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Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling
Published on: May 29, 2011
SAM domain-based protein oligomerization observed by live-cell fluorescence fluctuation spectroscopy
Brian D Slaughter1, Joseph M Huff, Winfried Wiegraebe
1The Stowers Institute for Medical Research, Kansas City, Missouri, United States of America.
Plos One
|April 24, 2008
Summary
Researchers visualized complex protein interactions in yeast, revealing how Sterile-alpha-motif (SAM) domain proteins Ste11 and Ste50 form distinct homo- and hetero-oligomers in live cells. This study advances understanding of cellular biochemistry and disease mechanisms.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Sterile-alpha-motif (SAM) domains are crucial protein interaction modules involved in diverse cellular processes like signal transduction and RNA binding.
- Mutations in SAM domains and their mediated oligomers are implicated in various human diseases.
- Observing heterogeneous SAM-mediated oligomers in vivo remains a significant challenge in live-cell biochemistry.
Purpose of the Study:
- To investigate the in vivo oligomerization and binding stoichiometry of SAM domain proteins Ste11 and Ste50 in live yeast cells.
- To characterize the composition of multi-component complexes formed by Ste11 and Ste50.
- To explore the dynamic behavior of these complexes in response to cellular signaling.
Main Methods:
- Utilized fluorescence fluctuation spectroscopy techniques, including Fluorescence Cross-Correlation Spectroscopy (FCCS) and 1-dimensional Photon Counting Histogram (1dPCH).
- Employed 2-dimensional Photon Counting Histogram (2dPCH) with endogenously expressed, fluorescently tagged (GFP or mCherry) Ste11 and Ste50 proteins.
- Investigated the effects of a SAM domain mutant of Ste50 on protein oligomerization.
Main Results:
- Confirmed SAM-mediated interactions and oligomerization of Ste11 and Ste50 using FCCS and 1dPCH.
- 2dPCH revealed a heterogeneous complex of Ste11 and Ste50 in yeast cytosol, consisting of a Ste11 dimer and a Ste50 monomer.
- Identified Ste50 existing as high-order oligomers independent of Ste11, with size reduction upon MAP kinase cascade activation.
- Demonstrated that a Ste50 SAM domain mutant disrupts both Ste50 oligomers and Ste11 dimerization.
Conclusions:
- Established a quantitative in vivo model for Ste11 and Ste50 homo- and hetero-oligomerization in yeast.
- Highlighted the utility of 2dPCH for dissecting complex molecular interactions in live genetic model organisms.
- Provided insights into the regulation of protein complex formation and its potential links to disease mechanisms.
