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RACK1 Is an Interaction Partner of ATG5 and a Novel Regulator of Autophagy.

The Journal of biological chemistry (2016-06-22)
Secil Erbil, Ozlem Oral, Geraldine Mitou, Cenk Kig, Emel Durmaz-Timucin, Emine Guven-Maiorov, Ferah Gulacti, Gokcen Gokce, Jörn Dengjel, Osman Ugur Sezerman, Devrim Gozuacik
ABSTRACT

Autophagy is biological mechanism allowing recycling of long-lived proteins, abnormal protein aggregates, and damaged organelles under cellular stress conditions. Following sequestration in double- or multimembrane autophagic vesicles, the cargo is delivered to lysosomes for degradation. ATG5 is a key component of an E3-like ATG12-ATG5-ATG16 protein complex that catalyzes conjugation of the MAP1LC3 protein to lipids, thus controlling autophagic vesicle formation and expansion. Accumulating data indicate that ATG5 is a convergence point for autophagy regulation. Here, we describe the scaffold protein RACK1 (receptor activated C-kinase 1, GNB2L1) as a novel ATG5 interactor and an autophagy protein. Using several independent techniques, we showed that RACK1 interacted with ATG5. Importantly, classical autophagy inducers (starvation or mammalian target of rapamycin blockage) stimulated RACK1-ATG5 interaction. Knockdown of RACK1 or prevention of its binding to ATG5 using mutagenesis blocked autophagy activation. Therefore, the scaffold protein RACK1 is a new ATG5-interacting protein and an important and novel component of the autophagy pathways.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
L-Lysine monohydrochloride, from non-animal source, meets EP, JP, USP testing specifications, suitable for cell culture, 98.5-101.0%
Sigma-Aldrich
Anti-ATG5 (N-terminal) antibody produced in rabbit, affinity isolated antibody, PBS solution
Sigma-Aldrich
Anti-LC3B antibody produced in rabbit, ~1 mg/mL, affinity isolated antibody, buffered aqueous solution
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Monoclonal Anti-β-Actin antibody produced in mouse, clone AC-15, ascites fluid
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Cycloheximide, ≥95% (HPLC)
Sigma-Aldrich
Saponin, used as non-ionic surfactant