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<p>Lipid-binding domains, traditionally isolated from natural proteins, are essential tools for probing membrane lipid dynamics and specialized cellular compartments. Despite diverse applications, a general strategy for their engineering remains elusive. Here we present a robust and high-throughput method for monitoring protein–lipid interactions, named the cell surface liposome binding (CLiB) assay. Using the assay, we conducted directed evolution of the PX domain from SnxA, isolating high-affinity variants specific for phosphatidylinositol 3,5-bisphosphate (PI(3,5)P<sub>2</sub>). Combining the CLiB assay with next-generation sequencing enabled parallel analysis of &gt;6,000 clones, comprehensively identifying key residues critical for lipid binding. An engineered variant, PX-SnxA<sup>GV</sup>, functioned as a lipid biosensor in yeast and mammalian cells, visualizing PI(3,5)P<sub>2</sub>-enriched membrane subdomains upon hyperosmotic shock and during microautophagy, thereby suggesting localized PI(3,5)P<sub>2</sub> synthesis within spatially restricted regions. This study provides a framework for on-demand generation of lipid-binding probes, facilitating the discovery of membrane compartments characterized by unique lipid compositions.</p>

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

  • 期刊论文

  • 发表日期：

    2026年7月2日

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  • Taki Nishimura,
  • Kotaro Tsuboyama,
  • Yuki Nakagaki,
  • Shiou-Ling Lu,
  • Yuki Ishino,
  • Nozomu Kono,
  • Takeshi Noda,
  • Eiji Yamamoto,
  • Noboru Mizushima

摘要

Lipid-binding domains, traditionally isolated from natural proteins, are essential tools for probing membrane lipid dynamics and specialized cellular compartments. Despite diverse applications, a general strategy for their engineering remains elusive. Here we present a robust and high-throughput method for monitoring protein–lipid interactions, named the cell surface liposome binding (CLiB) assay. Using the assay, we conducted directed evolution of the PX domain from SnxA, isolating high-affinity variants specific for phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2). Combining the CLiB assay with next-generation sequencing enabled parallel analysis of >6,000 clones, comprehensively identifying key residues critical for lipid binding. An engineered variant, PX-SnxAGV, functioned as a lipid biosensor in yeast and mammalian cells, visualizing PI(3,5)P2-enriched membrane subdomains upon hyperosmotic shock and during microautophagy, thereby suggesting localized PI(3,5)P2 synthesis within spatially restricted regions. This study provides a framework for on-demand generation of lipid-binding probes, facilitating the discovery of membrane compartments characterized by unique lipid compositions.