Fig. 3. yArf1-11 is a hyperactive mutant present on the ER and LDs.
a,b, Active yArf1 pull-down and detection experiments done with strains expressing yArf1 and yArf1-11 fused to GFP (a) or endogenous untagged yArf1 and yArf1-11 (b). Protein extracts from soluble (S100) or pellet (P100) fractions from yARF1 and yarf1-11 cells grown at 23 °C or shifted to 37 °C were incubated with equal amount of purified GST-tagged GAT domain of Gga2 (Gga2GAT). Sec61 and Anp1 were used as membrane marker and Pgk1 as cytosolic marker. s.e., short exposure; l.e., long exposure; PD, pull-down. c, Localization of WT yArf1 and yArf1-11 C-terminally fused to GFP. Cells were incubated either at 23 °C or shifted at 37 °C for 30 min. Mean and standard deviation are shown. Scale bar 5 µm. d, Co-localization of yArf1–GFP and yArf1-11–GFP with the ER marker Sec61 tagged with mCherry grown at 23 °C and 37 °C. Cells highlighted by dotted squares depict GFP and mCherry co-localization. Fluorescence intensities of each channel were measured on a circle drawn around the perinuclear ER and are shown here as arbitrary units (a.u.). Scale bar, 5 µm. e–h, TEM of yARF1 (e,f) and yarf1-11 (g,h) strains grown either at 23 °C (e,g) or shifted at 37 °C (f,h) for 30 min. yArf1 and yArf1-11 localizations were detected by immunogold labeling, and dotted squares show enlargements of specific Arf1 localizations. Scale bar, 500 nm. Scale bar magnification, 200 nm. i, Co-localization of yArf1–GFP and yArf1-11–GFP with the LD marker Erg6 tagged with mCherry grown at 23 °C or shifted to 37 °C for 30 min. Arrows indicate sites of co-localization between the yArf1/yArf1-11 and LD. Scale bar, 5 µm. Unprocessed blots are available in source data. See also Extended Data Fig. 2.