Higher‐order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle

Abstract

Selective autophagy is the mechanism by which large cargos are specifically sequestered for degradation. The structural details of cargo and receptor assembly giving rise to autophagic vesicles remain to be elucidated. We utilize the yeast cytoplasm‐to‐vacuole targeting (Cvt) pathway, a prototype of selective autophagy, together with a multi‐scale analysis approach to study the molecular structure of Cvt vesicles. We report the oligomeric nature of the major Cvt cargo Ape1 with a combined 2.8 Å X‐ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 Å cryo‐ EM structure. We show that the major dodecameric cargo prApe1 exhibits a tendency to form higher‐order chain structures that are broken upon interaction with the receptor Atg19 in vitro . The stoichiometry of these cargo–receptor complexes is key to maintaining the size of the Cvt aggregate in vivo . Using correlative light and electron microscopy, we further visualize key stages of Cvt vesicle biogenesis. Our findings suggest that Atg19 interaction limits Ape1 aggregate size while serving as a vehicle for vacuolar delivery of tetrameric Ams1. image This study provides a multi‐scale visualization of the cytoplasm‐to‐vacuole targeting (Cvt) pathway from cargo assembly to intact vesicle formation. It reveals the oligomeric organization of Cvt components and shows that higher‐order cargo receptor assemblies provide the membrane scaffold for selective autophagy. The EM visualization of the major Cvt cargo Ape1 reveals higher‐order chain‐like assemblies. The cryo‐ EM structure of the secondary Cvt cargo Ams1 shows that the N‐terminal four‐helix bundle domain is critical for the tetrameric assembly of Ams1. The interaction of the trimeric autophagy receptor Atg19 with Ape1 is key to maintaining the size of Cvt assemblies.