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Tracking yeast pheromone receptor Ste2 endocytosis using fluorogen-activating protein tagging
Anita Emmerstorfer-Augustin1, Christoph M Augustin2, Shadi Shams1
1Division of Biochemistry, Biophysics and Structural Biology, Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720-3202.
Molecular Biology of the Cell
|September 13, 2018
Summary
This study introduces fluorogen-activating protein (FAP) tagging for real-time observation of yeast pheromone receptor (Ste2) internalization. FAP-Ste2 imaging reveals new insights into endocytic adaptor roles during receptor trafficking.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Studying integral membrane protein trafficking in yeast is challenging due to the cell wall.
- Real-time visualization of specific protein pools at the cell surface is crucial for understanding dynamic processes like receptor internalization.
Purpose of the Study:
- To develop and validate a novel method, fluorogen-activating protein (FAP) tagging, for observing yeast pheromone receptor (Ste2) internalization in live cells.
- To investigate the mechanism of Ste2 internalization and the roles of specific endocytic adaptors.
Main Methods:
- Engineered a FAP-Ste2 chimera for specific cell surface labeling and fluorescence microscopy.
- Optimized FAP-Ste2 expression by exploring signal peptides and propeptide sequences.
- Deleted specific extracellular aspartyl proteases (Yps1 and Mkc7) to maintain FAP-Ste2 chimera integrity.
Main Results:
- FAP-Ste2 provided a brighter and more distinct plasma membrane signal compared to Ste2-GFP or Ste2-mCherry, with similar functional behavior.
- The FAP-Ste2 system enabled real-time observation of Ste2 internalization.
- New information was gained regarding the roles of cargo-selective endocytic adaptors, including Ldb19/Art1, Rod1/Art4, and Rog3/Art7.
Conclusions:
- FAP tagging is a viable and powerful tool for studying integral membrane protein trafficking in yeast.
- The FAP-Ste2 system offers novel insights into the Ste2 internalization pathway and the function of endocytic adaptors.
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