Fig. 5. Lysine myristoylation cycle is coupled to the catalytic cycle of ARF6.

a SIRT2 removes more lysine myristoylation from ARF6 T27N than from ARF6 Q67L in vitro. ARF6 proteins were purified from cells with endogenous NMT and SIRT2 KD and subjected to the ³²P-NAD⁺ assay with recombinant SIRT2. n = 3. b In-cell Alk12 labeling of ARF6 Q67L and T27N mutants during NMT2 OE showing more lysine acylation of ARF6 Q67L. n = 3. c In vitro NMT reaction on ARF6 Q67L and T27N showing more double acylation on ARF6 Q67L (ARF6-GTP). n = 4. d NMT2 preferentially binds ARF6-GTP. Flag-tagged ARF6 mutants and HA-NMT2 were overexpressed in HEK293T cells and subjected to the indicated pull downs. n = 3. e NMT2 is redistributed to the sites of ARF6 Q67L, but not ARF6 T27N. Confocal images of immunofluorescence in cells overexpressing FLAG-ARF6 mutants and HA-NMT2. Scale bars: 10 µm. f Model showing that NMT acts on active, but SIRT2 on inactive ARF6. For all experiments, error bars represent SEM and n represents biological replicates analyzed by unpaired two-tailed t test.