Plasma Membrane Organization DOI: 10.1002/anie.201306328 High-Fidelity Protein Targeting into Membrane Lipid Microdomains in Living Cells** Oliver Beutel, Jçrg Nikolaus, Oliver Birkholz, Changjiang You, Thomas Schmidt, Andreas Herrmann, and Jacob Piehler* 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. The formation of membrane domains based on the separa- tion of lipid mixtures into liquid-disordered (l d ) and liquid- ordered (l o ) phases has emerged as an important organizing principle of the plasma membranes of eukaryotic cells. [1] Submicroscopic l o domains in the plasma membrane termed lipid rafts have been suggested to be involved in numerous cellular processes including signal transduction, [2] membrane- protein trafficking [3] , and viral entry [4] and budding. [5] While the existence of a submicroscopic organization of lipids and proteins within the plasma membrane is well established, the particular properties of plasma membrane rafts and their functional roles in biological processes remain unclear. [6] Key challenges for unraveling the functional organization of lipid microdomains are the nanoscopic dimensions and the highly transient formation of l o phases in the plasma membranes of living cells. While biochemical isolation of l o phases from cells as “detergent-resistant membranes” is possible, this approach is not valid for exploring the intricate relationship between proteins and lipid rafts under native conditions. [7] To system- atically explore the role of lipid phase separation for membrane protein function, versatile bioanalytical tools for selectively probing specific lipid microdomains and manipu- lating their proteins and lipids within living cells are required. We aimed to develop an approach for selectively and efficiently targeting proteins into distinct lipid phases of the plasma membranes of living cells, where they could be employed as sensors or actuators for exploring lipid micro- domains. To this end, we synthesized multivalent chelator lipids based on a head group with three nitrilotriacetic acid (NTA) moieties grafted onto a cyclic scaffold to give tris- NTA, which has been demonstrated to bind His-tagged proteins with subnanomolar binding affinity. [8] For the targeting of proteins into l d domains, a lipid-like anchoring group containing a single cis double bond was conjugated to tris-NTA (tris-NTA SOA, [9] Figure 1a). Tris-NTA conjugated to a fully saturated lipid-like anchoring group (tris-NTA DODA, Figure 1b) was employed for targeting into l o phases. The partitioning of these lipids into lipid phases within artificial membranes was characterized quantitatively by using giant unilamellar vesicles (GUVs) and mica-supported membranes as model systems. After validating our approach, we used single-molecule tracking to investigate the targeting of proteins by tris-NTA SOA and tris-NTA DODA into submicroscopic lipid microdomains within the plasma mem- brane of living cells. Efficient incorporation of tris-NTA DODA and tris-NTA SOA into lipid membranes and specific binding of His-tagged maltose binding protein (MBP) labeled with DY647 ( DY647 MBP) was confirmed by probing binding to silica- supported membranes in real time using simultaneous total internal reflection fluorescence spectroscopy (TIRFS) and reflectance interference (RIf) detection. [10] To explore the potential role of binding stoichiometry, we tested His-tags with 6 (H6), 10 (H10), and 14 (H14) histidine residues. The tris-NTA head groups loaded with Ni II ions provide coordi- nation sites for six histidine residues and thus exactly match the H6 tag (Figure 1c), while the H10 and H14 tags provide an excess of histidine residues. Rapid and stable binding of His-tagged MBPs to a 1,2-dioleoyl-sn-glycero-3-phosphocho- line (DOPC) membrane doped with tris-NTA lipids loaded with Ni II ions was confirmed, as was efficient elution with [*] O. Beutel, O. Birkholz, Dr. C. You, Prof.Dr. J. Piehler Division of Biophysics, Department of Biology University of Osnabrück Barbarastrasse 11, 49076 Osnabrück (Germany) E-mail: piehler@uos.de Homepage: http://www.biophysik.uni-osnabrueck.de Dr. J. Nikolaus, Prof. Dr. A. Herrmann Institute of Biology/Biophysics, Humboldt University Berlin (Germany) Prof. Dr. T. Schmidt Physics of Life Processes, Leiden Institute of Physics (The Nether- lands) [**] We thank Gabriele Hikade and Hella Kenneweg for technical support. This project was supported by the Deutsche Forschungs- gemeinschaft (NanoSciERA/PI405-4 and SFB 944 to J.P.; SFB 765 and GK 1171 to A.H.) and by the Foundation for Fundamental Research on Matter (part of the Netherlands Organisation for Scientific Research) to T.S. (NanoSciERA/06NSE02). Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/anie.201306328. A ngewandte Chemi e 1311 Angew. Chem. Int. Ed. 2014, 53, 1311 –1315  2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim