Figure 8. Localization of RabO^RAB1 to autophagosomes. (A) AN7311/Trs85-GFP, expressed at physiological levels from an endogenously tagged allele, localizes to cytosolic puncta that also contain mCherry-Atg8. (B) Western blot analysis of GFP-RabO^RAB1 levels driven by the rabO^p and the alcA^p promoters. In the latter case, glucose represses the promoter, ethanol at 1% induces it, and fructose at 0.1% leads to basal levels of expression, which are similar to those driven by rabO^p. Anti-actin western blotting of the same samples was used as a loading control. (C) Strain co-expressing mCherry-RabO^RAB1 and GFP-Atg8. In nitrogen-fed cells cultured with 0.1% fructose as carbon source, mCherry-RabO^RAB1, expressed at approximately physiological levels under the control of the alcA^p driver, localizes to polarized Golgi cisternae. Images in (C–E) display the mCherry and GFP single channel images in inverted contrast, as indicated, and the “merge” of the two channels in color. In the “merge” images, the mCherry signal is shown in magenta, whereas the GFP signal is shown in green. Regions of signal overlap are white. (D) Localization of mCherry-RabO^RAB1 to cytosolic puncta and autophagosomes (APH) containing GFP-Atg8 in cells cultured under autophagy-inducing conditions. (E) Example of colocalization of mCherry-RabO^RAB1 and GFP-Atg8 in an expanding cup-shaped phagophore (PHG). For (D and E) the insets shown are at double magnification. (F) Several examples of autophagic structures labeled with GFP-RabO^RAB1, expressed at physiological levels under the control of the rabO promoter; cells were incubated in autophagy conditions. Autophagosomes are marked with a green arrowhead.