Identification of proteins interacting with the small GTPase Arl4D by liposome flotation assay and mass spectrometry

N-terminal myristoylation serves as an important modification for membrane insertion and concrete membrane anchoring of the small GTPases ADP-ribosylation factor (Arf) and ADP ribosylation factor-like (Arl). To simulate the binding topology of Arl4D on the membrane, we performed the liposome flotation assay in which N-terminally His-tagged Arl4D is loaded onto Ni-NTA-incorporated liposomes and the C-terminus of Arl4D is accessible to its interacting proteins. Liposomes containing Arl4D (active form Arl4D Q80L and inactive form Arl4D T35N) and Arl4D-interacting proteins derived from HeLa cell lysates were identified by a sucrose gradient in combination with mass spectrometry analysis. Specifically, we identified mitogen-activated protein kinase 1 (Erk2) as a specific interacting protein of Arl4D Q80L. We further demonstrate a novel role for Arl4D whereby the active form of Arl4D recruits Erk1/2 to the plasma membrane and promotes Pak1 T212 phosphorylation to promote cell migration.