High-Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells
High-Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells
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DOI：
10.1002/anie.201306328
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发表时间：
2014-01-27
影响因子：
16.6
通讯作者：
Piehler, Jacob
中科院分区：
文献类型：
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作者：
Beutel, Oliver;Nikolaus, Joerg;Piehler, Jacob
脂质类似物携带三个硝基三乙酸（tris-NTA）头基团，用于选择性靶向his标记的蛋白质进入液体有序（l(o)）或液体无序（l(d)）脂质相。与饱和烷基链结合的tris-NTA （tris-NTA DODA）结合的his标记蛋白被强烈划分为l(o)相，而与不饱和烷基链结合的tris-NTA （tris-NTA SOA）主要位于l(d)相。有趣的是，his标签介导的脂质交联被证明是tris-NTA DODA有效靶向到l(o)相所必需的。通过使用病毒脂质混合物和巨大的质膜囊泡，证实了l(o)相的稳健划分。此外，通过单分子跟踪证明了活细胞质膜内l(o)和l(d)结构域的高效蛋白靶向，从而建立了原位探索脂质微结构域的高度通用方法。
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.