Network organization of the human autophagy system

Abstract

Autophagy, the process by which proteins and organelles are sequestered in autophagosomal vesicles and delivered to the lysosome/vacuole for degradation, provides a primary route for turnover of stable and defective cellular proteins. Defects in this system are linked with numerous human diseases. Although conserved protein kinase, lipid kinase and ubiquitin-like protein conjugation subnetworks controlling autophagosome formation and cargo recruitment have been defined, our understanding of the global organization of this system is limited. Here we report a proteomic analysis of the autophagy interaction network in human cells under conditions of ongoing (basal) autophagy, revealing a network of 751 interactions among 409 candidate interacting proteins with extensive connectivity among subnetworks. Many new autophagy interaction network components have roles in vesicle trafficking, protein or lipid phosphorylation and protein ubiquitination, and affect autophagosome number or flux when depleted by RNA interference. The six ATG8 orthologues in humans (MAP1LC3/GABARAP proteins) interact with a cohort of 67 proteins, with extensive binding partner overlap between family members, and frequent involvement of a conserved surface on ATG8 proteins known to interact with LC3-interacting regions in partner proteins. These studies provide a global view of the mammalian autophagy interaction landscape and a resource for mechanistic analysis of this critical protein homeostasis pathway.

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Figure 1: Overview of the autophagy interaction network (AIN).
Figure 2: Autophagy subnetwork maps.
Figure 3: The ATG8 subnetwork.
Figure 4: Specificity within the ATG8 subnetwork.
Figure 5: RNAi analysis of the autophagy interaction network.
Figure 6: Functional integration of the autophagy interaction network.

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Acknowledgements

We thank I. Dikic for discussions and for sharing unpublished data, D. Bowman and J. Ringeling for assistance with Acapela software, N. Perrimon, S. Mohr and M. Ocana for access to the Opera microscope, and N. Gray for Torin1. This work was supported by grants to J.W.H. from Millennium Pharmaceuticals, the National Institutes of Health, and the Paul F. Glenn Foundation on Aging. C.B. is a Humboldt Postdoctoral Fellow.

Author information

C.B. and M.E.S. performed experiments, analysed data and co-wrote the paper. S.P.G. provided proteomic infrastructure support and interpreted data. J.W.H. directed the research, interpreted data and wrote the paper.

Correspondence to J. Wade Harper.

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Competing interests

J.W.H. is a consultant for Millennium Pharmaceuticals.

Supplementary information

Supplementary Information

This file contains Supplementary Methods, References and Supplementary Figures S1-S13 with legends. (PDF 8978 kb)

Supplementary Table 1

This file contains the cDNA constructs. (XLS 31 kb)

Supplementary Table 2

This file contains the primary LC-MS/MS data for 65 baits in the autophagy interaction network. (XLS 5354 kb)

Supplementary Table 3

This file contains the primary LC-MS/MS data for the ATG8 sub-network with and without the C-terminal Gly residue. (Sheet 1 and 2). (XLS 758 kb)

Supplementary Table 4

This file contains the primary LC-MS/MS data for sub-network proteomic analysis with and without Torin 1 treatment. (Sheet 1 and 2). (XLS 2877 kb)

Supplementary Table 5

This file contains the siRNA and RT-PCR primer sequences used in this study. (XLS 60 kb)

Supplementary Table 6

This file contains the normalized average intensity spot signals/cell for the RNAi autophagosome formation screen. (XLS 99 kb)

Supplementary Table 7

This file contains the curation of genes lacking Gene Ontologyc Process descriptors (Sheet 2), as well as the GO categories employed for this analysis (Sheet 1). (XLS 97 kb)

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