Analysis of the structural role of yeast aminopeptidase 1 in autophagic vesicle formation
Date Issued
2015
Date
2015
Author(s)
Su, Ming-Yuan
Abstract
In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, encapsulates precursor aminopeptidase I (prApe1) dodecamers in the form of a giant complex into a Cvt vesicle using the autophagic machinery, sorting it into the vacuole (the yeast lysosome) where it is proteolytically processed into its mature form, Ape1, by removal of an amino-terminal 45-amino acid propeptide. prApe1 is thought to serve as a scaffolding cargo vital for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex construction and autophagic receptor recognition. This dissertation presents the molecular architecture of Ape1 at 2.5 Å resolution and reveals its dodecameric architecture consisting of dimeric and trimeric units, which associate to form a large tetrahedron. The propeptide of prApe1 exhibits concentration-dependent oligomerization and forms a stable tetramer. Structure-based mutagenesis reveals that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar delivery in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, the 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is found to be essential for the assembly of the Cvt vesicle for vacuolar delivery. This dissertation provides the first mechanistic insight for understanding the autophagosomal biogenesis in selective macroautophagy.
Subjects
Ape1
autophagy
cytoplasm-to-vacuole targeting pathway
dodecamer
X-ray
Type
thesis
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