Figure 3.

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.