Increased phosphorylated S6K in the absence of buffy poises the animal for starvation-induced autophagy and is target of rapamycin (Tor) dependent. (A) Increased phosphorylated S6K is no longer detected in the buffy mutant when Tor signaling is blocked. Immunoblot of lysates from fed and 2 h-starved larvae of the indicated genotypes (all are homozygous with the exception of the heterozygous torΔP/tor+ control) using the indicated phosphospecific S6K antibody. The ratio of phosphorylated S6K to tubulin loading control is indicated below each lane, relative to wild-type fed. (B) Increased phosphorylated S6K in the buffy mutant does not promote growth. Left panel: the average mass of five staged pupae is similar for buffy mutant and wild-type animals. Mean ± SEM; n = 20 groups of five for both wild-type and buffy mutant. Right panel: the average fat body cell size is similar for buffy mutant and wild-type animals. Mean ± SEM; n = 97 fat body cells from 4 animals for buffy mutant and 119 fat body cells from 5 animals for wild-type. (C, D) LTR-positive punctae are not visible in fat bodies from da-Gal4; UAS-S6KSTDE larvae after 2 h of amino-acid starvation, but are after 4 h of amino-acid starvation (compare with (A)). (E) Quantification of density of LTR-positive punctae from 15 animals per indicated genotype after 2 and 4 h of starvation. Mean ± SEM. (F-H) Removal of a genomic copy of S6K reverts the precocious autophagy phenotype of the buffy mutant, as demonstrated by LTR staining of fat bodies from larvae starved for 2 h. (F) buffyH37/+; S6Kl-1/+ fat body with wild-type autophagy. (G) buffyH37 homozygous mutant fat body with robust autophagic response. (H) buffyH37/buffyH37; S6Kl-1/+ fat body demonstrating reversion of autophagic phenotype to wild-type. Images are representative of seven to ten animals per genotype.