Identification of autophagosome-associated proteins and regulators by quantitative proteomic analysis and genetic screens.
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.
Department of Biochemistry and Molecular Biology, University of Southern Denmark, 5230 Odense M, Denmark. joern.dengjel@frias.uni-freiburg.de
Jörn
J
Dengjel
Maria
M
Høyer-Hansen
Maria O
MO
Nielsen
Tobias
T
Eisenberg
Lea M
LM
Harder
Søren
S
Schandorff
Thomas
T
Farkas
Thomas
T
Kirkegaard
Andrea C
AC
Becker
Sabrina
S
Schroeder
Katja
K
Vanselow
Emma
E
Lundberg
Mogens M
MM
Nielsen
Anders R
AR
Kristensen
Vyacheslav
V
Akimov
Jakob
J
Bunkenborg
Frank
F
Madeo
Marja
M
Jäättelä
Jens S
JS
Andersen
D000596:Amino Acids / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000378:metabolism; D000998:Antiviral Agents / Q000494:pharmacology; D001343:Autophagy; D001943:Breast Neoplasms / Q000235:genetics* / Q000378:metabolism* / Q000473:pathology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005820:Genetic Testing; D049452:Green Fluorescent Proteins / Q000276:immunology / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007553:Isotope Labeling; D008247:Lysosomes / Q000378:metabolism; D018942:Macrolides / Q000494:pharmacology; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D040901:Proteomics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013217:Starvation; D014407:Tumor Cells, Cultured
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D000998:Antiviral Agents; D007166:Immunosuppressive Agents; D018942:Macrolides; D011506:Proteins; D049452:Green Fluorescent Proteins; C036978:concanamycin A; D020123:Sirolimus