(A) Vacuolar alkaline phosphatase activity was assayed in lysates prepared from a sec24∆iss1∆ deletion strain harboring sec24 alanine mutations. The activity of wild-type (WT) 2 hr after starvation was set as 100% and 0 time-point values were subtracted. Averages and s.e.m. are shown for 3 (or four for T324A/T325A) biological replicates. p-values=0.006 (T324A), 0.012 (T325A), 0.009 (T328), 0.008 (T324A/T328A), 0.02 (T324A/T325A), 0.01 (T325A/T328A), 0.006 (T324A/T325A/T328A), Student’s paired t-test. (B, C) As in (A) except activity was assayed in extracts from phosphomimetic mutations in sec24∆iss1∆ (B) or sec24∆ (C) deletion strains. Averages and s.e.m. are shown for three biological replicates. p-values=0.88 (T325E), 0.78 (T328E), 0.32 (T324E/T328E), 0.26 (T324E/T325E), Student’s paired t-test. (D) The translocation of GFP-Atg8 to the vacuole was examined 1 hr after nitrogen starvation at 25°C in sec24∆iss1∆ and sec24∆ deletion strains in either the presence of WT Sec24 or Sec24-3A. Representative images (left) and quantification from 300 cells (right) are shown. Scale bar 2 µm. WT was set to 100% for each experiment and had an average vacuolar localization of 76% (sec24∆iss1∆) and 86% (sec24∆). Averages and s.e.m. are shown for three biological replicates. p-values=0.006 (sec24∆iss1∆), 0.02 (sec24∆), Student’s paired t-test. (E) Cleavage of GFP-Atg8 was examined in sec24∆iss1∆ cells expressing Sec24 or Sec24-3A 1 hr after starvation at 25°C (left). The ratio of free GFP to GFP-Atg8 was quantitated. The cleavage in WT was set to 1 (right). Averages and s.e.m. are shown for three biological replicates. p-value = 0.015, Student’s unpaired t-test. (F) sec24∆iss1∆ cells expressing Sec24 (lanes 1–4) or Sec24-3A (lanes 5–8) were pulse-labeled for 4 min and chased for the indicated times (left). The p1 (ER), p2 (Golgi) and m (vacuolar) forms of CPY are labeled. Quantitation of the ratio of p2/p1 CPY for the 5 and 10 min time points are shown (right). Averages and s.e.m. are shown for three biological replicates. p-values=0.08 (5 min), 0.66 (10 min), Student’s unpaired t-test. *p<0.05; **p<0.01.