Origin and Function of the Autophagosome Membrane

  • Datum: 07.12.2015
  • Uhrzeit: 14:00 - 15:30
  • Vortragende(r): Randy Schekman
  • Ort: Central Building
  • Raum: Lecture Hall
  • Gastgeber: Ralph Bock
We studied the biogenesis of the autophagosome membrane by examining an early covalent event in autophagy, the lipidation of a cytosolic protein, LC3-I to form LC3-II. Using membranes from a cultured cell line deficient in lipidation, we demonstrated that the ERGIC was a major site for lipidation of LC3. Starvation induces the activation of a PI3 kinase to produce PI3P, which is required for the formation of LC3-II. We find that starved cells transfer a pool of COPII proteins from the ER to the ERGIC from which novel COPII vesicles bud that may be the immediate precursor of the phagophore membrane. Inflammation induces the abundant secretion of IL-1 β from macrophages and neutrophils by a process that appears to be independent of the normal secretory pathway. Co-expression of IL-1β and caspase 1 reconstituted the stress-induced secretion of mature IL-1β in HEK293 cells under conditions where cell lysis was minimal. Cells deficient in the lipidation of a cytosolic protein, LC-3, required for autophagy, accumulate IL-1β in the cytoplasm. Using cell fractionation experiments we found that IL-1β precursor and mature forms co-isolated with phagophore membranes marked by their content of lipidated LC-3. Using cells depleted of ATG2, a protein required for phagophore closure to produce a double-membrane mature authophagosome, we found that mature IL-1β, but not the precursor or control proteins were resistant to proteinase k, suggesting that IL-1β enters the lumen of the phagophore membrane rather than being engulfed into the cyoplasmic interior of the organelle. We have identified a targeting signal and a role for hsp90 in a translocation event that localizes IL-1β to the interior of the phagophore envelope. Fusion of the autophagosome with the plasma membrane would then result in the release of soluble IL-1β to the extracellular space.
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