Atg9-containing structures are small single-membrane vesicles. (A) Mobility of Atg9 puncta in vitro. ATG9-2×GFP atg11Δ atg17Δ pep4Δ cells and ANP1-GFP atg11Δ atg17Δ pep4Δ cells were spheroplasted, ruptured with a Dounce homogenizer, and centrifuged at 15,000 g for 15 min. The supernatant fraction was mixed with 40-nm red FluoSpheres and then observed as in Fig. 1 C. Green fluorescence (green channel [ch.]) and red fluorescence (red channel [ch.]) were acquired concurrently. (B–D) Histograms of the diffusion coefficients of Atg9 puncta prepared from growing cells (B) or rapamycin-treated cells (C) and of the Golgi protein Anp1-GFP (D) are shown. FS, FluoSpheres. The mean size of the FluoSpheres used in this study was 44.1 nm (Fig. S1 D). The histograms were fitted to Gaussian distributions; medians of the fitting curves are indicated at the top. The data shown are from a single representative experiment out of three repeats. (E) Immunoisolation of Atg9-containing structures. atg11Δ atg17Δ pep4Δ cells expressing Atg9-6×FLAG were converted to spheroplasts (nutrient [Nut.]) and then treated with rapamycin for 2 h (Rap.). The spheroplasts were ruptured by passage through membrane filters with 5-µm pores. After a centrifugation at 50,000 g for 15 min, the supernatants (S₅₀) were subjected to immunoisolation using the anti-FLAG antibody. The bound materials were eluted with 3×FLAG peptide and subjected to immunoblotting using antibodies against Atg9, Dpm1 (ER), Tim50 (mitochondria), Vph1 (vacuole), and Pgk1 (cytoplasm). Un, unbound fractions; E25×, eluted fractions concentrated 25-fold. (F) Size distribution profiles of the isolated Atg9-containing structures. The eluted fractions of E were subjected to DLS measurement. (G and H) Atg9-containing structures were immunoisolated from cells expressing both Atg9-6×FLAG and Atg9-3×BAP (3× biotinylated tag) and then subjected to negative staining EM. In G, the Atg9-containing structures were labeled with streptavidin-conjugated Qdots (Invitrogen). Arrowheads indicate Qdots. (I) CSE4-GFP cells were observed at 30 ms/frame. Arrowheads indicate kinetochore clusters consisting of ∼80 molecules of Cse4-GFP. Outlines indicate the edges of cells. (J) ATG9-2×GFP atg11Δ atg17Δ cells and CSE4-GFP cells were grown to logarithmic phase (nutrient) and then either starved for 2 h (Stv.) or treated with rapamycin for 2 h (Rap.). The fluorescence intensities of each GFP cluster (Atg9-2×GFP vesicle or Cse4-GFP kinetochore cluster) were quantified. Background signals of cytoplasmic regions were subtracted. The mean intensity of the Cse4-GFP kinetochore cluster is set to 80 U. Error bars indicate standard deviation. a.u., arbitrary unit.