Figure 4
Palmitoylation and intracellular localization of M2 with mutations in the amphiphilic helix that affect its biophysical properties. (A) Scheme of M2 and the sequence of the amphiphilic helix with the mutations inserted highlighted in red. The hydrophobic moment < μH > and overall hydrophobicity < H > of the helix were calculated with Heliquest (https://heliquest.ipmc.cnrs.fr/). (B) Helical wheel plots of the amphiphilic region of wild type M2 and the M2-AH mutants. Positive amino acids are in blue, negative amino acids in red, and hydrophobic amino acids in yellow. The arrow points to the hydrophobic face. The length of the arrow corresponds to the hydrophobic moment < μH > . The numbers indicate the net charge of the helix. (C) Confocal microscopy of mutants in transfected BHK21 cells. Cells were permeabilized and stained with antibodies against M2 (green), the cis-Golgi marker GM130 (red) and with DAPI (blue) to highlight the nucleus. The scale bar is 20 µm. (D) S-acylation: M2 wt and the mutants were expressed in 293 T cells. For the input samples different volume of the lysate was removed to adjust for the reduced expression level of some mutants. The remainder was divided into two aliquots that were adjusted to the same extent. One aliquot was not treated (− HA) and one treated with hydroxylamine (+ HA) to cleave cysteine-bound fatty acids before pulling down proteins with a free SH group. Samples were subjected to Western blotting with antibodies against M2. The results of two independent experiments are shown. (E) Quantification of these two and two other Acyl-RAC assays. The density of the bands with hydroxylamine (+ HA) bands was divided by the density of the input bands and normalized to M2 wild type (= 1)). The mean ± SD and the results from five independent experiments are shown. One-way ANOVA followed by multiple comparison Dunnett test was applied for statistical analysis. *P < 0.05, **P < 0.01 versus wild type.
