A novel prenyl-polybasic domain code determines lipid-binding specificity of the K-Ras membrane anchor

Figures & data

Figure 1. K-Ras membrane anchoring domain encodes a highly selective capacity for lipid sorting. K-Ras membrane anchor is composed of a hexa-lysine polybasic domain and an adjacent farnesyl methyl ester at the C-terminal end. The prenylated K-Ras PBD is more than just a charge sensor. The precise PBD amino acid sequence and the structure of the prenyl group combine to allow K-Ras anchor to sample well-defined conformational states, which results in distinct interactions with different lipid headgroups and acyl chains. Aggregation of a set of like anchors leads to assembly of nanoclusters with defined lipid compositions (shown as a heat map). The lipid composition of the resulting nanocluster shapes signal output (shown as a heat map of signaling pathway activity) [15].

Figure 2. Geranylgeranyl anchor modifies PBD interactions of the K-Ras anchor (A) Intact PM sheets of BHK cells expressing GFP-tagged farnesylated (Far) or geranylgeranylated (GG) K-RasG12V PBD mutants were attached to copper EM grids, fixed using 4% paraformaldehyde (PFA) and 0.1% glutaraldehyde and immunolabeled with 4.5nm gold nanoparticles coupled to anti-GFP antibody. Gold labels were then imaged using transmission EM at 100,000X magnification and digitized using ImageJ. An intact 1μm² PM sheet of a BHK cell expressing GFP-K-RasG12V-GG immunolabeled with 4.5nm gold conjugated to anti-GFP antibody is shown. (B) Univariate K-function was used to quantify the spatial distribution of gold particles on intact BHK PM sheets. Extent of nanoclustering, L(r)-r, was plotted against length scale, r. L(r)-r values above the 99% confidence interval (99%C.I.) of 1 (green line) indicate statistically meaningful clustering. (C) Peak values of the K-function curves shown in (B) were termed as L_max and used to summarize the spatial data. (D) Number of the gold particles within a PM area of 1μm² was counted and used as an estimate of K-Ras PM localization. At least 15 images were analyzed in each condition with mean ± SEM shown. Bootstrap tests were used to evaluate the clustering statistical significance between GFP-K-RasG12V-Far and GFP-K-RasG12V-GG PBD mutants (* indicates p <0.05). One-way ANOVA was used to evaluate the gold labeling data between GFP-K-RasG12V-Far and GFP-K-RasG12V-GG PBD mutants (* indicates p < 0.05) Spatial data of farnesylated K-Ras PBD mutants were previously shown [15].

Zhou, Y., et al., Lipid-Sorting Specificity Encoded in K-Ras Membrane Anchor Regulates Signal Output. Cell. 2017;168(1-2):p. 239–251. e16. https://doi.org/10.1016/j.cell.2016.11.059

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