Yeast model
Saccharomyces cerevisiae strain WLY176 was used to generate an ATG5 knockout strain (atg5∆) as previously described (Gueldener et al., 2002). The MKO strain YCY137 (SEY6210 atg1∆, 2∆, 4∆, 5∆, 6∆, 7∆, 8∆, 9∆, 10∆, 11∆, 12∆, 13∆, 14∆, 16∆, 17∆, 18∆, 19∆, 20∆, 21∆, 23∆, 24∆, 27∆, 29∆) (Cao et al., 2008), was used for in vivo reconstitution of Atg8 conjugation. Site-directed mutagenesis was performed to generate ATG5 amplicons with the E141D mutation as previously described (Liu and Naismith, 2008). A pRS406 empty plasmid was digested with Spel and SalI, and then ligated with a DNA fragment encoding either wild-type or mutant Atg5-PA. atg5∆ was transformed with an empty pRS406 vector, or plasmids encoding Atg5-PA WT or Atg5-PA E141D. Wild-type WLY176 colonies were transformed with empty pRS406 vector as a control. Colonies were grown on SMD-URA medium and starved in nitrogen-deficient medium. Pho8Δ60 and western blot analyses were performed as described previously (Noda and Klionsky, 2008; Shintani and Klionsky, 2004). Quantification was performed using ImageJ software. The pATG8∆R-ATG7-ATG10(414), pATG5(WT)-HA-ATG12(416) and pATG5(WT)-HA-ATG12-ATG16(416) plasmids were described previously (Cao et al., 2008). The pATG5(E141D)-HA-ATG12(416) and pATG5(E141D)-HA-ATG12-ATG16(416) plasmids were made by site-directed mutagenesis based on the wild-type constructs.