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. 2020 Jan 20;9:e47654. doi: 10.7554/eLife.47654

Figure 3. HMM analysis of KRAS4b HVR and mutations that adjusted the charges of the HVR.

(a) Table shows HaloTag HVR (of KRAS4b) constructs over-expressed in HeLa cells by transient transfection with charge-neutral and charge-reversed substitution mutations of lysine residues with varying net charge content (*CC). (b) Single molecule tracks are shown for WT HVR and each of the mutant HVRs, color-coded according to their residence time at the membrane. The color bar encodes time from 0.0 s to 0.6 s. (c and d) Diffusion coefficient and occupancy of the fast and slow diffusing species are plotted in red and black solid circles respectively for each charge-altered mutant. While the fast diffusing species exhibit a gradual, significant (p<0.05) increase in diffusion coefficient as the lysine charges are neutralized and then reversed, the slow species remain the same and the relative fraction of fast and slow diffusing species remain unchanged. (e) Normalized average residence time from more than 5000 tracks for each mutant is shown in the plot. Significant reduction (p<0.05) in residence time indicates impaired association between charge-reversed HVR and membrane. (f) Graph shows transition probabilities from fast to slow diffusive state for each charge-altered mutant.

Figure 3—source data 1. Diffusion coefficients and occupancy fractions obtained by HMM analysis (plotted in Figure 3c and d) of Halotag-KRAS4b HVR and the charge reversal mutants 5Ea, 5A, and 3A transiently overexpressed in HeLa cells.
Normalized residence time on the membrane and transition probabilities from the fast to slow diffusion states, plotted in Figure 3e and Figure 3f, are also reported for each of four replicates.

Figure 3.

Figure 3—figure supplement 1. Localization of KRAS4b HVR mutant constructs.

Figure 3—figure supplement 1.

HaloTag-fusion proteins linked to the KRAS4b HVR region were expressed in HeLa cells by transient transfection and visualized by confocal microscopy. Introducing charge deficient mutations in the polylysine region of the KRAS4b HVR reduces membrane localization, most notably in the 5Eb mutant.