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Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast
Published on: October 23, 2016
Plasma membrane localization is required for RGS4 function in Saccharomyces cerevisiae
S P Srinivasa1, L S Bernstein, K J Blumer
1Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid Ave., St. Louis, MO 63110, USA.
Summary
Regulator of G protein signaling 4 (RGS4) requires its N-terminal domain for plasma membrane localization and inhibition of yeast pheromone response. Palmitoylation is not essential for RGS4
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Regulator of G protein signaling 4 (RGS4) is a GTPase activating protein crucial for regulating G protein alpha subunit activity.
- Understanding RGS4's functional domains is essential for elucidating its role in cellular signaling pathways.
- The pheromone response pathway in Saccharomyces cerevisiae serves as a model system for studying G protein signaling.
Purpose of the Study:
- To identify the specific regions of RGS4 necessary for its function in vivo.
- To investigate the relationship between RGS4's plasma membrane localization and its signaling inhibition activity.
- To determine the role of palmitoylation in RGS4's membrane association and functional activity.
Main Methods:
- Assaying deletion mutants of RGS4 for functional activity in the yeast pheromone response pathway.
- Utilizing green fluorescent protein (GFP) fusions to assess protein localization.
- Investigating the effects of cysteine residue mutations on RGS4 localization and function.
Main Results:
- Deletion of the N-terminal 33 amino acids (aa) of RGS4 resulted in loss of function and plasma membrane localization.
- The N-terminal 33 aa of RGS4 were sufficient to localize a soluble protein to the plasma membrane, identifying it as a membrane anchorage domain.
- Mutation of cysteine residues (Cys-2 and Cys-12), potential palmitoylation sites, did not affect RGS4's plasma membrane localization or signaling inhibition in yeast.
- Non-palmitoylation features of the N-terminal domain are responsible for RGS4's plasma membrane association and inhibitory function.
Conclusions:
- RGS4's N-terminal domain is critical for its plasma membrane localization and function in inhibiting the yeast pheromone response pathway.
- Plasma membrane localization is a prerequisite for RGS4's signaling inhibition activity.
- Palmitoylation is not the primary mechanism driving RGS4's plasma membrane association or its functional activity in this system.
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