Quantitative Measurement of Protein Relocalization in Live Cells

ALAN BUSH; ALEJANDRO COLMAN LERNER

BIOPHYSICAL JOURNAL

CELL PRESS

Lugar: United States; Año: 2013 vol. 104 p. 727 - 736

0006-3495

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 Gbg 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 (Kde) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first acti- vated. Kde changed during the first minutes from a high affinity of