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Biophys J

Title:		Quantitative measurement of protein relocalization in live cells
Author(s):		Bush A; Colman-Lerner A;
Address:		"Instituto de Fisiologia, Biologia Molecular y Neurociencias, Consejo Nacional de Investigaciones Cientificas y Tecnicas, Universidad de Buenos Aires, Buenos Aires, Argentina"
Journal Title:		Biophys J
Year:		2013
Volume:		104
Issue:		3
Page Number:		727 - 736
DOI:		10.1016/j.bpj.2012.12.030
ISSN/ISBN:		1542-0086 (Electronic) 0006-3495 (Print) 0006-3495 (Linking)
Abstract:		"Microscope cytometry provides a powerful means to study signaling in live cells. Here we present a quantitative method to measure protein relocalization over time, which reports the absolute fraction of a tagged protein in each compartment. Using this method, we studied an essential step in the early propagation of the pheromone signal in Saccharomyces cerevisiae: recruitment to the membrane of the scaffold Ste5 by activated Gbetagamma dimers. We found that the dose response of Ste5 recruitment is graded (EC50 = 0.44 +/- 0.08 nM, Hill coefficient = 0.8 +/- 0.1). Then, we determined the effective dissociation constant (K(de)) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. K(de) changed during the first minutes from a high affinity of < 0.65 nM to a steady-state value of 17 +/- 9 nM. During the same period, the total number of binding sites decreased slightly, from 1940 +/- 150 to 1400 +/- 200. This work shows how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system"
Keywords:		"Adaptor Proteins, Signal Transducing/chemistry/*metabolism Binding Sites Cell Membrane/chemistry/metabolism Data Interpretation, Statistical Feedback, Physiological Kinetics Mitogen-Activated Protein Kinases/metabolism Protein Binding Protein Transport Sa;"
Notes:		"MedlineBush, Alan Colman-Lerner, Alejandro eng R01 GM097479/GM/NIGMS NIH HHS/ 1R01GM097479-01/GM/NIGMS NIH HHS/ Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't 2013/02/28 Biophys J. 2013 Feb 5; 104(3):727-36. doi: 10.1016/j.bpj.2012.12.030"