Calpain mobilizes Atg9/Bif-1 vesicles from Golgi stacks upon autophagy induction by thapsigargin

Abstract

CAPNS1 is essential for stability and function of the ubiquitous calcium dependent proteases micro- and milli-calpain. Upon the inhibition of the endoplasmic reticulum Ca2+ ATPase by 100nM thapsigargin, both micro-calpain and autophagy are activated in human U2OS osteosarcoma cells in a CAPNS1 dependent manner. As reported for other autophagy triggers, thapsigargin treatment induces Golgi fragmentation and fusion of Atg9/Bif-1 containing vesicles with LC3 bodies in control cells. On the opposite, CAPNS1 depletion is coupled to an accumulation of LC3 bodies and Rab5 early endosomes. Moreover, Atg9 and Bif-1 stay in the GM130-positive Golgi stacks and Atg9 fails to interact with the endocytic route marker transferrin receptor and to the core autophagic protein Vps34 in CAPNS1 depleted cells. Ectopic expression of a Bif-1 point mutant resistant to calpain processing is coupled to endogenous p62 and LC3-II accumulation. Altogether these data indicate that calpain allows Atg9/Bif-1 vesicles dynamic flux from the Golgi toward the budding autophagosome.