Figure 5.

Atg9 binds to Arp2. (A) Atg9 is coimmunoprecipitated with Arp2. Either wild-type cells containing chromosomally TAP-tagged Arp2 (Arp2-TAP, WT) or atg9Δ cells with chromosomally tagged Arp2-TAP (IRA020) were used for affinity isolation as described in Materials and Methods. Eluted polypeptides were separated by SDS-PAGE and detected with anti-Atg9 antiserum. The same amounts of the total lysate (T), immunoprecipitate (IP), and flow through (F) were loaded per gel lane. (B) Arp2 is coimmunoprecipitated with Atg9. A strain expressing chromosomally tagged Atg9-protein A (FRY171) and plasmid-based Arp2-GFP or a control strain (SEY6210) only expressing plasmid-based Arp2-GFP and protein A driven by the CUP1 promoter were used for affinity isolation as described in A. Protein bands were detected with anti-YFP antibody. (C) Endogenous Atg9 is not coimmunoprecipitated with Arp2 in the absence of Atg1 or Atg11. The indicated atg1Δ (IRA023) or atg11Δ (IRA028) Arp2-tagged strains were used for affinity isolation as in A and probed with anti-Atg9 antiserum. (D) Atg9 is not coimmunoprecipitated with Atg1. A strain with chromosomally TAP-tagged Atg1 (UNY102) served as a control for Atg9 affinity isolation. Eluted polypeptides were separated by SDS-PAGE and visualized by immunoblotting as described in A.