Project: In Vitro Mimics of the Autophagosome
The Making of an Autophagosome
Organelle growth in other systems is controlled by protein machineries that manipulate membrane architecture by applying nanoscale forces along or across the membrane surfaces such that simple paradigms in membrane bending or membrane fusion are repeated across much of cell biology. The autophagosome however appears surprisingly devoid of otherwise ubiquitous trafficking related machinary including SNAREs, coat protein complexes, Arfs, etc. As such, how the autophagosome grows, how the cup-like shape of the Pre-autophagosomal Structure (PAS or isolation membrane) is determined, how it closes and eventually fuses into the lysosome are all unknown and likely rely upon novel membrane-directed protein machineries.
In vitro reconstitution of autophagosome-associated proteins is a powerful approach towards elucidating protein function. We use a variety of membrane supports, including small, large, and giant unilamellar liposomes as well as supported bilayers to explore how various autophagy proteins tether, fuse, bend, and otherwise manipulate membrane architecture. Our most recent work in this direction has been to reconsider the hypothesis that the lipidated form of the autophagosomal marker, Atg8, is a membrane fusogen (1). Using approaches we previously applied to SNARE and SNARE-associated membrane-fusing proteins (2), as well as novel chemical ligation strategies for artificial lipidation (3), we have concluded that Atg8 is likely just a membrane tether (1). If it is also responsible for membrane fusion, the the pre-autophagosomal membrane to which it binds must have a unique composition or architecture. Work to establish these features is underway in our lab.
- Nair et al., 2011. SNARE proteins are required for macroautophagy. Cell, 146, 290-302
- Shen et al., 2007. Selective activation of cognate SNAREpins by Seci/Munci8 proteins. Cell, 128, 183-195
- Jotwani et al., 2011. Approaches to the study of Arg8 functions in vitro. Methods of Cell Biology. Lipids Volume.