(A) Schematic explaining the principle of the prApe1 protease protection assay.
(B) The WT (SEY6210), vam3Δ (CWY40), atg1Δ (WHY1), and ymr1Δ (YMR1Δ) cells were converted to spheroplasts and starved for 3 hr in SD-N. Total cell extracts from lysed spheroplasts were centrifuged and the pellet fraction was either not treated, or mixed with proteinase K (PK) and/or TX-100 detergent before being incubated on ice for 30 min. After protein precipitation, samples were analyzed by immunoblots with anti-prApe1 antibodies [38]. The experiment was repeated four times with identical results.