Extended Data Fig. 9: In vivo lipid-binding-pocket mutants.
From: Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain
a, Lipid sorting coefficients of TF-PI(4,5)P2 and TF-PS for PS-binding-impaired mutant (pil1-K66A/R70A) and PI(4,5)P2-binding-impaired mutant (pil1-K130A/R133A) in +1% PI(4,5)P2/+sterol lipid nanotubes. A significant decrease in PI(4,5)P2 sorting is observed in the PI(4,5)P2-binding impaired mutant relative to the WT (WT vs K130A p = 0.04058), but not in the PS-binding-impaired mutant (WT vs K66A p = 0.71233, K66A vs K130A p = 0.15098). PS sorting is impaired in both the PS- and the PI(4,5)P2-binding impaired mutants (WT vs K66A p = 8.417e-7, WT vs K130A p = 2.425e-5, K66A vs K130A p = 0.03931). Box indicates interquartile range (IQR) from Q1 (25%) to Q3 (75%) quartiles. Lower and upper whiskers show from Q1 and Q3 quartiles to minimum and maximum data points, respectively. Horizontal line shown inside the box indicates the median [Statistical significance: p-values obtained applying two-sample t-test with all conditions following normal distribution at 0.01 tested by Shapiro–Wilk, Kolmogorov–Smirnov, and Anderson–Darling normality tests. n is the number of independent tested nanotubes, N = 2 for all conditions, being the number of experimental repetitions. Black rhombuses show data points obtained at day 1 and grey circles show data points obtained for day 2]. b, FRAP of TF-cholesterol for sterol-binding-impaired mutant (Pil1-F33A/Y40A/F42A/F50A), with and without Pil1 protein in +1% PI(4,5)P2/+sterol lipid nanotubes. The mobile fraction of sterols is increased in the sterol-binding-impaired mutant, confirming the reduction of sterol binding by this mutant. Solid lines indicate a mean of n number of measured nanotubes with standard deviation shown. c, Eisosome morphology in lsp1Δ yeast expressing Pil1-GFPenvy lipid-binding-pocket mutation variants and Nce102–mScarlet-I (summed stacks). Merge represents summed stacks of Pil1-GFPenvy (cyan) and mScarlet-I (magenta) signals. d, Central confocal slice of lsp1Δ yeast expressing Pil1-GFPenvy variants and Nce102–Scarlet-I highlighting cytosolic ingression of eisosomes in sterol-binding-impaired mutant Pil1F33A/Y40A/F42A/F50A. e, Thresholded fraction of Nce102–mScarlet-I that colocalizes with indicated Pil1-GFPenvy lipid-binding-pocket mutants in single cells (Manders’ M1 colocalization coefficient). The shaded area represents the probability for data points of the population to take on this value. f, Growth of serial dilutions of lsp1Δ cells expressing Pil1-GFPenvy lipid-binding-pocket mutants on Complete Supplement Mixture media (CSM) with the indicated treatments. (DMSO: dimethylsulfoxide, Atorva: atorvastatin, Nys: nystatin, Myr: myriocin). All tagged/mutant proteins in c–f are expressed from their endogenous locus. Scale bars, 2 μm.
