Autophagic activity of cells expressing deleted variants of Atg14p. Cells grown in SD + CA + AdeTrp medium at 30°C were collected during logarithmic phase growth (starvation–) and at 4 h after transfer to SD (–N) medium (starvation+). Lysates were prepared by the alkaline-trichloroacetic acid (TCA) method (A and B) or using glass beads (C). (A) Maturation of API was monitored by immunoblotting with an anti-API antibody. The amount of each Atg14p variant (arrowheads) was estimated by immunoblotting with an anti-HA antibody (HA-7). (B) Autophagy-dependent API maturation was estimated in cells lacking ATG11. Each variant was expressed from a single-copy plasmid. The asterisks indicate autophagy-dependent API maturation. (C) Autophagic activity was estimated using an ALP assay. The means ± SD of results from triplicate measurements are shown.

Interaction of Atg14p with Vps30p and Vps34p is mediated by coiled-coils I and II. Proteins were coimmunoprecipitated from the supernatants of Triton X-100-treated lysates using anti-GFP (A) and anti-Vps30 (B) antibodies. Isolated proteins (E, elution fraction) were resuspended in 1/10 (A) or 1/40 (B) volume of the starting supernatant fraction (I, input); equal (A) or 1.5× volume (B) was loaded onto SDS-PAGE. (A) Atg14p interacts with Vps30p and Vps34p at coiled-coil I and II regions. Atg14-I, Atg14-II, and Atg14-III were expressed from multicopy plasmids, and the other variants were expressed form single-copy plasmids. (B) Vps34p and Vps15p coimmunoprecipitated with Vps30p in a manner dependent on the variants of Atg14p expressed from single-copy plasmids in Δvps38Δatg14 cells. Arrow and asterisks indicate Vps15p and nonspecific bands, respectively. (C) In this schematic representation of Atg14p-mediated complex formation, the regions required for this interaction are indicated.

Vps38p and autophagy. (A) SEY6210 strain cells at logarithmic phase growth were transferred to S (–NC) medium in the presence of 1 mM PMSF. After 4 h of culture in S (–NC) medium, WT cells (a), Δatg14 cells (b), and Δvps38 cells (c) were observed by phase-contrast microscopy. (B) API maturation was monitored in Δatg14 cells overexpressing Vps38-HA-GFP. Cells, grown in SD + CA + AdeTrp medium at 30°C, were collected during logarithmic growth (starvation –) or 4 h after transfer to SD (–N) medium (starvation +). Lysates were subjected to immunoblotting with anti-API and anti-HA (HA-7) antibodies. (C) The accumulation of autophagic bodies was examined in Vps38-HA-GFP-overexpressing Δatg14 cells, on the BJ2168 background which lacks vacuolar proteases. WT cells (a), Δatg14 cells (b), Δatg14 cells expressing Atg14-FL from a single-copy plasmid (c), and Δatg14 cells expressing Vps38-HA-GFP from a multicopy plasmid (d) were transferred to S (–NC) medium. After 4 h of culture in S (–NC) medium, cells were observed by phase-contrast microscopy. (D) We investigated the localization of Vps38p. Diploid cells expressing chromosomal Vps38-GFP and API-RFP (YOK89) were cultured in SD + CA + AdeTrpUra medium and subjected to fluorescence microscopy. Two representative fields are shown. Bars, 5 μm.

Localization of Vps30p. (A) Diploid cells expressing chromosomal Vps30-GFP and API-RFP were observed by fluorescence microscopy. The arrows indicate the structures to which Vps30-GFP and API-RFP colocalize. (B) Haploid cells expressing chromosomal Vps30-GFP (left). Vps30-GFP was dispersed throughout the cytoplasm, which was confirmed by cell fractionation analysis of WT and Δatg14Δvps38 cells expressing an untagged Vps30p (right). Dpm1p and Kex2p were used as markers for the ER and late Golgi, respectively. Bars, 5 μm.

Distinct distribution of the two PI3-kinase complexes. (A) The intracellular localization of each component of the PI3-kinase complexes is shown in green, whereas the outline of the cell is detailed in black. (B) The distribution of the PI3-kinase complexes is summarized. “X” represents an unknown component(s).

Atg14p deletion series. (A) The presence of three coiled-coil regions (I, II, and III) were predicted within Atg14p (COILS; http://www.ch.embnet.org/software/COILS_form.html). (B) The deleted variants of Atg14p are shown schematically. HA-GFP, with a linker sequence (two repeats of GlyGlyGlySer) before both the HA and GFP sequences was fused to the C terminus of each variant. The coiled-coils are shown in gray. Two amino acid residues (SerSer, corresponding to the ligation site, XbaI/SpeI) were inserted at the position denoted by the asterisks.

Minimal region essential for Atg14p function in autophagy. Cells grown in SD + CA + AdeTrp medium at 30°C were collected during logarithmic growth (starvation–) and 4 h after transfer to SD (–N) (starvation+) (A and B). Lysates were prepared by the alkaline-TCA method (A) or using glass beads (B). (A) Maturation of API was monitored by immunoblotting with an anti-API antibody. The amount of each Atg14p variant (arrowheads) expressed from a multicopy plasmid was estimated by immunoblotting with an anti-HA antibody (HA-7). (B) The extent of autophagy was estimated by ALP assay. The means ± SD of results from triplicate measurements are shown. (C) The accumulation of autophagic bodies was examined on the BJ2168 background, which lacks vacuolar proteases. Cells in logarithmic phase growth were transferred to S (–NC) medium. After 4 h of culture in S (–NC) medium, cells were observed by phase-contrast microscopy. WT cells bearing an empty vector (a) and Δatg14 cells bearing an empty vector (b) or expressing Atg14-FL from a single-copy plasmid (c), Atg14-ΔIII from a single-copy plasmid (d), Atg14-I·II·III from a single-copy plasmid (e), Atg14-I·II from a single-copy plasmid (f), Atg14-FL from a multicopy plasmid (g), Atg14-I·II·III from a multicopy plasmid (h), or Atg14-I·II from a multicopy plasmid (i) are shown. Bar, 5 μm. (D) Cell viability under nitrogen-starvation conditions was measured. Cells were grown to logarithmic phase in SD + CA + AdeTrp and then transferred to SD (–N) medium. Cell viability was determined by phloxine B staining, as described in Materials and Methods.

PAS-localization of Atg14p variants. (A) Atg14-FL, Atg14-I·II·III, and Atg14-I·II expressed from single-copy plasmids colocalized with API, a PAS marker. Vacuolar fluorescence was significantly reduced in cells expressing either Atg14-I·II·III or Atg14-I·II. (B) Atg14-FL expressed from a single copy-plasmid in Δatg14Δvps38 cells (YOK435). Bars, 5 μm.

Localization of Vps34p. Diploid cells expressing chromosomal Vps34-GFP and API-RFP were observed by fluorescence microscopy. The arrows indicate the structures to which Vps34-GFP and API-RFP colocalize. Bar, 5 μm.

Localization of Vps15p. Within the fluorescence images of diploid cells expressing chromosomal Vps15-GFP and API-RFP, the arrows indicate the structures at which Vps15-GFP and API-RFP colocalize. Bar, 5 μm.