Dataset Information

Evening Complex associated proteins in Arabidopsis by AP-MS

ABSTRACT: To determine how clock components are integrated with cellular pathways, affinity purification and mass spectrometry (AP-MS) were used to identify proteins that co-precipitate with the evening complex, which is critical regulator of clock, growth, light and flowering pathways.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Alvarez Sophie

LAB HEAD: Dmitri Nusinow

PROVIDER: PXD002606 | Pride | 2016-03-15

REPOSITORIES: Pride

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Publications

Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry.

Huang He H Alvarez Sophie S Bindbeutel Rebecca R Shen Zhouxin Z Naldrett Michael J MJ Evans Bradley S BS Briggs Steven P SP Hicks Leslie M LM Kay Steve A SA Nusinow Dmitri A DA

Molecular & cellular proteomics : MCP 20151106 1

Many species possess an endogenous circadian clock to synchronize internal physiology with an oscillating external environment. In plants, the circadian clock coordinates growth, metabolism and development over daily and seasonal time scales. Many proteins in the circadian network form oscillating complexes that temporally regulate myriad processes, including signal transduction, transcription, protein degradation and post-translational modification. In Arabidopsis thaliana, a tripartite complex ...[more]

PMID: 26545401

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Project description:Circadian rhythms are daily oscillations in metabolism and physiology and are generated by the circadian clock. In fruit fly Drosophila, the circadian clock is generated by a transcription-translation feedback loop in which the positive arm components Clock and Cycle activate the expression of the Period and Timeless genes of negative arm, as well as other circadian clock-regulated genes. After being retained in the cytoplasm, the Period and Timeless proteins then migrate to the nucleus to inhibit the Clock/Cycle transactivity by protein-protein interactions (PPIs). The endogenous circadian clock is synchronized with the geological (solar) clock via photoreceptors. Drosophila Cryptochrome protein functions as a circadian photoreceptor. In the early morning, exposure of Cryptochrome to light causes a conformational change in it which results in the formation of new PPIs. Light-activated Cryptochrome interacts with the core clock protein Timeless and the E3 ubiquitin ligase-substrate adaptor protein Jetlag, which results in the ubiquitylation of Timeless by Jetlag-E3 ligase complex and then degradation of Timeless within minutes by proteasome system. Rapid degradation of Timeless and then its partner protein Period, because of its instability in the absence of Timeless, relieves the inhibition on the Clock/Cycle transcription factors suddenly. Therefore, Clock/Cycle-driven expression of circadian clock-regulated genes are induced again, which is the restart of the circadian oscillation or the resetting of the clock. Following Timeless degradation, Cryptochrome is also degraded so the photoreceptor mechanism does not start a new resetting signal until all the required factors are re-synthesized in a circadian manner. Light-dependent degradation of Drosophila Cryptochrome can be observed in Drosophila S2 cell line in culture. In this project, we aimed at finding the interactome of Cryptochrome protein in Drosophila S2 cell line under light and in the dark using proximity labeling method. Because of the fast kinetics of Cryptochrome degradation, we chose the enzymes that can saturate in less than one hour. TurboID (TID) and APEX2 enzymes label proteins with biotin in the proximity even though they work with different mechanisms. They were fused to Cryptochrome protein, and proximity labeling was performed in the dark or under light. We have identified novel light-dependent or -independent interactors of Drosophila Cryptochrome and confirmed some of them using classical coimmunoprecipitation technique.

Dataset Information

Evening Complex associated proteins in Arabidopsis by AP-MS

Publications

Identification of Evening Complex Associated Proteins in Arabidopsis by Affinity Purification and Mass Spectrometry.

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