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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 (Kde) between Ste5 and membrane sites during the first few minutes when the negative feedback from the MAPK Fus3 is first activated. Kde 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. © 2013 Biophysical Society.

Registro:

Documento: Artículo
Título:Quantitative measurement of protein relocalization in live cells
Autor:Bush, A.; Colman-Lerner, A.
Filiación:Instituto de Fisiología, Biología Molecular y Neurociencias, Departamento de Fisiología, Biología Molecular y Celular, Universidad de Buenos Aires, Buenos Aires, Argentina
Palabras clave:FUS3 protein, S cerevisiae; mitogen activated protein kinase; Saccharomyces cerevisiae protein; signal transducing adaptor protein; STE5 protein, S cerevisiae; article; binding site; cell membrane; chemistry; feedback system; kinetics; metabolism; protein binding; protein transport; Saccharomyces cerevisiae; statistical analysis; Adaptor Proteins, Signal Transducing; Binding Sites; Cell Membrane; Data Interpretation, Statistical; Feedback, Physiological; Kinetics; Mitogen-Activated Protein Kinases; Protein Binding; Protein Transport; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins
Año:2013
Volumen:104
Número:3
Página de inicio:727
Página de fin:736
DOI: http://dx.doi.org/10.1016/j.bpj.2012.12.030
Título revista:Biophysical Journal
Título revista abreviado:Biophys. J.
ISSN:00063495
CODEN:BIOJA
CAS:mitogen activated protein kinase, 142243-02-5; Adaptor Proteins, Signal Transducing; FUS3 protein, S cerevisiae, 2.7.11.24; Mitogen-Activated Protein Kinases, 2.7.11.24; STE5 protein, S cerevisiae; Saccharomyces cerevisiae Proteins
Registro:https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063495_v104_n3_p727_Bush

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Citas:

---------- APA ----------
Bush, A. & Colman-Lerner, A. (2013) . Quantitative measurement of protein relocalization in live cells. Biophysical Journal, 104(3), 727-736.
http://dx.doi.org/10.1016/j.bpj.2012.12.030
---------- CHICAGO ----------
Bush, A., Colman-Lerner, A. "Quantitative measurement of protein relocalization in live cells" . Biophysical Journal 104, no. 3 (2013) : 727-736.
http://dx.doi.org/10.1016/j.bpj.2012.12.030
---------- MLA ----------
Bush, A., Colman-Lerner, A. "Quantitative measurement of protein relocalization in live cells" . Biophysical Journal, vol. 104, no. 3, 2013, pp. 727-736.
http://dx.doi.org/10.1016/j.bpj.2012.12.030
---------- VANCOUVER ----------
Bush, A., Colman-Lerner, A. Quantitative measurement of protein relocalization in live cells. Biophys. J. 2013;104(3):727-736.
http://dx.doi.org/10.1016/j.bpj.2012.12.030