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Reconstituting Autophagosome Biogenesis in vitro

Objective

Autophagy is a catabolic pathway that delivers cytoplasmic material to lysosomes for degradation. Under vegetative conditions, the pathway serves as quality control system, specifically targeting damaged or superfluous organelles and protein-aggregates. Cytotoxic stresses and starvation, however, induces the formation of larger autophagosomes that capture cargo unselectively. Autophagosomes are being generated from a cup-shaped precursor membrane, the isolation membrane, which expands to engulf cytoplasmic components. Sealing of this structure gives rise to the double-membrane surrounded autophagosomes. Two interconnected ubiquitin (Ub)-like conjugation systems coordinate the expansion of autophagosomes by conjugating the autophagy related (Atg)-protein Atg8 to the isolation membrane. In an effort to unravel the function of Atg8, we reconstituted the system on model membranes in vitro and found that Atg8 forms together with the Atg12–Atg5-Atg16 complex a membrane scaffold which is required for productive autophagy in yeast. Humans possess seven Atg8-homologs and two mutually exclusive Atg16-variants. Here, we propose to investigate the function of the human Ub-like conjugation system using a fully reconstituted in vitro system. The spatiotemporal organization of recombinant fluorescent-labeled proteins with synthetic model membranes will be investigated using confocal and TIRF-microscopy. Structural information will be obtained by atomic force and electron microscopy. Mechanistic insights, obtained from the in vitro work, will be tested in vivo in cultured human cells. We belief that revealing 1) the function of the human Ub-like conjugation system in autophagy, 2) the functional differences of Atg8-homologs and the two Atg16-variants Atg16L1 and TECPR1 and 3) how Atg16L1 coordinates non-canonical autophagy will provide essential insights into the pathophysiology of cancer, neurodegenerative, and autoimmune diseases.

Field of science

  • /medical and health sciences/basic medicine/physiology/pathophysiology
  • /natural sciences/biological sciences/biochemistry/biomolecules/proteins
  • /medical and health sciences/clinical medicine/oncology/cancer

Call for proposal

ERC-2014-STG
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Funding Scheme

ERC-STG - Starting Grant

Host institution

INSTITUT PASTEUR
Address
Rue Du Docteur Roux 25-28
75724 Paris Cedex 15
France
Activity type
Research Organisations
EU contribution
€ 1 185 057,50

Beneficiaries (2)

INSTITUT PASTEUR
France
EU contribution
€ 1 185 057,50
Address
Rue Du Docteur Roux 25-28
75724 Paris Cedex 15
Activity type
Research Organisations
MAX-PLANCK-GESELLSCHAFT ZUR FORDERUNG DER WISSENSCHAFTEN EV

Participation ended

Germany
EU contribution
€ 314 668,50
Address
Hofgartenstrasse 8
80539 Muenchen
Activity type
Research Organisations