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Free keywords:
autophagy cargo
autophagy receptor
correlative light and electron
microscopy
electron cryomicroscopy
fluorescence light microscopy
selective autophagy
X-ray crystallography
yeast aminopeptidase-i
selective autophagy
saccharomyces-cerevisiae
correlated fluorescence
structure prediction
quaternary structure
alpha-mannosidase
atg proteins
vacuole
cytoplasm
Biochemistry & Molecular Biology
Cell Biology
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 angstrom X-ray and negative stain EM structure, as well as the secondary cargo Ams1 with a 6.3 angstrom 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.
