Figure 8.

The Arp2/3 complex is necessary for selective autophagy. (A) Precursor Ape1 maturation is blocked in the arp3-2 (RLY193), arc15-10 (Y4963), arc35-6 (BGY0809), and arc40-118 (BGY0883) strains. The cells were grown to mid-log phase at 24°C, and then they were incubated for 3 h either at 24 or 37°C. Precursor Ape1 maturation was monitored with pulse-chase radiolabeling followed by immunoprecipitation with antiserum to Ape1. The positions of precursor and mature Ape1 are indicated. (B) GFP-Atg8 processing is delayed in some strains with mutations in Arp2/3 complex subunits. The wild-type, arp3-2, arc15-10, arc35-6, and arc40-118 strains transformed with a GFP-Atg8 plasmid [pGFPAUT7(414) or pGFPAUT7(416)] were grown in SMD to mid-log phase at permissive temperature and preincubated for 30 min either at 24 or 37°C before being shifted to starvation conditions (SD-N). At the indicated time points, aliquots were taken and protein extracts were analyzed by Western blot by using anti-GFP antibody. The positions of GFP-Atg8 and free GFP are indicated along with the percentage of free GFP. (C) Mutations in Arp2/3 complex regulatory proteins block selective autophagy. The wild-type (SEY6210) and atg9Δ (JKY007) strains were grown to mid-log phase at 30°C, and protein extracts were prepared for controls. The pan1-3 (YAS1115), las17-11 (DDY1960), las17ΔWCA (DDY3045) and las17ΔWCA pan1Δ855-1480 (DDY2885) strains were grown to mid-log phase at 24°C, shifted to 37°C for 60 min, and examined by Western blot as described in A.