Abstract
In Saccharomyces cerevisiae, a constitutive biosynthetic transport pathway, termed the cytoplasm-to-vacuole targeting (Cvt) pathway, sequesters precursor aminopeptidase I (prApe1) dodecamers in the form of a large complex into a Cvt vesicle using autophagic machinery, targeting 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 critical for the assembly of the Cvt vesicle by presenting the propeptide to mediate higher-ordered complex formation and autophagic receptor recognition. Here we report the X-ray crystal structure of Ape1 at 2.5 Å resolution and reveal 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 demonstrates that disruption of the inter-subunit interface prevents dodecameric assembly and vacuolar targeting in vivo despite the presence of the propeptide. Furthermore, by examining the vacuolar import of propeptide-fused exogenous protein assemblies with different quaternary structures, we found that 3-dimensional spatial distribution of propeptides presented by a scaffolding cargo is essential for the assembly of the Cvt vesicle for vacuolar delivery. This study describes a molecular framework for understanding the mechanism of Cvt or autophagosomal biogenesis in selective macroautophagy.
Highlights
Selective macroautophagy plays a critical role in the clearance of defective aggregates and organelles that cause neurodegeneration or other diseases.[1,2] Many kinds of targeted cargos of selective autophagy have been described; they include mitochondria, peroxisomes, ribosomes, the endoplasmic reticulum, aggregated proteins, and pathogens.[3,4,5,6] The autophagic receptors recruit the autophagic machinery, which forms double-membrane vesicles known generally as autophagosomes, for cargo engulfment and subsequent delivery to the lysosome/vacuole
Our results suggest that the spatial distribution of the propeptides presented on a 3-dimensional scaffolding cargo is essential for proper assembly of the cytoplasm-to-vacuole targeting (Cvt) vesicle to ensure vacuolar delivery
Overall structure of Ape[1] For the structural analysis of Ape[1], we have expressed in bacterial cells recombinant full-length prApe[1] and a truncated version corresponding to the mature form without the N-terminal propeptide region, Ape[1]
Summary
Selective macroautophagy (hereafter autophagy) plays a critical role in the clearance of defective aggregates and organelles that cause neurodegeneration or other diseases.[1,2] Many kinds of targeted cargos of selective autophagy have been described; they include mitochondria, peroxisomes, ribosomes, the endoplasmic reticulum, aggregated proteins, and pathogens.[3,4,5,6] The autophagic receptors recruit the autophagic machinery, which forms double-membrane vesicles known generally as autophagosomes (additional terms are sometimes used depending on the specific cargo, such as mitophagosomes, etc.), for cargo engulfment and subsequent delivery to the lysosome/vacuole. The autophagic machinery is employed in import of selected cargos to the lysosome/vacuole for degradation, but is involved in important cellular pathways such as the secretion of cytokines, endosomal transport, as well as lysosomal trafficking and activation of hydrolytic enzymes by the cytoplasm-to-vacuole targeting (Cvt) pathway.[7,8,9] it is essential to understand the structural mechanism of cargo recognition by the autophagic receptors and their role in autophagosome formation, which may provide insight into curing diseases by targeting the autophagic machinery. Together with Atg[11], the Atg19-bound Ape[1] complex ( referred to as a Cvt complex) facilitates the formation of Cvt
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