title: Proteomic profiling of autophagosome cargo in Saccharomyces cerevisiae.
abstract: Macroautophagy (autophagy) is a bulk protein-degradation system ubiquitously conserved in eukaryotic cells. During autophagy, cytoplasmic components are enclosed in a membrane compartment, called an autophagosome. The autophagosome fuses with the vacuole/lysosome and is degraded together with its cargo. Because autophagy is important for the maintenance of cellular homeostasis by degrading unwanted proteins and organelles, identification of autophagosome cargo proteins (i.e., the targets of autophagy) will aid in understanding the physiological roles of autophagy. In this study, we developed a method for monitoring intact autophagosomes ex vivo by detecting the fluorescence of GFP-fused aminopeptidase I, the best-characterized selective cargo of autophagosomes in Saccharomyces cerevisiae. This method facilitated optimization of a biochemical procedure to fractionate autophagosomes. A combination of LC-MS/MS with subsequent statistical analyses revealed a list of autophagosome cargo proteins; some of these are selectively enclosed in autophagosomes and delivered to the vacuole in an Atg11-independent manner. The methods we describe will be useful for analyzing the mechanisms and physiological significance of Atg11-independent selective autophagy.
authors.affiliation: Bioimaging Center, Graduate School of Frontier Sciences, University of Tokyo, Kashiwa, Chiba, Japan.
authors.forename: Kuninori
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Shingo
authors.initials: S
authors.lastname: Nakamura
authors.affiliation: Frontier Research Center, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Morimoto
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Kiyonaga
authors.initials: K
authors.lastname: Fujii
authors.affiliation: Laboratory of Molecular Structure, Institute of Microbial Chemistry (BIKAKEN), Tokyo, Japan.
authors.forename: Nobuo N
authors.initials: NN
authors.lastname: Noda
authors.affiliation: Department of Structural Biology, Faculty of Advanced Life Science, Hokkaido University, Sapporo, Hokkaido, Japan.
authors.forename: Fuyuhiko
authors.initials: F
authors.lastname: Inagaki
authors.affiliation: Frontier Research Center, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan.
authors.forename: Yoshinori
authors.initials: Y
authors.lastname: Ohsumi
mesh_terms: D001343:Autophagy; D000071183:Autophagy-Related Proteins; D002853:Chromatography, Liquid; D003593:Cytoplasm / Q000378:metabolism; D019286:Endopeptidase K / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006706:Homeostasis; D015151:Immunoblotting; D008856:Microscopy, Fluorescence; D010588:Phagosomes / Q000201:enzymology*; D025341:Principal Component Analysis; D020543:Proteome / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D033921:Vesicular Transport Proteins / Q000378:metabolism
chemical_list: C502923:Atg11 protein, S cerevisiae; D000071183:Autophagy-Related Proteins; D020543:Proteome; D029701:Saccharomyces cerevisiae Proteins; D033921:Vesicular Transport Proteins; D049452:Green Fluorescent Proteins; D019286:Endopeptidase K