UC Berkeley

Autophagy is a ubiquitous process in the cell in which cytoplasmic cargo gets engulfed by the autophagosome, a double membrane structure, and delivered to the lysosome for degradation. Defects in the process are detrimental to cellular homeostasis and can lead to human diseases such as cancer and neurodegenerative diseases. A hallmark of autophagy is when a member of the ATG8 protein family is covalently attached to a membrane lipid, phosphatidylethanolamine (PE). LC3 lipidation closely resembles ubiquitin conjugation where an E3 like protein complex, ATG12–5-16L1, binds to an E2 like protein, ATG3, charged with an ATG8 protein. The whole protein machinery is localized to the growing autophagosome by WIPI2 binding to PI3P in the membrane and ATG16L1 simultaneously. While the process resembles a ubiquitin conjugation cascade, the structural details of LC3 lipidation occurring on a membrane have not been described. Here I determine structural details of the LC3 conjugation machinery using a variety of structural techniques. I determined the X-ray crystal structure of WIPI2d bound to the interacting region of ATG16L1. Hydrogen Deuterium Exchange Mass Spectrometry (HDX MS) along with cryo-electron microscopy (Cryo-EM) show the entire assembly on membranes is a dynamic protein complex. These dynamics limit structural determination of the full-length complex. However, our results divulge structural information of the LC3 lipidation machinery on a membrane.