A Yeast-Based In Vivo Assay Complements In Vitro and In Silico Approaches to Characterize Sterol-Binding by Saccharomyces cerevisiae Lam/Ltc Proteins

Cellular membranes maintain distinct lipid compositions, with sterols enriched in the plasma membrane despite their synthesis in the endoplasmic reticulum (ER). This distribution relies on vesicular and non-vesicular transport, the latter facilitated by lipid transfer proteins (LTPs) at membrane contact sites. In yeast, the Lam/Ltc family of LTPs is critical for sterol transport. To characterize their sterol-binding properties in a cellular context, we developed a yeast-based in vivo assay using Saccharomyces cerevisiae. Here, sterol-binding proteins, fused to signal sequences, extract and export radiolabeled cholesterol from the luminal compartment of the secretory pathway to the culture medium, offering a qualitative measure of binding capacity. We demonstrate that yeast Lam/Ltc proteins (Ysp1, Ysp2, Lam4, Lam5, Lam6) and their StARkin domains efficiently extract sterols, complementing the activity of known LTPs (Pry1, NPC2, Osh4, STARD1). In vitro microscale thermophoresis (MST) and in silico docking confirmed low micromolar to nanomolar sterol affinities. Notably, Lam6 binds phosphatidylserine through its GRAM domain, with synergistic binding to membranes containing both phosphatidylserine and ergosterol. While limited by its qualitative nature and luminal specificity, the in vivo sterol binding assay complements in vitro methods, providing a robust tool to study LTPs in a cellular environment. These findings enhance our understanding of Lam/Ltc protein function and highlight the assay's potential for characterizing known and orphan LTPs.