MilliporeSigma
  • Home
  • Search Results
  • Atg17/FIP200 localizes to perilysosomal Ref(2)P aggregates and promotes autophagy by activation of Atg1 in Drosophila.

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

Autophagy (2014-01-15)
Péter Nagy, Manuéla Kárpáti, Agnes Varga, Karolina Pircs, Zsolt Venkei, Szabolcs Takáts, Kata Varga, Balázs Erdi, Krisztina Hegedűs, Gábor Juhász
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Anti-HA antibody produced in rabbit, affinity isolated antibody, buffered aqueous solution
Sigma-Aldrich
Anti-Rat IgG (whole molecule)–Alkaline Phosphatase antibody produced in rabbit, affinity isolated antibody, buffered aqueous glycerol solution
Millipore
Monoclonal Anti-HA−Agarose antibody produced in mouse, clone HA-7, purified immunoglobulin, PBS suspension
Sigma-Aldrich
Sample Buffer, Laemmli 2× Concentrate
Sigma-Aldrich
Goat Anti-Rabbit IgG Antibody, Alkaline Phosphatase conjugate, 0.6 mg/mL, Chemicon®
Sigma-Aldrich
Goat Anti-Mouse IgG Antibody, Alkaline Phosphatase conjugate, Chemicon®, from goat
Sigma-Aldrich
Anti-c-Myc antibody, Mouse monoclonal antibody produced in mouse, clone 9E10, purified from hybridoma cell culture
Sigma-Aldrich
Anti-ATG5 (N-terminal) antibody produced in rabbit, affinity isolated antibody, PBS solution
Sigma-Aldrich
Freund′s Adjuvant, Complete, cell suspension
Sigma-Aldrich
Freund′s Adjuvant, Incomplete, liquid
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)