Abstract
Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation. Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1–kinase complex. However, the underlying molecular mechanism remains unclear. Here we reconstitute this early step in autophagy from purified components in vitro. We find that on assembly from its cytoplasmic subcomplexes, the Atg1–kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. The scaffolding protein Atg17 targets the Atg1–kinase complex to autophagic membranes by specifically recognizing the membrane protein Atg9. This interaction is inhibited by the two regulatory subunits Atg31 and Atg29. Engagement of the Atg1–Atg13 subcomplex restores the Atg9-binding and membrane-tethering activity of Atg17. Our data help to unravel the mechanism that controls Atg17-mediated tethering of Atg9-vesicles, providing the molecular basis to understand initiation of autophagosome-biogenesis.
Highlights
Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation
In contrast to the recombinant Atg1-MITdomain, which dimerized in solution[12,25], full-length Atg[1] was monomeric (Supplementary Fig. 1c)
Since Atg[17] functions upstream of Atg[8], we investigated whether Atg17mono localizes to the phagophore assembly site (PAS) by analysing mCherry-Atg17- and GFP-Atg8-puncta formation in cells expressing Atg[17] or Atg17mono (Fig. 5c)
Summary
Autophagosomes are double-membrane vesicles that sequester cytoplasmic material for lysosomal degradation Their biogenesis is initiated by recruitment of Atg9-vesicles to the phagophore assembly site. This process depends on the regulated activation of the Atg1–kinase complex. We find that on assembly from its cytoplasmic subcomplexes, the Atg1–kinase complex becomes activated, enabling it to recruit and tether Atg9-vesicles. To which we will refer to as autophagy in the following, cytoplasmic cargo is engulfed by a cup-shaped membrane, termed phagophore. Autophagy is initiated at the phagophore assembly site (PAS) in yeast, to which Atg-proteins and donor-membranes are recruited in a spatiotemporally coordinated manner[7,8]. On deactivation of TORC1 Atg[13] becomes partially dephosphorylated and functional Atg1PC assembles at the PAS24
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