The HARPER Lab

Department of Cell Biology

Macro and Selective Autophagy

The human autophagy interaction network examined by mass spectrometry.
Ferritin delivery to lysosomes via the NCOA4 cargo adaptor protein

We have used proteomic tools in conjunction with the CompPASS system to analyze the interaction landscape of the human autophagy system. Autophagy is a process by which cellular proteins and organelles are sequestered into a double-lipid bilayer structure called the autophagosome and are then delivered to the lysosome for degradation. The turnover of cellular proteins through this pathway provides both recycled building blocks such as amino acids, and also is responsible for the turnover of most of the highly stable proteins in the cell. The autophagy system is critical for the ability of the cell to respond to stress, particularly in the context of cancer. Our proteomic analysis has identified an interaction network containing more than 400 proteins, derived from 32 primary autophagy related proteins and 33 new secondary bait proteins. Extensive validation and functional studies have revealed numerous aspects of how the machinery works together to build autophagosomes and deliver them to the lysosome. RNAi experiments examining a sub-set of genes in the pathway have revealed dozens of genes that affect the production of autophagosomes or flux through the autophagy pathway. See Behrends et al, Nature, 2010.

A major question concerns the identity and functions of cargo adaptors, in particular those that are involved in selective autophagy. Moreover, the identity of specific proteins that are targeted to the lysosome via autophagy for selective degradation is largely unknown. We have purified autophagosomes and employed a quantitative proteomic approach to identify resident autophagosomal proteins, identifying more than 200 proteins that are intimately associated with autophagosomes. Exploration of one of these - NCOA4 - led to the discovery of a pathway that targets ferritiin for lysosomal degradation when cells need iron. Ferritin is a critical protein for ior homeostasis. 24 copies of ferriton form a cage which binds ~4000 iron atoms and when iron levels are too high, ferritin is induced and captures excess iron, thereby protecting cells from the Fenton reaction which produces toxic oxidative species. However, when iron levels are reduced, ferritin is targeted to the lysosome, where it is degraded, thereby releasing the iron for its incorportation into important iron-dependent proteins. NCOA4 functions as an essential component by linking ferritin to the autophagosome, which is then targeted to the lysosome for degradation. In cells which lack NCOA4, ferritin isnt collected in the autophagosome and therefore isn't targeted to the lysosome. Mechanistically, NCOA4 interacts with the autophagosomal protein ATG8, which is a key step in the process. These studies suggest a new framework inwhich to understand the links between autophagy and iron control, a process that is linked to numerous diseases, including neurodegeneration. See: Mancias et al, Nature, 2014.