1. Targeting of Nbp1 to the inner nuclear membrane is essential for spindle pole body duplication
Recombinant Nbp1‐(1–103)–sfGFP–His binds to liposomes. (A) SDS–PAGE analysis of purified Nbp1 proteins used for liposome‐binding assays. (B) Liposome binding analysed by using FACS. Fluorescence‐labelled liposomes were incubated with 11 μg recombinant Nbp1 protein in 100 μl buffer, sorted via flow cytometry and GFP fluorescence was detected. The relative GFP fluorescence of Nbp1‐(1–103)–sfGFP–His bound to liposomes was set to 100 (importance of AH for liposome binding was confirmed in three independent FACS experiments). (C) Binding of Nbp1‐(15–103)–sfGFP–His (−helix) and Nbp1‐(1–103)–sfGFP–His (+helix) to liposomes as analysed by the flotation assay. About 7.5 μg of each protein was incubated with liposomes extruded through a polycarbonate filter of 400 nm pore size. Lipid‐bound proteins from the top fraction T of the sucrose gradient were analysed by immunoblotting with an anti‐Penta‐His antibody (15% of each of the top fractions was used) in comparison with the input (I, 3.75% of each assay was used). (D) Binding of Nbp1‐(1–103)–sfGFP–His to liposomes depends on the lipid composition. In contrast to Nbp1‐(1–103)–nls1–sfGFP–His, Nbp1‐(1–103)–sfGFP–His bound better to PC (89 mol%)/PE (10 mol%)/PE‐rhodamine (1 mol%) liposomes than to PC liposomes. Two independent FACS experiments are shown; c.F.U. (corrected fluorescence units; Temmerman and Nickel, 2009). (E) Binding of Nbp1‐(1–103)–sfGFP–His protein to liposomes of different curvature. Nbp1‐(1–103)–sfGFP–His (about 5 μg) was incubated with PC‐liposomes extruded through polycarbonate filters of decreasing pore size (2: 400 nm, 3: 100 nm, 4: 50 nm) or without liposomes (assay 1). Lipid‐bound proteins from the top fraction (T, 15% of each of the top fraction was set in) of the sucrose gradient were analysed by immunoblotting with an anti‐Penta‐His antibody in comparison with the input (I, 1.875% of each assay was used).
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EMBO J, Volume: 30, Issue: 16, Pages: , First published: 22 July 2011, DOI: ( /emboj )