title: Quantitative measurement of protein relocalization in live cells.
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 Gβγ 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.
authors: Instituto de Fisiología, Biología Molecular y Neurociencias, Consejo Nacional de Investigaciones Científicas y Técnicas, Universidad de Buenos Aires, Buenos Aires, Argentina.
authors: Alan
authors: A
authors: Bush
authors: Alejandro
authors: A
authors: Colman-Lerner
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D003627:Data Interpretation, Statistical; D025461:Feedback, Physiological; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011485:Protein Binding; D021381:Protein Transport; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C080321:STE5 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C075580:FUS3 protein, S cerevisiae; D020928:Mitogen-Activated Protein Kinases