Quantitative Measurement of Protein Relocalization in Live Cells

ALAN BUSH; ALEJANDRO COLMAN-LERNER

Taormina

Workshop; International Synthetic and Systems Biology Summer School (SSBSS); 2014

SSBSS

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. With it, we studied an essential step in the early propagation of the pheromone signal in S. 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 of 0.44±0.08 nM, nHill of 0.8±0.1). Then, we determined the effective dissociation constant (Kde) between Ste5 and membrane sites during the first minutes, when the negative feedback from the MAPK Fus3 is first activated. Kde changes during the first minutes from a high affinity of less than 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 exemplifies how careful quantification of a protein relocalization dynamic can give insight into the regulation mechanisms of a biological system.