Reconstitution of the Atg12–Atg5 conjugation system. MKO, MKO (ATG3), and atg12Δ cells transformed with various plasmids were grown in selective SMD medium, collected at mid-log phase, and subjected to Western blot analysis using an anti-HA antibody. 0.2 OD600 units of cells were loaded in each lane. Pgk1 was used as a loading control. Plasmids expressing HA-tagged Atg12 (pHA-ATG12(416)); Atg7 and Atg10 (pATG7-ATG10(414)); Atg5 and HA-tagged Atg12 (pATG5-HA-ATG12(416)); Atg5, HA-Atg12, and Atg16 (pATG5-HA-ATG12-ATG16(416)); and Atg8, Atg4, Atg7, and Atg10 (pATG8-ATG4-ATG7-ATG10(414)) were used as indicated. With Atg5, Atg12, Atg7, and Atg10 expressed in the MKO strain, a small amount of Atg12–Atg5 conjugate formed (lane 5). Additional expression of Atg16 improved the conjugation efficiency (lane 6). The presence of Atg8, Atg4, and Atg3 from the Atg8 conjugation system further facilitated the formation and/or enhanced the stability of Atg12–Atg5 (lanes 7 and 8).
