Recent evidence suggests that autophagy facilitates the unconventional secretion of the pro-inflammatory cytokine interleukin 1β (IL-1β). Here, we reconstituted an autophagy-regulated secretion of mature IL-1β (m-IL-1β) in non-macrophage cells. We found that cytoplasmic IL-1β associates with the autophagosome and m-IL-1β enters into the lumen of a vesicle intermediate but not into the cytoplasmic interior formed by engulfment of the autophagic membrane. In advance of secretion, m-IL-1β appears to be translocated across a membrane in an event that may require m-IL-1β to be unfolded or remain conformationally flexible and is dependent on two KFERQ-like motifs essential for the association of IL-1β with HSP90. A vesicle, possibly a precursor of the phagophore, contains translocated m-IL-1β and later turns into an autophagosome in which m-IL-1β resides within the intermembrane space of the double-membrane structure. Completion of IL-1β secretion requires Golgi reassembly and stacking proteins (GRASPs) and multi-vesicular body (MVB) formation.

DOI: http://dx.doi.org/10.7554/eLife.11205.001