Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 inDrosophila

Abstract

Phagophore-derived autophagosomes deliver cytoplasmic material to lysosomes for degradation and reuse. Autophagy mediated by the incompletely characterized actions of Atg proteins is involved in numerous physiological and pathological settings including stress resistance, immunity, aging, cancer, and neurodegenerative diseases. Here we characterized Atg17/FIP200, the Drosophila ortholog of mammalian RB1CC1/FIP200, a proposed functional equivalent of yeast Atg17. Atg17 disruption inhibits basal, starvation-induced and developmental autophagy, and interferes with the programmed elimination of larval salivary glands and midgut during metamorphosis. Upon starvation, Atg17-positive structures appear at aggregates of the selective cargo Ref(2)P/p62 near lysosomes. This location may be similar to the perivacuolar PAS (phagophore assembly site) described in yeast. Drosophila Atg17 is a member of the Atg1 kinase complex as in mammals, and we showed that it binds to the other subunits including Atg1, Atg13, and Atg101 (C12orf44 in humans, 9430023L20Rik in mice and RGD1359310 in rats). Atg17 is required for the kinase activity of endogenous Atg1 in vivo, as loss of Atg17 prevents the Atg1-dependent shift of endogenous Atg13 to hyperphosphorylated forms, and also blocks punctate Atg1 localization during starvation. Finally, we found that Atg1 overexpression induces autophagy and reduces cell size in Atg17-null mutant fat body cells, and that overexpression of Atg17 promotes endogenous Atg13 phosphorylation and enhances autophagy in an Atg1-dependent manner in the fat body. We propose a model according to which the relative activity of Atg1, estimated by the ratio of hyper- to hypophosphorylated Atg13, contributes to setting low (basal) vs. high (starvation-induced) autophagy levels in Drosophila.

Loss of Drosophila Vps16A enhances autophagosome formation through reduced Tor activity

“…9,11 Indirect immunofluorescent labeling and microscopy was carried out as described previously, using chicken anti-GFP (Invitrogen, A10262), rabbit anti-Cp1/cathepsin L (Abcam, ab58991), rat antiAtg8a primary and appropriate secondary antibodies: Alexa568 conjugated anti-rat (Life Technologies, A11077), Alexa 488 conjugated anti-chicken (Life Technologies, A11039), and Alexa546 conjugated anti-rabbit (Invitrogen, A11035). 3,9,11 Gel electrophoresis and western blots were done as before, using mouse anti-aTub84B/a-tubulin (DSHB, AA4.3-s), mouse anti-P-S6k/ Phospho-RPS6KB1 (Thr389) (Cell Signaling Technology, 9206), rabbit anti-P-Thor/Phospho-EIF4EBP1 (Thr37/46) (Cell Signaling Technology, 2855), rabbit anti-Atg8a, rat anti-Atg13, rat anti-Atg1 and rat anti-Atg9 primary and appropriate alkaline phosphatase-conjugated secondary antibodies: anti-rabbit (Sigma-Aldrich, A3812), anti-rat (Jackson ImmunoResearch, 112-055-003), and anti-mouse (Millipore, AP124A). 3,9,11,33 …”

“…3,9,11 Gel electrophoresis and western blots were done as before, using mouse anti-aTub84B/a-tubulin (DSHB, AA4.3-s), mouse anti-P-S6k/ Phospho-RPS6KB1 (Thr389) (Cell Signaling Technology, 9206), rabbit anti-P-Thor/Phospho-EIF4EBP1 (Thr37/46) (Cell Signaling Technology, 2855), rabbit anti-Atg8a, rat anti-Atg13, rat anti-Atg1 and rat anti-Atg9 primary and appropriate alkaline phosphatase-conjugated secondary antibodies: anti-rabbit (Sigma-Aldrich, A3812), anti-rat (Jackson ImmunoResearch, 112-055-003), and anti-mouse (Millipore, AP124A). 3,9,11,33 …”

“…Mutant and overexpression lines used in this study were UASRheb, hs-Gal4, Df(3R)BSC507, Df(3R)ED5339, Df(3L) Exel8098, Df(2L)lt45 (all obtained from the Bloomington Stock Center), UAS-gig/Tsc2 RNAi[KK100646] (obtained from the Vienna Drosophila RNAi Center), CG32350/Vps11[LL06553], lt [11], Vps16A[d32], 11 Syx17[LL06330], 9 hs-Flp, Act>CD2> Gal4, UAS-mCD8-GFP (all recombined together on chromosome X), 3 and UAS-Venus-raptor (this study). Expression of Venus-raptor was mediated by crossing transgenic flies to a hsGal4 stock, and heat shocking early L3-stage larvae for 30 0 at 37 C, followed by a 4 h recovery.…”

“…Vps16A null mutant clones were generated using the mosaic analysis with a repressible marker (MARCM) method, by crossing FRT82B Vps16A[d32] mutants with hs-Flp; QUAS-mCD8-GFP; ET49-QF, FRT82B tub-QS flies and heat shocking 0 to 4 h embryos in a 37 C water bath for 1 h, as before. 3,11 Molecular cloning and PCR Raptor coding sequences were PCR amplified from genomic DNA using primers ATT ACA AGT GCC GTT CGC GCT AAG T and ATC TTA AGA TTG TTC TCT ACC TGG GGT GGG and blunt cloned into XmnI-EcoRV digested, dephosphorylated pENTR1A (Invitrogen, 11813-011), followed by recombination into pTVW (DGRC, 1091). Transgenic flies were established by standard embryo transformation.…”

“…Starvation or growth factor withdrawal inhibits Tor, and as a result it blocks cell growth and activates Atg1, which then promotes autophagosome formation by phosphorylating other Atg proteins such as Atg13 in Drosophila. 2,3 It has been established that the activation of Tor takes place on lysosomes, and that Tor is recruited to these organelles through binding to the lysosome-anchored Ragulator complex. 4,5 Its activity also depends on sensing the presence of amino acids inside the lysosome, in association with the vacuolar H CATPase.…”