The membrane-bound immunophilin Zonda is an autophagy regulator required at early stages of autophagy for VPS34 activation

MARIANA MELANI; AYELÉN VALKO; ELEONORA M. SORIANELLO; PABLO WAPPNER

Chia-Cerdeña

Conferencia; Autophagy signalling and progression in health and disease; 2015

ABSTRACT: The first distinguishable autophagy-specific membranous structure, the omegasome, forms when the VPS34 autophagy nucleation complex (VPS34, BECN1/ATG6, ATG14 and VPS15) mediates local deposition of phospatidylinositol-3-phospate (PI3P) in a specific domain of the ER. The molecules and mechanisms that mediate targeting of this complex and localized deposition of PI3P remain however poorly defined. We have identified a membrane-bound immunophilin, that we have named Zonda, which plays a critical role at early stages of autophagy. Zonda is required for autophagy as evaluated by several different markers that include: ATG8 nucleation, Lysotracker and Lamp1 accumulation and Ref2-P degradation. Moreover, Zonda overexpression can induce ATG8 nucleation. Zonda subcellular distribution responds to nutrients availability. Zonda displays a reticular distribution under nutrient-rich conditions, soon after nutrient deprivation Zonda nucleates in omega-shaped structures, where it colocalizes with PI3P, visualized by a FYVE-GFP probe, suggesting that Zonda is part of omegasomes. This is supported by observations in which ATG5 and ATG8 particles are contained within Zonda-labeled omegasomes. Later in autophagy Zonda becomes part of autophagosomes and autophagolysosomes, as shown by colocalization experiments with ATG8 and Lamp1. We have identified that Zonda nucleation fully depends on ATG1 activity but not on VPS34, suggesting that Zonda operates downstream of ATG1, and upstream of VPS34. In support of this, we found that Zonda is required for autophagy-specific activation of VPS34. We conclude that Zonda is a novel component of the autophagy cascade essential for VPS34-dependent PI3P deposition and possibly omegasome formation.