Detecting protein-protein interaction in live yeast by flow cytometry.
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.


CONCLUSIONS
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.
Dye
Billy T
BT
Howard Hughes Medical Institute, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706, USA.
Schell
Kathleen
K
Miller
David J
DJ
Ahlquist
Paul
P
D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D015415:Biomarkers / Q000737:chemistry / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000378:metabolism; D008164:Luminescent Proteins / Q000737:chemistry* / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*
D001426:Bacterial Proteins; D015415:Biomarkers; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins