PMC

Atg20 interacts with Atg11 via coiled-coil domain 2. Detergent extracts of atg11Δ (AHY001) cells expressing myc-Atg11 with or without PrtA-Atg20 (A) or myc-Atg11ΔCC2 with PrtA-Atg20 (B) were incubated with IgG-Sepharose. Precipitated proteins were subjected to SDS-PAGE followed by immunoblotting with anti-myc antibody. T and IP lanes show total lysates and IgG precipitates, respectively. (C) atg11Δ cells expressing CFP-Atg11 or CFP-Atg11ΔCC2 with Atg20-YFP were grown to mid-log phase and observed by fluorescence microscopy. DIC, differential interference contrast.

Mapping of the binding sites of Atg components within Atg11. Two-hybrid analysis of Atg1 lacking the kinase domain (Δ1–325), Atg11, Atg17, Atg19, and Atg20 with wild-type Atg11 or Atg11 mutants. The two-hybrid atg11Δ (YTS121) strain was transformed with plasmids containing BD-Atg1, BD-Atg11, BD-Atg17, or BD-Atg20 and AD-Atg11 (or AD-Atg11 with deletions), or AD-Atg19 and BD-Atg11 (or BD-Atg11 with deletions), and transformants were grown at 30°C for 7 d for all combinations with the exception of AD-Atg19 and BD-Atg11, which were grown for 2 d. + and – show an ability and inability, respectively, for cells with plasmids to grow on plates lacking uracil, leucine, and adenine.

Localization of Atg components at the PAS with Atg11 mutants. atg11Δ (AHY001) cells transformed with a plasmid expressing GFP-Atg1, atg11Δ (YTS192) cells expressing GFP-Atg8, or atg11Δ vps38Δ (TYY014) cells with Atg20-YFP containing the empty vector or a plasmid carrying the wild-type or indicated mutant Atg11 were grown in rich medium. The percentage of cells containing a punctate dot corresponding to the fluorescent protein are indicated. More than 200 cells were quantified in three individual experiments by fluorescence microscopy, and the error bars represent the SD.

The Atg1–Atg13 complex and Atg1 kinase activity affect Atg11 homo-oligomer disassembly. (A) Atg11 homo-oligomers accumulate in the absence of Atg1 or Atg13. atg11Δ (AHY001), atg1Δ (WHY1), atg13Δ (CWY233), or atg20Δ (D3Y109) cells with plasmids expressing CFP-Atg11ΔCC4 and wild-type Atg11, or atg1Δ cells with a plasmid expressing CFP-Atg11ΔCC4 and the Atg1^K54A mutant and wild-type Atg11 were observed with a fluorescence microscope, and 200–250 cells containing fluorescent punctate dots were quantified. Error bars indicate the SD of three independent experiments. (B) The absence of Atg1 or Atg13 does not increase the stability of the PAS. Wild-type (SEY6210), atg1Δ (WHY1), atg13Δ (CWY233), or atg20Δ (D3Y109) cells transformed with a plasmid expressing GFP-Atg8 were observed and cells containing punctate dots were quantified. Error bars indicate the SD of three independent experiments.

Atg11 interacts with the Cvt complex via Atg19. Atg11 does not localize to a punctate structure in the absence of prApe1 (A) or Atg19 (B). (A) Wild-type (SEY6210) or ape1Δ (YTS107) cells expressing CFP-Atg11 and YFP-Atg19 were grown to mid-log phase and observed by fluorescence microscopy. DIC, differential interference contrast. (B) Wild-type or atg19Δ (SSY31) cells expressing CFP-Atg11 and YFP-Ape1 were grown to mid-log phase and observed by fluorescence microscopy. (C) Atg11 interacts with the Cvt complex via Atg19. Detergent extracts of atg19Δ or ape1Δ atg19Δ (YTS108) cells expressing HA-Atg11 and protein A-fused Atg19 (PrtA-Atg19) were incubated with IgG-Sepharose. Precipitated proteins were subjected to SDS-PAGE followed by immunoblotting with anti-HA antibody or anti-Ape1 antiserum. T and IP lanes show total lysates and IgG precipitates, respectively.

Atg11 forms a homo-oligomer at the PAS. (A) atg11Δ (AHY001) cells with a plasmid encoding CFP-Atg11ΔCC4 or wild-type Atg11 under control of the CUP1 promoter were grown to mid-log phase and observed by fluorescence microscopy. DIC, differential interference contrast. (B) Atg11 CC4 can act in trans for interaction with Atg19 and localization to the PAS. atg11Δ cells expressing CFP-Atg11ΔCC4 and wild-type Atg11 with YFP-Atg19 or YFP-Atg8 were grown to mid-log phase and examined by fluorescence microscopy. (C) Atg11 CC2 is unable to act in trans. atg11Δ or atg1Δ atg11Δ (YTS157) cells with plasmids containing CFP-Atg11ΔCC4 and Atg11ΔCC2 were grown to mid-log phase and observed by fluorescence microscopy. (D) Atg11ΔCC2 is defective in homo-oligomer formation. Detergent extracts of atg11Δ cells expressing wild-type HA-Atg11 or HA-Atg11ΔCC2 and myc-Atg11 were incubated with anti-HA antibody. Precipitated proteins were subjected to SDS-PAGE followed by immunoblotting with anti-myc or anti-HA antibody.

Atg11 interacts with multiple Atg proteins. (A) Schematic drawing of interactions involving Atg11 and its interacting proteins (except for the Ape1 complex, drawn in approximately relative scales). The proteins shown in yellow are specific to import of prApe1, whereas those shown in green are needed primarily for specific types of autophagy such as the Cvt pathway and/or pexophagy. The protein depicted in purple is required only for nonspecific autophagy, whereas those shown in blue are needed for both specific and nonspecific types of autophagic sequestration. 8, Atg8; PAS, pre-autophagosomal structure. (B) Atg11 is predicted to have four coiled-coil domains. Double-headed arrows show the interactions of Atg11 with Atg components based on previous and present studies. See the text for details and references.

Coiled-coil domains 2 and 3 of Atg11 are required for the transport of prApe1 to the PAS. (A) Total cell extracts of atg11Δ (AHY001) cells expressing wild-type or mutant Atg11 proteins were separated by SDS-PAGE followed by immunoblotting with anti-Ape1 antiserum. (B) Deletion of Atg11 CC2 or CC3 prevents localization of prApe1 to the PAS. atg11Δ cells expressing GFP-Ape1 and wild-type or mutant Atg11 proteins were grown to mid-log phase, labeled with FM 4-64, and examined by fluorescence microscopy. (C) Atg11ΔCC2 and ΔCC3 mutants can interact with Atg19. Detergent extracts of atg11Δ cells expressing mutant HA-Atg11 and PrtA-Atg19 were incubated with IgG-Sepharose. Precipitated proteins were subjected to SDS-PAGE followed by immunoblotting with anti-HA antibody. T and IP lanes show total lysates and IgG precipitates, respectively. (D) Colocalization of Atg11ΔCC2 and ΔCC3 with Atg19. atg11Δ cells expressing wild-type or mutant CFP-Atg11 and YFP-Atg19 were grown to mid-log phase and examined by fluorescence microscopy. (E) Atg11ΔCC2 and ΔCC3 do not localize to the PAS. atg11Δ cells expressing wild-type or mutant CFP-Atg11 and YFP-Atg8 were grown to mid-log phase and examined by fluorescence microscopy. DIC, differential interference contrast.

Atg19 interacts with the C terminus of Atg11. (A) Mapping of the Atg19 binding site within Atg11 by a yeast two-hybrid assay. A schematic of Atg11 is shown indicating the location of the coiled-coil domains. The two-hybrid atg11Δ (YTS121) strain was transformed with plasmids containing the activation domain (AD)-fused Atg19 and the binding domain (BD)-fused wild-type or mutant Atg11, and transformants were grown on plates lacking uracil, leucine, and adenine at 30°C for 2 d. (B) Atg19 binds the C terminus of Atg11. Detergent extracts of atg11Δ (AHY001) cells expressing wild-type or mutant HA-Atg11 and PrtA-Atg19 were incubated with IgG-Sepharose. Precipitated proteins were subjected to SDS-PAGE followed by immunoblotting with anti-HA antibody. T and IP lanes show total lysates and IgG precipitates, respectively. (C) The ability to bind Atg19 is not sufficient for Atg11 to facilitate prApe1 maturation. Total cell extracts of atg11Δ cells expressing wild-type or mutant Atg11 were separated by SDS-PAGE followed by immunoblotting with anti-Ape1 antiserum. (D) Multiple Atg11 domains are required to recruit prApe1 to the PAS. atg11Δ cells expressing GFP-Ape1 and wild-type Atg11 or Atg11 mutant proteins were grown to mid-log phase and labeled with FM 4-64. (E) Colocalization of Atg11 with Atg19 requires CC4. atg11Δ cells expressing wild-type or mutant CFP-Atg11 and YFP-Atg19 were grown to mid-log phase and examined by fluorescence microscopy. DIC, differential interference contrast.