title: The PI 3-kinase regulator Vps15 is required for autophagic clearance of protein aggregates.
abstract: Autophagy is involved in cellular clearance of aggregate-prone proteins, thereby having a cytoprotective function. Studies in yeast have shown that the PI 3-kinase Vps34 and its regulatory protein kinase Vps15 are important for autophagy, but the possible involvement of these proteins in autophagy in a multicellular animal has not been addressed genetically. Here, we have created a Drosophila deletion mutant of vps15 and studied its role in autophagy and aggregate clearance. Homozygous Deltavps15 Drosophila died at the early L3 larval stage. Using GFP-Atg8a as an autophagic marker, we employed fluorescence microscopy to demonstrate that fat bodies of wild type Drosophila larvae accumulated autophagic structures upon starvation whereas vps15 fat bodies showed no such response. Likewise, electron microscopy revealed starvation-induced autophagy in gut cells from wild type but not Deltavps15 larvae. Fluorescence microscopy showed that Deltavps15 mutant tissues accumulated profiles that were positive for ubiquitin and Ref(2)P, the Drosophila homolog of the sequestosome marker SQSTM1/p62. Biochemical fractionation and Western blotting showed that these structures were partially detergent insoluble, and immuno-electron microscopy further demonstrated the presence of Ref(2)P positive membrane free protein aggregates. These results provide the first genetic evidence for a function of Vps15 in autophagy in multicellular organisms and suggest that the Vps15-containing PI 3-kinase complex may play an important role in clearance of protein aggregates.
authors.affiliation: Centre for Cancer Biomedicine, University of Oslo, Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo, Norway.
authors.forename: Karine
authors.initials: K
authors.lastname: Lindmo
authors.forename: Andreas
authors.initials: A
authors.lastname: Brech
authors.forename: Kim D
authors.initials: KD
authors.lastname: Finley
authors.forename: Sébastien
authors.initials: S
authors.lastname: Gaumer
authors.forename: Didier
authors.initials: D
authors.lastname: Contamine
authors.forename: Tor Erik
authors.initials: TE
authors.lastname: Rusten
authors.forename: Harald
authors.initials: H
authors.lastname: Stenmark
mesh_terms: D000818:Animals; D001343:Autophagy / Q000502:physiology*; D004268:DNA-Binding Proteins; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000033:anatomy & histology / Q000196:embryology; D056827:Endosomal Sorting Complexes Required for Transport; D005216:Fat Body / Q000166:cytology / Q000378:metabolism; D017353:Gene Deletion; D007814:Larva / Q000378:metabolism / Q000648:ultrastructure; D009154:Mutation; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013217:Starvation; D025801:Ubiquitin / Q000378:metabolism; D058571:Vacuolar Sorting Protein VPS15
chemical_list: D004268:DNA-Binding Proteins; D029721:Drosophila Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D009687:Nuclear Proteins; D019869:Phosphatidylinositol 3-Kinases; D017346:Protein Serine-Threonine Kinases; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D058571:Vacuolar Sorting Protein VPS15; C527373:Atg8a protein, Drosophila; C080596:ref(2)P protein, Drosophila