Detecting protein-protein interaction in live yeast by flow cytometry

Area of Science:

• Molecular Biology
• Cell Biology
• Biophysics

Background:

• Saccharomyces cerevisiae is a key model organism for studying protein-protein interactions.
• Fluorescence Resonance Energy Transfer (FRET) between cyan and yellow fluorescent proteins (CFP and YFP) serves as a marker for molecular proximity.
• Flow cytometry allows for high-throughput analysis of cellular properties.

Purpose of the Study:

• To demonstrate the utility of flow cytometry for observing FRET in live yeast cells.
• To use FRET as an indicator of protein-protein interactions mediated by the Tom70p N-terminal transmembrane domain (NTM).
• To employ probability binning for statistical validation of FRET observations.

Main Methods:

• Coexpression of CFP and YFP fusion proteins containing the Tom70p NTM in yeast.
• Analysis of FRET in live yeast cells using a multilaser flow cytometer.
• Application of probability binning to statistically confirm FRET signals.

Main Results:

• FRET was detected exclusively in yeast cells expressing wild-type Tom70p NTM fusions of both CFP and YFP.
• Mutations known to disrupt NTM interaction abolished FRET signals.
• Probability binning confirmed statistically significant differences between experimental and control samples.

Conclusions:

• Flow cytometric FRET analysis is a powerful and efficient technique for studying protein-protein interactions in yeast.
• This method allows for the analysis of a large number of individual cells, offering advantages over other techniques.
• The application of flow cytometry to FRET in yeast provides a novel approach for proteomic studies in this widely used model organism.