Fig. 6: Lysine myristoylation regulates ARF6 membrane localization.

a Confocal microscopy of overexpressed Flag-ARF6 and HA-NMT2 showing a decreased membrane localization of ARF6 K3R compared to ARF6 WT and colocalization of ARF6 with NMT2 at the plasma membrane and cytosol but not ERC in HEK293T cells. Scale bars: 10 µm. b Subcellular fractionation results in cells with HA-NMT2 expression and Alk12 treatment showing that K3 myristoylation promotes ARF6 membrane localization. The K3R mutant shows a decreased membrane and increased cytosolic localization compared to ARF6 WT. n = 2. c Subcellular fractionation results in cells with HA-NMT2 expression, SIRT2 KD, and Alk12 treatment demonstrating that lysine myristoylation promotes ARF6 membrane localization in different catalytic states. n = 2. d ARF6 K3R mutation does not affect ARF6 Q67L localization but inhibits ARF6 T27N localization to plasma membrane and ERC as indicated by colocalization with TfR in SIRT2 KD cells. These cells do not overexpress NMT and thus we were examining the effect of endogenous lysine myristoylation. Each point represents one cell (Q67L = 17, Q67L/K3R = 19, T27N = 12, T27N/K3R = 18). Scale bars: 10 µm. e SIRT2 localizes to ERC, recycling, and early endosomes as indicated by colocalization with TfR. Each point represents one cell (63 cells). Scale bars: 10 µm. All experiments in this Fig. were performed in HEK293T cells. Colocalization quantifications are presented as Pearson’s correlation coefficients. For all experiments, error bars represent SEM and n represents biological replicates analyzed by unpaired two-tailed t test.