Characterization of Protein-Membrane Interactions in Yeast Autophagy

Abstract

Cells rely on autophagy to degrade cytosolic material and maintain homeostasis. During autophagy, content to be degraded is encapsulated in double membrane vesicles, termed autophagosomes, which fuse with the yeast vacuole for degradation. This conserved cellular process requires the dynamic rearrangement of membranes. As such, the process of autophagy requires many soluble proteins that bind to membranes to restructure, tether, or facilitate lipid transfer between membranes. Here, we review the methods that have been used to investigate membrane binding by the core autophagy machinery and additional accessory proteins involved in autophagy in yeast. We also review the key experiments demonstrating how each autophagy protein was shown to interact with membranes.

Keywords: autophagy; membrane binding proteins; yeast.

Figure 1

Types of synthetic bilayers of various sizes and curvature that are used in in vitro experiments. The type of synthetic bilayers varies depending on the method. For example, SUVs and LUVs are often used in liposome sedimentation or floatation assays, GUVs are often used in fluorescence microscopy, and SLBs are used in SPR. In all cases, the composition of the synthetic bilayer can be generated using lipid mixtures, such as Folch or YPL, or with synthetic lipids. Created with BioRender.com (accessed on 8 June 2022).

Figure 2

Summary of membrane interactions in yeast autophagy. Atg1-initiation complex: Atg17 (and Atg11 in selective autophagy) tethers Atg9-vesicles, which is inhibited by Atg29-Atg31 binding. Vacuolar protein Vac8 binds to the initiation complex to tether the PAS to the vacuole. The C-terminal MIT domain of Atg1 and the disordered region of Atg13 bind to SUVs. Atg2-Atg18 complex: Atg18 binds to PI3P, while Atg2 transfers lipids between the ER and the isolation membrane. PI3K complex: The Y-shaped complex binds membrane via the Vps34 activation loop and the Vps30/Atg6 aromatic finger in the BARA domain to phosphorylate PI to PI3P. Atg8-conjugation system: Atg3 and the Atg12~Atg5-Atg16 complex all bind to membrane in vitro and are involved in Atg8-lipidation. The C terminus of Atg8 is covalently attached to PE in the isolation membrane. Additional autophagy factors Atg20 and Atg24 form a heterodimer that binds to PI3P and the isolation membrane in vitro. Atg23 can bind to and tether membrane to organize transmembrane Atg9-containing vesicles. The following PBD IDs are used in this figure Atg9 (7JLP), Atg1/13 2014 (4P1N), Atg17 (4HPQ), PI3K (5DFZ), Atg18 (6KYB), Atg8 (2ZPN), Atg3 (2DYT), Atg12~Atg5-Atg16 (3W1S), and Vac8 (6KBM). Created with BioRender.com.