Wild-type, apg12-1 mutant and Δapg12cells were cultured in nitrogen-starvation medium containing 1 mM PMSF. After incubation for 6 h, cells were observed under a phase-contrast microscope. Arrows indicate autophagic bodies.c, Wild-type (squares) and Δapg12 (circles) were cultured in nitrogen-starvation medium and their viability was determined by phloxine B staining.
d, Quantification of autophagic activity of wild-type and Δapg12 cells by alkaline phosphatase (ALP) assay before (black bars) and after (white bars) nitrogen starvation for 4 h. Error bars indicate s.d. of three independent experiments.
a, b, Lysates from Δapg12 cells carrying only vector or 3 × HA-APG12 (a), and Δapg5, apg7-1, apg10-1 and Δapg1 cells carrying 3 × HA-Apg12 plasmid (b) were immunoblotted using anti-HA antibody. The positions of 3 × HA-Apg12 and the larger product (asterisks) are indicated.
c, Immunoblot analysis of wild-type and Δapg12 cells harbouring HA-APG5 plasmid.d, Δapg5 Δapg12cells were co-transformed with Myc-APG12 and HA-APG5. Their lysates were immunoprecipitated with anti-Myc or anti-HAantibodies and detected by immunoblotting using anti-Myc antibody. The position of the crossreacting IgG heavy chain is indicatedb, Δapg12 cells were transformed with the mutant plasmids and their lysates were immunoblotted with anti-HA antibody.
c, Autophagic activity was measured as described for Fig. 1d.
d, Transport of pro-API to the vacuole was examined byimmunoblottingwith anti-API antiserum. The positions of pro-API and mature API are indicated.Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted with anti-HA (b)Autophagic activity was determined by alkaline phosphatase assay (c).Δapg5 cells were transformed with vector alone, wild-type APG5 or APG5K149R, and then immunoblotted anti-API (d).c, In vitro conjugationof Apg5 andApg12. A cell lysate of Δapg12 carrying HA-APG5 (lane 1) was incubated with an equal amount of lysate from Δapg5 carrying HA-APG12(2 µ plasmid) (lane 2) at 30 °C with (lane 4-6) or without (lane 7) 5 mM ATP. Samples were mixed with SDS-sample buffer at the times indicated.Spheroplasts were generated from cells expressing eitherHA-Apg12 or HA-Apg5. Their lysates were mixed and layered on top of a 10-step (18-54 % w/w) sucrose gradient, and centrifuged at 174,000g for 2.5 h (ref. 29). Fifteen fractions were collected and the positions of free Apg5, free Apg12 and Apg5/Apg12conjugate were examined by western blotting. The peak fractions of alcohol dehydrogenase (ADH)(cytosol), ALP (vacuole), Kex2 (Golgi) and Sec12 (endoplasmic reticulum) are indicated by arrows.