TY - JOUR

T1 - Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens

AU - Dengjel, Jörn

AU - Høyer-Hansen, Maria

AU - Nielsen, Maria O

AU - Eisenberg, Tobias

AU - Harder, Lea Mørch

AU - Schandorff, Søren

AU - Farkas, Thomas

AU - Kirkegaard, Thomas

AU - Becker, Andrea C

AU - Schroeder, Sabrina

AU - Vanselow, Katja

AU - Lundberg, Emma

AU - Nielsen, Mogens Møller

AU - Kristensen, Anders Riis

AU - Akimov, Vyacheslav

AU - Bunkenborg, Jakob

AU - Madeo, Frank

AU - Jaattela, Marja

AU - Andersen, Jens S

PY - 2012

Y1 - 2012

N2 - Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

AB - Autophagy is one of the major intracellular catabolic pathways, but little is known about the composition of autophagosomes. To study the associated proteins, we isolated autophagosomes from human breast cancer cells using two different biochemical methods and three stimulus types: amino acid deprivation or rapamycin or concanamycin A treatment. The autophagosome-associated proteins were dependent on stimulus, but a core set of proteins was stimulus-independent. Remarkably, proteasomal proteins were abundant among the stimulus-independent common autophagosome-associated proteins, and the activation of autophagy significantly decreased the cellular proteasome level and activity supporting interplay between the two degradation pathways. A screen of yeast strains defective in the orthologs of the human genes encoding for a common set of autophagosome-associated proteins revealed several regulators of autophagy, including subunits of the retromer complex. The combined spatiotemporal proteomic and genetic data sets presented here provide a basis for further characterization of autophagosome biogenesis and cargo selection.

KW - Amino Acids

KW - Antibodies, Monoclonal

KW - Antiviral Agents

KW - Autophagy

KW - Breast Neoplasms

KW - Electrophoresis, Polyacrylamide Gel

KW - Female

KW - Genetic Testing

KW - Green Fluorescent Proteins

KW - Humans

KW - Immunoprecipitation

KW - Immunosuppressive Agents

KW - Isotope Labeling

KW - Lysosomes

KW - Macrolides

KW - Phagosomes

KW - Proteins

KW - Proteomics

KW - Saccharomyces cerevisiae

KW - Sirolimus

KW - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

KW - Starvation

KW - Tumor Cells, Cultured

U2 - 10.1074/mcp.M111.014035

DO - 10.1074/mcp.M111.014035

M3 - Journal article

C2 - 22311637

VL - 11

SP - M111.014035

JO - Molecular and Cellular Proteomics

JF - Molecular and Cellular Proteomics

SN - 1535-9476

IS - 3

ER -