Figure 3. Phosphorylation of Sec24 regulates autophagosome frequency during starvation.

(A) Representative images from WT cells expressing GFP-Atg8 treated with 400 ng/ml rapamycin for 1 hr at 25°C (left top) or untreated (left bottom). Deconvolved images are shown. Scale bar, 1 µm. 100 GFP-Atg8 puncta were measured in WT cells treated with or without rapamycin (right). Averages and s.e.m. are shown for three biological replicates; p=0.0002, Student’s unpaired t-test. (B) WT Sec24 and Sec24-3A expressed in the sec24Δiss1Δ deletion strain, and WT (+Atg11) and atg11Δ cells (-Atg11) expressing GFP-Atg8 were imaged and the number of puncta per cell was quantitated from 300 cells. Averages and s.e.m. are shown for three biological replicates; p-values=0.81 (Sec24-3A), 0.009 (-Atg11), Student’s unpaired t-test. (C) As in (B) except cells were treated with 400 ng/ml rapamycin for 1 hr at 25°C. Averages and s.e.m. are shown for three biological replicates; p-values=0.02 (Sec24-3A), 0.68 (-Atg11), Student’s unpaired t-test. (D) As in (C) only the diameter of 100 GFP-Atg8 puncta was measured from cells expressing WT Sec24 and Sec24-3A in sec24Δiss1Δ deletion strains treated with 400 ng/ml rapamycin for 1 hr at 25°C. Averages and s.e.m. are shown for four biological replicates; p-value = 0.057, Student’s unpaired t-test. (E) Representative images of autophagic bodies in cells expressing Sec24 (left) and Sec24-3A (right) in sec24Δiss1Δpep4Δ deletion strains after 1.5 hr of nitrogen starvation at 30°C. Scale bar represents 500 nm. (F) Histogram showing the distribution of the number of autophagic bodies per vacuole section in Sec24 and Sec24-3A. The number of autophagic bodies was quantitated for 78 vacuole sections for each strain (left). p-value = 0.00012; Mann-Whitney Test. Box plot of the number of autophagic bodies per vacuole section. Bars show data between the lower and upper quartiles, the median is a horizontal line within the box. Whiskers indicate the smallest and largest observations (right). (G) The diameter of autophagic bodies was determined. For Sec24 N = 398, for Sec24-3A N = 342. Averages with error bars as s.e.m. are shown. *p<0.05; **p<0.01; ***p<0.001.

DOI: http://dx.doi.org/10.7554/eLife.21167.009

Figure 3—figure supplement 1. GFP-Atg8 puncta formation is affected in the sec12-4 mutant during autophagy induction.

(A) Cells with (left) and without (right) Iss1 were lysed as described in the Materials and methods and Ape1 processing was assayed by western blot analysis. Ape1 is processed in the sec24 alanine mutants to the same extent as wild-type. (B) WT and sec12-4 mutant cells expressing GFP-Atg8 were treated with 400 ng/ml rapamycin (left) or untreated (right) for 1 hr at 37°C. The number of GFP-Atg8 puncta per cell was determined for 300 cells. Averages and s.e.m. are shown for three biological replicates; p-value = 0.002 (rapamycin) 0.51 (nutrient rich), Student’s unpaired t-test. **p<0.01. (C) The size of GFP-Atg8 puncta from WT and sec12-4 cells treated with rapamycin was determined for 100 puncta. Averages and s.e.m. are shown for four biological replicates; p-value = 0.13, Student’s unpaired t-test.

DOI: http://dx.doi.org/10.7554/eLife.21167.010