Detecting protein-protein interaction in live yeast by flow cytometry.
Cytometry A
; 63(2): 77-86, 2005 Feb.
Article
en En
| MEDLINE
| ID: mdl-15651008
BACKGROUND: The yeast Saccharomyces cerevisiae is the most commonly used organism for studying protein- protein interactions. In this report we demonstrate the use of flow cytometry in observing fluorescence resonance energy transfer (FRET) between cyan and yellow fluorescent fusion proteins (CFP and YFP, respectively) as a marker for protein interaction in live yeast cells. Probability binning is also employed to provide a statistical confirmation of our observations. METHODS: We coexpressed CFP and YFP fusions containing the N-terminal transmembrane domain (NTM) of Tom70p in yeast and analyzed FRET in live cells with a multilaser flow cytometer. The Tom70p NTM was previously shown to be sufficient for mitochondrial localization and protein-protein interaction (Millar and Shore, 1994, J Biol Chem 269:12229-12232). RESULTS: FRET was observed only in cells that expressed CFP and YFP fusions that each contained the wild-type NTM. The introduction of mutations previously shown to disrupt NTM interaction eliminated FRET. Probability binning confirmed that differences between the FRET channels of experimental and control samples were statistically and physiologically significant. CONCLUSION: Flow cytometric analysis of FRET in yeast is a powerful technique for studying protein-protein interactions. The use of flow cytometry allows FRET data to be gathered from a large number of individual cells, thus providing important advantages unavailable to other techniques. Its application to yeast presents a new method to a popular system widely used in proteomic studies.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Saccharomyces cerevisiae
/
Proteínas Bacterianas
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Transferencia Resonante de Energía de Fluorescencia
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Proteínas Fluorescentes Verdes
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Citometría de Flujo
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Colorantes Fluorescentes
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Proteínas Luminiscentes
Idioma:
En
Revista:
Cytometry A
Año:
2005
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos