Fig 5.

The ATG8-65 homozygous knockout was sensitive to starvation. (A, left) Schematic showing the ATG8-2 genomic locus before (upper) or after (lower) homologous recombination with the neo4 knockout cassette. Numbered boxes represent three exons. Probe 1 (1,093 bp) was used to detect the insertion accuracy of the knockout cassette. Probe 2 (0.7 kb) was used to ensure the existence of the neo knockout cassette. (Right) Southern analysis of PacI-digested genomic DNA isolated from ATG8-2^−/− (2-4 and 3-8) or wild-type (CU427 and CU428) strains. KO (knockout) bands, 4.5 kb; WT (wild-type) bands, 1.5 kb; M (marker), DIG labeling marker. (B, left) Schematic showing the ATG8-65 genomic locus before (upper) and after (lower) knockout cassette replacement. The representation of numbered boxes and functions of probe 1 (1,055 bp) and probe 2 (0.7 kb) are the same as for panel A. (Right) Southern analysis of HindIII-digested genomic DNA from ATG8-65^−/− (2-2 and 3-1) or WT strains. KO bands, 3.1 kb; WT bands, 1.1 kb; M, DIG labeling marker. (C) Remaining cells in ATG8 knockout or wild-type starvation pools. Cells starved in 10 mM Tris buffer for 0, 1, 2, 4, 6, 8, 12, and 18 days were sampled (200 μl) from starvation pools and fixed in 2% paraformaldehyde. Total cell numbers represent the averages of three independent samples taken from the same starvation pool. (D) Percentages of wells with viable cells in a 96-well plate seeded with starved ATG8 knockout or wild-type cells. A 25-μl aliquot of starved cells (containing an average of 30 input cells at the beginning of starvation) was sampled from the same starvation pools and at the same time point as shown in panel C and transferred to 96-well plates with rich SPP medium (50 μl) for incubation at 30°C. Three days later, wells were examined for the presence of viable cells. Percentages represent averages from three 96-well plates. Panels C and D share the same labeling as that shown at the right side of panel D. (E and F) Mating ability of ATG8 knockout and wild-type starved cells. Vegetative cells grown in Neff medium were directly diluted with Tris buffer to generate a 10-fold-diluted Neff solution for starvation. Cells of different mating types were either mixed immediately after dilution (E) or later after overnight prestarvation (F). Cells were fixed at 0, 1, 2, 3, 4, 5, 6, and 7 h postmixing to calculate the pairing rate. Panels E and F share the same labeling as shown at the right side of panel F. Percentages represent the average values of three independent samples from two individual experiments.