High‐Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells

Abstract Lipid analogues carrying three nitrilotriacetic acid (tris‐NTA) head groups were developed for the selective targeting of His‐tagged proteins into liquid ordered (l o ) or liquid disordered (l d ) lipid phases. Strong partitioning into the l o phase of His‐tagged proteins bound to tris‐NTA conjugated to saturated alkyl chains (tris‐NTA DODA) was achieved, while tris‐NTA conjugated to an unsaturated alkyl chain (tris‐NTA SOA) predominantly resided in the l d phase. Interestingly, His‐tag‐mediated lipid crosslinking turned out to be required for efficient targeting into the l o phase by tris‐NTA DODA. Robust partitioning into l o phases was confirmed by using viral lipid mixtures and giant plasma membrane vesicles. Moreover, efficient protein targeting into l o and l d domains within the plasma membrane of living cells was demonstrated by single‐molecule tracking, thus establishing a highly generic approach for exploring lipid microdomains in situ.