A new GUV-based assay to reconstitute membrane tetheringin vitrodemonstrates the vesicle tethering ability of the selective autophagy scaffold Atg11

Abstract

Membrane tethering is essential for the generation of organelle contact sites and to anchor incoming vesicles to their target membranes before vesicle fusion. During autophagy in yeast, tethering of 30 nm vesicles to cargo is one of the first steps in the generation of the isolation membrane that engulfs the cargo to be degraded. While membrane tethering is critical for cellular function, many of the current biochemical techniques to assay for membrane tethering rely on indirect readouts and are limited in their ability to monitor protein localization at sites of tethering. As such, we developed a fluorescence-microscopy-basedGUV liposome tethering assay (GLT) to directly visualize membrane tethering and monitor protein localization at tethering sites simultaneously. We initially used GLT with engineered membrane tethers to demonstrate the ease of use, versatility and sensitivity of the assay. We also demonstrated that the selective autophagy scaffolding protein Atg11 can bind, and tether negatively charged membranes but is unable to bind GUVs mimicking the charge of the outer mitochondrial membrane. Atg11 instead requires the selective autophagy receptor Atg32 to be recruited to mitochondrial membranes. Lastly, we demonstrate that Atg11 bound to Atg32 on GUVs can tether negatively charged vesicles to GUVs. Collectively, our results reconstitute one of the first steps during the initiation of mitochondrial autophagy and highlight the versatility of GLT to study a range of membrane tethering events biochemically.

Conserved C-Terminal Tail Is Responsible for Membrane Localization and Function of Pseudomonas aeruginosa Hemerythrin

“…Images were analyzed using Fiji, and statistical analysis was performed using GraphPad Prism (version 5.0a). Protein densities on GUVs were performed as previously described . Protein densities were normalized to the highest value and plotted as percentage normalized densities.…”

“…Protein densities on GUVs were performed as previously described. 45 Protein densities were normalized to the highest value and plotted as percentage normalized densities. Data from three independent experiments were pooled, and statistical analysis was performed using Mann−Whitney's test.…”