Figure 6. Impaired Recruitment of Vam7 to the PAS Leads to Defects in Autophagosome-Vacuole Fusion and Autophagy Activity.
(A) atg11Δ (XLY135) cells with empty vector, and atg11Δ atg17Δ (XLY136) cells with either empty vector, pCu-Atg17 (WT), pCu-Atg17F317D, or pCu-Atg17I325D were grown in SMD -Ura to mid-log phase. The cells were then shifted to SD-N medium for 4 h and autophagy activity was analyzed by the Pho8Δ60 assay. The activity of atg11Δ atg17Δ (XLY136) cells with pCu-Atg17 (WT) after starvation was set to 100% and other values were normalized. The graph shows the average activity obtained from three independent experiments. Error bars represent the SD. **, p<0.01.
(B) A schematic model of the prApe1 protease protection assay. PK, proteinase K.
(C) vam7Δ (XLY137) cells with empty vector, and atg11Δ atg17Δ (XLY136) cells with either empty vector, pCu-Atg17 (WT) or pCu-Atg17 (F317D) were grown in SMD-Ura medium to mid-log phase. The cells were then shifted to SD-N medium for 4 h. Total cell lysates from lysed spheroplasts were centrifuged at 300 x g to pellet cell debris. Then the cell lysates were further centrifuged at 5000 x g to obtain a prApe1-enriched membrane fraction, which was split into four aliquots and subjected to different treatments: no addition, 0.2% Triton X-100 (TX-100), proteinase K (PK), or proteinase K with 0.2% Trition X-100 on ice for 30 min. After protein precipitation, samples were analyzed by western blot. S.E., short exposure; L.E., long exposure.
(D) The quantification of percentage of protected prApe1 from three independent experiments. The percentage is calculated from amount of prApe1 after PK treatment divided by total level of Ape1 without any treatment. *, p<0.05.
(E) The atg17Δ vam7Δ GFP-ATG8 (XLY142) cells with pCu-Atg17 (WT), and the atg11Δ atg17Δ GFP-ATG8 (XLY147) cells with pCu-Atg17 (F317D) were grown in SMD-Ura to mid-log phase. Cells were then shifted to SD-N medium for 4 h to induce autophagy before samples were frozen and analyzed by correlative light and electron microscopy. The top two panels show samples observed by TEM tomography, while the bottom panel shows results from STEM tomography. The bottom right corner of each electron tomographic image shows the same cell visualized by fluorescence microscopy. An overlay of the fluorescent signal on the tomographic volume of the same cell is also shown. Tomographic models of the outlined autophagic structures are shown to the right; the lower model is the same as the top one, but is rotated 90° around the x-axis. Scale bars, 300 nm.