FIGURE 1:

Snf1 is required for localization of the TGN–endosomal clathrin adaptors Gga2 and Ent5 during prolonged glucose starvation. (A) AMPK pathway proteins are required for Ent5 hyperphosphorylation. Cellular lysates were prepared from indicated cells before or 2 h after glucose starvation. The cell lysates were then probed with an antibody to Ent5. Arrowheads indicate hyperphosphorylated Ent5. (B) Snf1 is required for adaptor localization during prolonged glucose starvation. Wild-type (WT) and snf1∆ cells expressing Gga2-GFP (top) or Ent5-GFP (bottom) from their endogenous loci were imaged before (+glucose), within 10 min (acute starvation), and after 2 h of glucose starvation (prolonged starvation). Scale bar, 5 μm. (C) Quantification of the number of puncta per cell for cells in B. **p < 0.01; n.s., not significant. Charts show box plots of the data. The line indicates the median, the edges of the box indicate the 25 and 75% percentiles, and the whiskers extend to the extremes of the data not considered outliers. (D) Snf1 is activated within 5 min of glucose starvation. Cellular lysates were prepared from cells expressing Snf1-myc from its endogenous locus at the indicated time points before or after glucose starvation. Active Snf1 was detected with a phospho-AMPK, and total cellular Snf1 was detected with an anti-myc antibody.