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Ypt1 and TRAPP interactions: optimization of multicolor bimolecular fluorescence complementation in yeast
Zhanna Lipatova1, Jane J Kim, Nava Segev
1Department of Biochemistry and Molecular Genetics, College of Medicine, University of Illinois at Chicago, Molecular Biology Research Building, 900 South Ashland Avenue, Chicago, IL, 60607, USA.
This study introduces multicolor bimolecular fluorescence complementation (BiFC) in yeast to visualize protein interactions in intracellular trafficking and autophagy. The method maps interactions between Ypt GTPases and TRAPP GEF subunits, revealing their roles in cellular transport pathways.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Ypt/Rab GTPases are crucial for intracellular vesicular transport, acting as molecular switches.
- Guanine-nucleotide exchange factors (GEFs), like TRAPP, activate Ypt GTPases.
- Specific TRAPP complexes (TRAPP I, II, III) regulate distinct Ypt-mediated transport pathways, including secretion, Golgi transport, and autophagy.
Purpose of the Study:
- To develop and optimize multicolor bimolecular fluorescence complementation (BiFC) for studying protein interactions in yeast.
- To investigate the interactions between Ypt GTPases and TRAPP subunits.
- To determine the cellular sites of these protein interactions within yeast.
Main Methods:
- Development of a multicolor BiFC plasmid set optimized for yeast.
- Utilization of yeast strains expressing red fluorescent compartmental markers.
- Co-localization of BiFC signals with compartmental markers to define interaction sites.
Main Results:
- Successful implementation of multicolor BiFC for in vivo protein interaction analysis in yeast.
- Visualization of Ypt-TRAPP subunit interactions in the context of intracellular trafficking and autophagy.
- Mapping of protein interaction sites within specific cellular compartments.
Conclusions:
- Multicolor BiFC is a powerful tool for dissecting protein interaction networks in yeast.
- This approach provides insights into the regulation of Ypt GTPase function by TRAPP complexes.
- The findings contribute to understanding the molecular mechanisms of intracellular transport and autophagy.
