated G-protein γ subunit (Gγcyto) lacking the ability to localize to the inner leaflet of the plasma membrane. However, the previous systems were adapted to use only soluble cytosolic proteins as targets. Recently, membrane proteins have been found to form the principal nodes of signaling involved in diseases and have attracted a great deal of interest as primary drug targets. Here, we describe new protocols for the Gγ recruitment systems that are specifically designed to use membrane proteins as targets to overcome previous limitations. These systems represent an attractive approach to exploring novel interacting candidates and affinity-altered protein variants and their interactions with proteins on the inner side of the plasma membrane, with high specificity and selectivity.

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confidence: 99%

Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets

Kaishima

Ishii

Fukuda

et al. 2015

Sci Rep

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“…This system takes advantage of Ras-activation by membrane-localized hSos in yeast (Aronheim et al 1997;Kruse et al 2006). PEP/PAS2 should be particularly well suited for this test, since it is capable to rescuing mutations in its yeast homolog YJL097w/PHS1 (Bellec et al 2002).…”

Section: Discussionmentioning

confidence: 99%

“…The yeast strain cdc25H, with the temperature-sensitive cdc25-2 mutation, will then be capable of growing even at the restrictive temperature. SRS (and RRS) has been successfully used to analyze protein interactions in humans, animals and yeast (Kruse et al 2006). Although there have been pioneer studies for the use of SRS in interaction analyses of plant and plant virus proteins (Kim et al 2006;Frischmuth et al 2004), the method is not well established in plants.…”

Section: Introductionmentioning

confidence: 99%

“…Several alternative systems have been developed to overcome these limitations. These include the split-ubiquitin system (Johnsson and Varshavsky 1994;Laser et al 2000;Möckli et al 2007), the expressed transactivator system (Hirst et al 2001), the G-protein fusion assay (Ehrhard et al 2000), the Sos-recruitment system (SRS), and variants of it including the (reverse) Ras-recruitment system (RRS) (Aronheim et al 1997;Broder et al 1998;Hubsman et al 2001;Kruse et al 2006). SRS and RRS use the activation of the mitogenic Ras pathway in the budding yeast Saccharomyces cerevisiae as a selection mechanism.…”

Section: Introductionmentioning

confidence: 99%

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The Sos-recruitment system as a tool to analyze cellular localization of plant proteins: membrane localization of Arabidopsis thaliana PEPINO/PASTICCINO2

Schönhofer-Merl

Torres-Ruiz

2010

Mol Genet Genomics

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We have explored a modified cytosolic yeast-two-hybrid Sos-recruitment system (SRS) in order to test for membrane localization of a protein. In this system, membrane localization is assessed by rescue of a yeast strain carrying a temperature-sensitive mutation in the CDC25 gene (cdc25-2) at restrictive temperature. The homologous human Sos (hSos) is capable to replace cdc25-2 provided that it is attached to the membrane because only then hSos is functional. This can be achieved when hSos is artificially fused to a protein containing trans-membrane domains (Tms). GFP/YFP fusion construct analyses of the Arabidopsis thaliana PEPINO/PASTICCINO2 (PEP/PAS2) protein have previously shown disparate cellular localizations although this protein possesses clear Tms. Analysis of N-terminal and C-terminal hSos-PEP/PAS2 fusions respectively suggests, that PEP/PAS2 is an integral membrane protein with cytosolic N- and C-termini. This implies that the protein has an even number of Tms and that the first Tm, a signal peptide, is not cleaved off. Our study shows that SRS is suitable to test for protein membrane localization and possibly for more detailed topological analysis of membrane proteins.

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“…The Sos recruitment system takes advantage of Ras-activation through membrane-localized hSos in yeast (Kruse et al, 2006;Aronheim et al, 2006). The SUMF2 subtypes are particularly well suited for this test, as they can rescue mutations in the yeast homolog YJL097w/PHS1 (Bellec et al, 2002).…”

Section: Discussionmentioning

confidence: 99%

Sos recruitment system for the analysis of the interaction between sulfatase-modifying factor 2 subtypes and interleukin-13

Zhang¹,

Liang²,

Chun³

et al. 2013

Genet. Mol. Res.

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ABSTRACT. Interleukin (IL)-13 is a central mediator in allergic asthma. Our previous results have indicated that sulfatase-modifying factor 2 (SUMF2) interacts with IL-13 and inhibits its secretion. In this study, we investigated the interactions between SUMF2 subtypes and 2 types of IL-13. Wild type IL-13 (wh-IL-13) and its mutated counterpart (mh-IL-13) were analyzed and cloned into pSos yeast expression vectors. Protein was expressed in host cdc25H yeast strains. A quartet of agar growth plates was prepared for the yeast twohybrid system, which was used to detect IL-13 and SUMF2 subtype interactions. Both yeast expression vectors, pSos/whIL-13 and pSos/ whIL-13, and recombinant expression vectors for the 5 subtypes of SUMF2 (pMyr/SUMF2-Vx) were constructed. Our data showed that all of the SUMF2 subtypes bound to whIL-13 and mhIL-13 in the CytoTrap system. Five SUMF2 subtypes -SUMF2-V2, SUMF2-V3, 5665©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 12 (4): 5664-5672 (2013) SUMF2 subtype interaction with IL-13 SUMF2-V4, SUMF2-V5, and SUMF2-V7 -interacted with whIL-13 and mhIL-13. These subtypes may contribute to allergic asthma by mediating IL-13 release.

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Protein‐protein interaction screening with the Ras‐recruitment system

Cited by 4 publications

References 59 publications

Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets

Gγ recruitment systems specifically select PPI and affinity-enhanced candidate proteins that interact with membrane protein targets

The Sos-recruitment system as a tool to analyze cellular localization of plant proteins: membrane localization of Arabidopsis thaliana PEPINO/PASTICCINO2

Sos recruitment system for the analysis of the interaction between sulfatase-modifying factor 2 subtypes and interleukin-13

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