Cell surface liposome binding (CLiB) allows lipid-binding probe engineering via high-throughput screening.

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Lipid-binding domains are crucial for studying membrane dynamics.
• Engineering these domains for specific lipid interactions remains challenging.
• A generalizable strategy for creating novel lipid-binding probes is needed.

Purpose of the Study:

• To develop a high-throughput method for engineering lipid-binding domains.
• To isolate high-affinity variants for phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2).
• To create a functional lipid biosensor for visualizing PI(3,5)P2 in living cells.

Main Methods:

• Developed the cell surface liposome binding (CLiB) assay for monitoring protein-lipid interactions.
• Employed directed evolution on the PX domain of SnxA.
• Combined CLiB assay with next-generation sequencing for high-throughput screening (>6,000 clones).

Main Results:

• Isolated high-affinity PX domain variants specific for PI(3,5)P2.
• Identified critical amino acid residues for lipid binding through comprehensive analysis.
• Engineered variant PX-SnxA^GV demonstrated PI(3,5)P2 visualization in yeast and mammalian cells.
• Observed PI(3,5)P2-enriched membrane subdomains during hyperosmotic shock and microautophagy.

Conclusions:

• The CLiB assay provides a robust framework for engineering custom lipid-binding probes.
• The study demonstrates the utility of engineered probes for discovering membrane compartments with specific lipid compositions.
• Localized PI(3,5)P2 synthesis is suggested within spatially restricted regions during cellular stress and autophagy.