Figure 4. Closed Autophagosomes Accumulate in the Absence of Ymr1.
(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.
