Figure 5.
Atg9 is in a complex with itself. (A) A yeast two-hybrid analysis reveals that Atg9 can bind itself. pGAD-ATG9, pGDBU-ATG9 and the vectors without inserts were cotransformed in the appropriate combinations into the PJ69-4A strain. Interactions were monitored by the ability of cells to grow on plates lacking histidine (-his). (B) Atg9 affinity isolation. The Atg9-PA pep4Δ (FRY172) strain carrying either an empty plasmid (pRS416) or one expressing Atg9-GFP (pCuGFPATG9(416)), and the pep4Δ (TVY1) strain expressing Atg9-GFP were used to prepare detergent-solubilized extracts. Protein A fusions together with the associated proteins were affinity purified (IP) with IgG sepharose beads. Eluted polypeptides were separated by SDS-PAGE and visualized by immunoblotting with antibodies to PA (Dako, Glostrup, Denmark) and GFP (Covance Research Products, Berkeley, CA). (C-F) The Atg9-3HA (PSY278) strain expressing the ATG9-triple HA fusion under the control of the authentic ATG9 promoter was grown at 30°C in YPD medium to mid log phase and cells prepared for immuno-electron microscopy as described in Materials and Methods. (C) and (D) represent images of Atg9 (6 nm gold particles) colocalizing with porin (12 nm gold particles) around the mitochondrial surface, whereas (E) and (F) illustrate examples of cytosolic Atg9 clusters. White bars, 0.1 μm. (G) Atg9 is not concentrated to the MAMs. The ATG9-GFP (FRY162) strain carrying both the promSPO7RFP424 and pYES-mtBFP plasmids was grown overnight to an early log phase in a selective medium with galactose as the sole carbon source before being transferred for 3 h to medium containing glucose. Cells were then fixed as described in Materials and Methods and imaged. Mito, mitochondria. Open and closed arrowheads mark the locations of Atg9 colocalizing or not colocalizing with MAMs, respectively.