title: Structure of yeast Ape1 and its role in autophagic vesicle formation.
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.
authors.affiliation: a Institute of Biological Chemistry; Academia Sinica ; Taipei , Taiwan.|b Institute of Biochemical Sciences; College of Life Science; National Taiwan University ; Taipei , Taiwan.
authors.forename: Ming-Yuan
authors.initials: MY
authors.lastname: Su
authors.affiliation: a Institute of Biological Chemistry; Academia Sinica ; Taipei , Taiwan.
authors.forename: Wen-Hsin
authors.initials: WH
authors.lastname: Peng
authors.affiliation: a Institute of Biological Chemistry; Academia Sinica ; Taipei , Taiwan.
authors.forename: Meng-Ru
authors.initials: MR
authors.lastname: Ho
authors.affiliation: a Institute of Biological Chemistry; Academia Sinica ; Taipei , Taiwan.|b Institute of Biochemical Sciences; College of Life Science; National Taiwan University ; Taipei , Taiwan.
authors.forename: Shih-Chieh
authors.initials: SC
authors.lastname: Su
authors.affiliation: c Institute of Cellular and Organismic Biology; Academia Sinica ; Taipei , Taiwan.
authors.forename: Yuan-Chih
authors.initials: YC
authors.lastname: Chang
authors.affiliation: a Institute of Biological Chemistry; Academia Sinica ; Taipei , Taiwan.|b Institute of Biochemical Sciences; College of Life Science; National Taiwan University ; Taipei , Taiwan.
authors.forename: Guang-Chao
authors.initials: GC
authors.lastname: Chen
authors.affiliation: a Institute of Biological Chemistry; Academia Sinica ; Taipei , Taiwan.|b Institute of Biochemical Sciences; College of Life Science; National Taiwan University ; Taipei , Taiwan.
authors.forename: Chung-I
authors.initials: CI
authors.lastname: Chang
mesh_terms: D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism*; D001343:Autophagy; D018360:Crystallography, X-Ray; D022162:Cytoplasmic Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D008958:Models, Molecular; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D055503:Protein Multimerization; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D014617:Vacuoles / Q000378:metabolism
chemical_list: D010455:Peptides; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D000626:Aminopeptidases; C019689:APE1 protein, S cerevisiae
keywords: Ape1; Atg19; Cvt; X-ray crystallography; autophagy