Abstract
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.
Highlights
Macroautophagy is an evolutionarily conserved lysosomal pathway involved in the turn
The data presented above provide a framework of autophagosome-associated proteins and will help in characterizing underlying cellular processes regulating the biogenesis and cargo selection of autophagosomes
Employing protein correlation profiling (PCP)-SILAC, we grouped the identified proteins in three clusters per organelle preparation for cells treated with concanamycin A, rapamycin, and HBSS
Summary
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved lysosomal pathway involved in the turn-. Identification of Autophagosome-associated Proteins and Regulators form (LC3-I) to a phosphatidylethanolamine-conjugated form (LC3-II), which associates with both membranes of the autophagosome [13] This process is frequently used as an autophagy marker because the change in the LC3 staining pattern from diffuse to dotted can be readily visualized. Even though the ubiquitin-proteasome system and autophagy have long been viewed as complementary degradation systems with no point of intersection, it was shown recently that autophagy can act compensatorily when the ubiquitin-proteasome system is impaired in Drosophila melanogaster [21] These data suggest that there might be a link between the two major cellular proteolysis pathways [22,23,24]
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