TY - JOUR
T1 - Identification of evening complex associated proteins in arabidopsis by affinity purification and mass spectrometry
AU - Huang, He
AU - Alvarez, Sophie
AU - Bindbeutel, Rebecca
AU - Shen, Zhouxin
AU - Naldrett, Michael J.
AU - Evans, Bradley S.
AU - Briggs, Steven P.
AU - Hicks, Leslie M.
AU - Kay, Steve A.
AU - Nusinow, Dmitri A.
N1 - Funding Information:
We thank Doug Allen, Rebecca Bart, Dhruv Patel and Takato Imaizumi for critically reading the manuscript, Ivan Baxter and Toby Kellog for useful discussions, and Sarah Huss for technical assistance. This work was supported by various grants. The National Science Foundation (NSF) supported instrumentation, DBI-0922879 for acquisition of the LTQ-Velos Pro Orbitrap LC-MS/MS and DBI-1337680-acquisition of a Leica SP8-X confocal microscope. The NSF (IOS-0924023) supported S.P.B. The Kay laboratory was supported by the NIH R01 GM50006 and GM67837 to S.A.K. D.A.N. acknowledges support from the National Institutes of Health (NRSA GM083585), the Donald Danforth Plant Science Center Institutional start up funds and the National Science Foundation (IOS 1456796).
PY - 2016/1
Y1 - 2016/1
N2 - 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 composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. Thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. Thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways.
AB - 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 composed of EARLY FLOWERING 4 (ELF4), EARLY FLOWERING 3 (ELF3), and LUX ARRHYTHMO (LUX), named the evening complex, modulates daily rhythms in gene expression and growth through transcriptional regulation. However, little is known about the physical interactions that connect the circadian system to other pathways. We used affinity purification and mass spectrometry (AP-MS) methods to identify proteins that associate with the evening complex in A. Thaliana. New connections within the circadian network as well as to light signaling pathways were identified, including linkages between the evening complex, TIMING OF CAB EXPRESSION1 (TOC1), TIME FOR COFFEE (TIC), all phytochromes and TANDEM ZINC KNUCKLE/PLUS3 (TZP). Coupling genetic mutation with affinity purifications tested the roles of phytochrome B (phyB), EARLY FLOWERING 4, and EARLY FLOWERING 3 as nodes connecting the evening complex to clock and light signaling pathways. These experiments establish a hierarchical association between pathways and indicate direct and indirect interactions. Specifically, the results suggested that EARLY FLOWERING 3 and phytochrome B act as hubs connecting the clock and red light signaling pathways. Finally, we characterized a clade of associated nuclear kinases that regulate circadian rhythms, growth, and flowering in A. Thaliana. Coupling mass spectrometry and genetics is a powerful method to rapidly and directly identify novel components and connections within and between complex signaling pathways.
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U2 - 10.1074/mcp.M115.054064
DO - 10.1074/mcp.M115.054064
M3 - Article
C2 - 26545401
AN - SCOPUS:84955472770
VL - 15
SP - 201
EP - 217
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 1
ER -