Lengths of the IMs reflect the magnitude of autophagy. (A) RFP–prApe1-expressing cells harboring pRS314[GFP-ATG8] and pYEX-BX[prAPE1] were grown in SDCA medium containing CuSO₄ and treated with rapamycin for 6 hours. Scale bar: 2 µm. (B) Lengths of IMs, estimated from the images in A. Fluorescence intensities of GFP–Atg8 were measured using the ‘linescan’ function of the MetaMorph software, and FWHM (full width at half maximum) was calculated from each graph. Wild-type (n = 10), atg1Δ (n = 8), atg2Δ (n = 17), atg18Δ (n = 19), atg1^D211A (n = 12) and atg1^K54A (n = 13) cells were used. Error bars indicate standard deviations. *P<0.05; **P<0.001; N.S. (not significant) indicates P>0.05 (two-tailed Student's t-test). (C) Autophosphorylation of Atg1. Cells were grown in YEPD medium and incubated in the presence of rapamycin for 6 hours; cell lysates were prepared by the alkaline lysis method. Aliquots of cell lysates (0.05 OD₆₀₀ units) were subjected to immunoblot analysis using an anti-Atg1 antiserum. The slower-migrating bands represent phosphorylated Atg1 (P-Atg1), and the faster-migrating bands represent unphosphorylated Atg1 (Atg1). (D) Activity of bulk autophagy, estimated by the alkaline phosphatase assay. Cells harboring pTN3 were grown in SDCA medium and transferred to SD(–N) medium. Collected cells were subjected to the alkaline phosphatase assay (Noda et al., 1995). **P<0.001; N.S. (not significant) indicates P>0.05 (two-tailed Student's t-test).

Fine mapping of Atg proteins on the IMs. Cells expressing C-terminally GFP-fused Atg/Vps proteins were transformed with pRS314[2×mCherry-Atg8] and pYEX-BX[prApe1]. The cells were grown in SDCA medium containing CuSO₄ and treated with rapamycin. Fluorescence images of mCherry–Atg8 (Ch–Atg8) and GFP-fused Atg/Vps proteins (GFP) were merged (Merge). Fluorescence profiles along the IMs from the distal end to the vacuole are shown from left to right (Fluorescence). Based on these images, the localization of Atg/Vps proteins on IMs can be classified into three categories, as indicated. Arrowheads indicate the VICS. Scale bar: 2 µm.

The Atg2–Atg18 complex and Atg9 are colocalized at the edge of the IM. Cells were grown in SDCA medium containing CuSO₄ and treated with rapamycin for 3 hours. (A) Images of cells harboring pYEX-BX[prApe1] and chromosomally integrated Atg2-2×GFP and Atg18-3×mCherry. Arrows indicate colocalized dots. Asterisks indicate positions of the GAC. Scale bar: 2 µm. (B) Images of cells harboring pYEX-BX[prApe1] and chromosomally integrated Atg9-2×GFP and Atg18-3×mCherry. Arrows indicate colocalized dots. Asterisks indicate positions of the GAC. Scale bar: 2 µm.

ER is localized adjacent to the IM. Cells were grown in SDCA medium containing CuSO₄ and treated with rapamycin for the indicated number of hours. (A) Cells harboring pRS314[2×mCherry-Atg8] and pYEX-BX[prApe1] as well as chromosomally integrated Dpm1-GFP. Asterisks indicate positions of the GAC. Scale bar: 2 µm. (B) Cells chromosomally expressing Sec71TMD-GFP harboring pRS314[2×mCherry-Atg8] and pYEX-BX[prApe1]. Asterisks indicate positions of the GAC. Scale bar: 2 µm. (C) Three-dimensional images of cells harboring pMO13 (GFP-HDEL) and pRS424[P_CUP1-prApe1] as well as chromosomally integrated 2×mCherry-Atg8 (Ch-Atg8). An asterisk indicates the position of the GAC. Arrows indicate sites at which the IM and the ER are associated. Scale bar: 1 µm.

The IM is associated with ERES. Cells were grown in SDCA medium containing CuSO₄ and treated with rapamycin. (A) Three sets of three-dimensional images of cells harboring pRS314[2×mCherry-Atg8] and pYEX-BX[prApe1] as well as chromosomally integrated Sec13-2×GFP. ERES labeled with Sec13 were localized in close proximity to the IM (arrows). Asterisks indicate positions of the GAC. Scale bar: 1 µm. (B) The Atg2–Atg18 complex is closely localized to ERES at the IM edge. Three sets of three-dimensional images of cells harboring pYEX-BX[prApe1] as well as chromosomally integrated Atg18-2×GFP and Sec13-2×mCherry (Sec13-Ch). ERES labeled with Sec13 were localized in close proximity to Atg18 at two or three sites per cell (arrows). Asterisks indicate positions of the GAC. Scale bar: 1 µm.