Figure 3.

LatA blocks pexophagy. (A) Actin filaments are required for peroxisome elimination by pexophagy. The wild-type (BY4742) and atg1Δ (atg1Δ) strains transformed with a plasmid expressing GFP-SKL were grown in conditions that induce peroxisomes, washed, and resuspended in SD-N medium for 6 h in the presence or absence of 200 μg/ml LatA. Cells were imaged before and after starvation. (B) Quantification of peroxisome accumulation in LatA-treated cells. Peroxisomes were counted in ∼30 cells prepared for the imaging shown in A. The experiment was repeated twice, and error bars represent the peroxisome number deviation between cells. (C) LatA inhibits the proteolytic degradation of peroxisomal proteins during pexophagy. The Pex14-GFP (IRA001) and Pex14-GFP atg1Δ (IRA002) strains were grown and treated as in A. Protein extracts were prepared from cells before and after nitrogen starvation and resolved by SDS-PAGE, and after Western blot, membranes were probed with anti-GFP monoclonal antibody (Covance, Princeton, NJ). The positions of the full-length Pex14-GFP fusion protein and free GFP are indicated.