Figure 5.
Atg27 localizes to mitochondria and the Golgi complex. The Atg27-HA (WLY1) strain was grown in YPD to OD600 = 1.0 and converted into spheroplasts. The spheroplasts were osmotically lysed and separated into pellet (P13) and supernatant (S13) fractions after a 13,000 × g centrifugation. The P13 pellet fraction was separated on a sucrose density gradient (18–54%) and centrifuged for 18 h at 176,000 × g as described in Materials and Methods. A total of 13 fractions were collected from the top to the bottom of the gradient and were subjected to immunoblot analysis with antiserum against Atg27-HA, Atg9, Pma1 (plasma membrane), Por1 (mitochondria), Mnn1 (Golgi complex), and Pho8 (vacuole).