Figure 1. PpAtg30 Is Essential for Micropexophagy and Macropexophagy, but Not for the Cvt or Autophagy Pathway.

(A) Wild-type (SJCF247) and Ppatg30Δ (SJCF332) cells expressing BFP-SKL were transferred from methanol to glucose or ethanol medium to induce micropexophagy or macropexophagy, respectively. Merged images show vacuoles stained by FM4-64 (red) and peroxisomes labeled with BFP-SKL (blue). Bars = 2 μm.

(B) Visualization of AOX activity after glucose adaptation for 10 hr (micropexophagy) and ethanol adaptation for 30 hr (macropexophagy) in wild-type (PPY12), Ppatg8Δ (SJCF257), and Ppatg30Δ (SJCF44) cells.

(C) Ppatg30Δ (SJCF44) and Ppatg30Δ cells expressing PpAtg30-HA (SJCF590) were grown in methanol medium overnight and shifted to glucose medium. Five OD (optical density at 600 nm) of cells were lysed and analyzed as described in the Experimental Procedures. AOX, PpPex3, and PpPex17 were used to follow peroxisome degradation. The β subunit of F0/F1 ATPase served as loading control. Filled and open arrowheads show unmodified and modified PpAtg30, respectively.

(D) Processing of PpApe1-CFP by the Cvt and autophagy pathways. Wild-type (SJCF483) and Ppatg30Δ (SJCF651) cells expressing PpApe1-CFP were grown in glucose medium (+N, Cvt pathway) and switched to nitrogen starvation medium for 4 hr (−N, autophagy pathway). PpApe1-CFP was detected in cell lysates. Precursor (pr) and mature (m) forms, respectively, of PpApe1 are shown.

(E) Wild-type (SJCF547), Ppatg30Δ (SJCF826), Ppatg9Δ (SJCF843), Ppatg11Δ (SJCF777), and pep4 prb1(SJCF557) cells expressing GFP-PpAtg8 were grown in glucose medium (+N) and shifted to SD-N for 6 hr (−N). Protein extracts were immunoblotted with anti-GFP antibody.

(F) Ppatg30Δ cells remain viable in nitrogen starvation conditions. Wild-type (PPY12), Ppatg30Δ (SJCF44), and Ppvps15Δ cells were grown toapproximately1 OD/ml in YPD medium, washed, and resuspended in nitrogen starvation medium at 0.5 OD/ml. Viability was assessed as described in the Experimental Procedures.