Fig. 2: GTP dependent membrane targeting of AArfs.

a Heim12F4_9-5 displayed membrane targeting when expressed in yeast, and the targeting was dependent on GTP binding and its N-terminal helixe. Representative live cell fluorescent microscopy images are shown. DIC, differential interference contrast. b Four other AArfs displayed GTP dependent membrane targeting in yeast. Representative live cell fluorescent microscopy images are shown. c Heim12F4_9-5 and GerdYT_6_3-5 displayed GTP dependent membrane targeting in Hela cells. Cells were mildly fixed. Representative confocal microcopy images are shown. d Principle of liposome floatation assay. Liposomes float together with associated proteins to the top in a density gradient upon centrifugation. Proteins alone do not float. e, f Heim12F4_9-5-His-MBP floated to top fractions with liposomes of both archaeal and eukaryotic compositions. Heim12F4_9-5-His-MBP was purified from E. coli. Archaeal lipid was extracted from cultured Haloferax volcanii. Eukaryotic composition consisted of synthetic phospholipids and sterol. e representative immunoblots. f quantification of protein levels. Mean ± standard deviation, n = 3. a.u., arbitrary unit. g–k Heim12F4_9-5 association with liposomes was dependent on GTP loading, and independent of myristoylation. Purified full length Heim12F4_9-5, Heim12F4_9-5 lacking amphipathic helix (Heim12F4_9-5ΔN), Sar1, and Arf6 were subject to liposome floatation assay using liposomes of eukaryotic composition. g representative silver stained gels. h–k, quantification of protein levels. Mean ± standard deviation, n = 3. a.u., arbitrary unit. White scale bar, 2 μm. Yellow scale bar, 20 μm. Source data are provided in the Source Data.