Figure 6
Palmitoylation and intracellular localization of M2 mutants with disrupted amphiphilic helix and replacement of a glycine in the transmembrane region. (A) Scheme and sequence of M2, wild type and mutants with one or three Pro inserted into the helix and with the Gly34Ala exchange in the TM. (B) Confocal microscopy of mutants in transfected BHK 21 cells. Cells were permeabilized and stained with antibodies against the cis-Golgi marker GM130 (red) and with DAPI (blue) to highlight the nucleus. The scale bar is 20 µm. (C) 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. (D) Quantification of 5C and two other independent experiments. The density of the bands with hydroxylamine (+ HA) bands was divided by density of the input bands and normalized to wild type (= 1)). The mean ± SD and the results from four independent experiments are shown. One-way ANOVA followed by multiple comparison Dunnett test was applied for statistical analysis. ns not significant, ***P < 0.001 versus wild type.
