Protein Interactions

We are interested in identifying the protein interaction partners of RTE1 and RTH in order to elucidate the function of RTE1/RTH and gain insight into how RTE1 regulates ethylene receptor function. To achieve this, we are employing various approaches including the yeast "split ubiquitin" assay and tandem affinity purification (TAP)-tagging in Arabidopsis.

We are also characterizing a novel protein (coined "D2"), which was identified on the basis of its interaction with both the ETR1 ethylene receptor and the CTR1 protein kinase in the yeast two-hybrid assay.

Project Participants:

• Split Ub screens with RTE1 and RTH (Jianhong Chang, Chunhai Dong, Jeff Liesch)
• TAP-tagging of RTE1 (Chunhai Dong)
• Cyto-trap (Chris McClellan)
• D2 (Mandy Kendrick)

Split Ubiquitin Details

Preliminary results indicate that RTE1 and RTH interact with ER-localized Sec62-NubI on the basis of native split ubiquitin affinity. RTE1 and RTH do not interact substantially with SSO1 (PM), SNC1 (Golgi->PM), or SED5 (Golgi), suggesting that Arabidopsis RTE1 and RTH are localized to the ER in yeast.

Split Ubiquitin Links

• The Split-Ubiquitin Sensor: Measuring Interactions and Conformational Alterations of Proteins In Vivo. Christoph Reichel and Nils Johnsson
• Analysis of membrane protein interactions using yeast-based technologies. Igor Stagljar and Stanley Fields
• A genetic system based on split-ubiquitin for the analysis of interactions between membrane proteins in vivo. Igor Stagljar*, Chantal Korostenskydagger , Nils JohnssonDagger , and Stephan te Heesen
• Igor Stagljar Lab
• Dualsystems