Phosphorylation of Atg9 regulates movement to the phagophore assembly site and the rate of autophagosome formation

Yuchen Feng,Steven K Backues,Misuzu Baba,Jin-Mi Heo,J Wade Harper,Daniel J Klionsky

doi:10.1080/15548627.2016.1157237

Abstract

ABSTRACTMacroautophagy is primarily a degradative process that cells use to break down their own components to recycle macromolecules and provide energy under stress conditions, and defects in macroautophagy lead to a wide range of diseases. Atg9, conserved from yeast to mammals, is the only identified transmembrane protein in the yeast core macroautophagy machinery required for formation of the sequestering compartment termed the autophagosome. This protein undergoes dynamic movement between the phagophore assembly site (PAS), where the autophagosome precursor is nucleated, and peripheral sites that may provide donor membrane for expansion of the phagophore. Atg9 is a phosphoprotein that is regulated by the Atg1 kinase. We used stable isotope labeling by amino acids in cell culture (SILAC) to identify phosphorylation sites on this protein and identified an Atg1-independent phosphorylation site at serine 122. A nonphosphorylatable Atg9 mutant showed decreased autophagy activity, whereas the phosphomimetic mutant enhanced activity. Electron microscopy analysis suggests that the different levels of autophagy activity reflect differences in autophagosome formation, correlating with the delivery of Atg9 to the PAS. Finally, this phosphorylation regulates Atg9 interaction with Atg23 and Atg27.

Highlights

Autophagy refers to a group of highly conserved cellular processes in which cytoplasmic components are degraded within the lysosome in most of the more complex eukaryotes or the vacuole in yeast and plants
We carried out a SILAC analysis to identify phosphorylation sites on Atg[9] that might affect its movement to the phagophore assembly site (PAS), and decided to focus on modifications that were independent of direct Atg[1] kinase function, because recent studies have already identified Atg1-dependent sites in this protein.[14]
In addition to nonselective autophagy, our data showed that the phosphorylation of serine 122 (S122) is important for selective autophagy

Summary

Introduction

Autophagy refers to a group of highly conserved cellular processes in which cytoplasmic components are degraded within the lysosome in most of the more complex eukaryotes or the vacuole in yeast and plants. Macroautophagy (hereafter called autophagy) is the major process in which random cytoplasm is sequestered within a double-membrane structure, the phagophore, which eventually matures into a vesicle termed the autophagosome; generation of the autophagosome is a distinguishing feature between macroautophagy and microautophagy because the latter process involves direct uptake at the vacuole limiting membrane without the use of a phagophore or autophagosome. After fusion of the autophagosome with the vacuole, and degradation in the vacuole lumen, the breakdown products are released back into the cytosol.[2] Autophagy can be selective or nonselective. Nonselective autophagy is used for the turnover of bulk cytoplasm, whereas selective autophagy targets damaged or superfluous organelles, including mitochondria and peroxisomes.[3,4]

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Conclusion