Quantitative measurement of protein relocalization in live cells
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 earl...
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2013
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| Acceso en línea: | https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063495_v104_n3_p727_Bush http://hdl.handle.net/20.500.12110/paper_00063495_v104_n3_p727_Bush |
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paper:paper_00063495_v104_n3_p727_Bush2023-06-08T14:31:12Z Quantitative measurement of protein relocalization in live cells 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 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. 2013 https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063495_v104_n3_p727_Bush http://hdl.handle.net/20.500.12110/paper_00063495_v104_n3_p727_Bush |
| institution |
Universidad de Buenos Aires |
| institution_str |
I-28 |
| repository_str |
R-134 |
| collection |
Biblioteca Digital - Facultad de Ciencias Exactas y Naturales (UBA) |
| topic |
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 |
| spellingShingle |
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 Quantitative measurement of protein relocalization in live cells |
| topic_facet |
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 |
| description |
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. |
| title |
Quantitative measurement of protein relocalization in live cells |
| title_short |
Quantitative measurement of protein relocalization in live cells |
| title_full |
Quantitative measurement of protein relocalization in live cells |
| title_fullStr |
Quantitative measurement of protein relocalization in live cells |
| title_full_unstemmed |
Quantitative measurement of protein relocalization in live cells |
| title_sort |
quantitative measurement of protein relocalization in live cells |
| publishDate |
2013 |
| url |
https://bibliotecadigital.exactas.uba.ar/collection/paper/document/paper_00063495_v104_n3_p727_Bush http://hdl.handle.net/20.500.12110/paper_00063495_v104_n3_p727_Bush |
| _version_ |
1768545031997620224 |