Higher-order assemblies of oligomeric cargo receptor complexes form the membrane scaffold of the Cvt vesicle.
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.
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Chiara
C
Bertipaglia
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Sarah
S
Schneider
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Hamburg Unit, European Molecular Biology Laboratory, Hamburg, Germany.
Arjen J
AJ
Jakobi
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Abul K
AK
Tarafder
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Yury S
YS
Bykov
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Andrea
A
Picco
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Wanda
W
Kukulski
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Jan
J
Kosinski
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Wim Jh
WJ
Hagen
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Arvind C
AC
Ravichandran
Hamburg Unit, European Molecular Biology Laboratory, Hamburg, Germany.
Matthias
0000-0003-2486-5239
M
Wilmanns
Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
Marko
M
Kaksonen
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Heidelberg, Germany.
John Ag
JA
Briggs
Structural and Computational Biology Unit, European Molecular Biology Laboratory, Heidelberg, Germany carsten.sachse@embl.de.
Carsten
0000-0002-1168-5143
C
Sachse
D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism; D001343:Autophagy; D000071183:Autophagy-Related Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001692:Biological Transport; D003593:Cytoplasm / Q000378:metabolism; D008566:Membranes / Q000378:metabolism; D008954:Models, Biological; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C434480:ATG19 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D011956:Receptors, Cell Surface; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae
X‐ray crystallography; autophagy cargo; autophagy receptor; correlative light and electron microscopy; electron cryomicroscopy; fluorescence light microscopy; selective autophagy