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.

p62 filaments capture and present ubiquitinated cargos for autophagy

“…During selective autophagy, the cargos themselves drive the local assembly of autophagosomes and keep the membrane close to the cargo (Sawa‐Makarska et al , ; Lazarou et al , ; Bertipaglia et al , ; Fracchiolla et al , ; Zaffagnini & Martens, ). Here, we show that within the p62 clusters in vitro, the p62 filaments are largely immobile.…”

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

“…Like immuno-EM, room-temperature CLEM also requires chemically fixed and dehydrated cells, which can distort or obscure important structural features (Lucic et al, 2013, Afzelius and Maunsbach, 2004). Nevertheless, room-temperature CLEM has been instrumental in the visualization of numerous bacterial and mammalian cellular events that would otherwise have been challenging or impossible to capture prior to the advent of this method (Grabenbauer et al, 2005, Muller-Reichert et al, 2007, Bertipaglia et al, 2016, Darcy et al, 2006, Schorb et al, 2016, Avinoam et al, 2015, Kukulski et al, 2011, Kukulski et al, 2012, Redemann and Muller-Reichert, 2013, Kapoor et al, 2006). …”

“…To visualize fluorescence inside frozen-hydrated cells, cryogenic LM (cryo-LM) stages are used (Schlimpert et al, 2012, Bertipaglia et al, 2016, Schorb and Briggs, 2014, Briegel et al, 2010, Schellenberger et al, 2014, Schorb et al, 2016). Unfortunately, because the sample has to be kept frozen, long-working-distance air-objective lenses with low numerical apertures are used instead of oil-immersion lenses.…”

Next-generation electron microscopy in autophagy research

“…2E) (22). The x-ray structure of the mature particle, also a dodecamer, was determined at 2.5 and 2.8 Å (22, 23), and the negative stain EM structure at 24 Å (23). The structures of the precursor and mature particle are sufficiently similar that the crystallographic dodecamer could be docked into the EM reconstruction of prApe1.…”

“…The structure shows that the geometry of propeptide presentation on the dodecamer surface is important for selective autophagic uptake (22). The cryo-EM structure of another autophagic cargo, α-mannosidase I (Ams1) has been determined at 6.3 Å resolution (23). Ams1 is a tetramer and is considered a secondary cargo.…”