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.

Figure 1. Overview of the autophagy interaction network (AIN)

HCIPs within the autophagy network are shown for 32 primary baits (solid squares) and 33 secondary baits (open squares). Sub-networks are color-coded. Interacting proteins are indicated by gray circles.

Figure 2. Autophagy sub-network maps

Common interacting proteins with sub-threshold WD^N-scores were included if HCIP criteria were fulfilled in ≥ 1 IP-MS/MS experiment. Solid squares, primary baits; open squares, secondary baits; grey circles, HCIPs; dotted lines, interactions found in BIOGRID or MINT databases. a, UBL transfer cascade network, b, PIK3C3-BECN1 network, c, SH3GLB1 network, d, ULK1-AMPK network, e, ATG2 network, and f, NSF network. g, Effect of autophagy activation by Torin1 on bait-HCIP association. BN-NSAF, bait normalized-normalized spectral abundance factor.

Figure 3. The ATG8 sub-network

a, Proteomic analysis of MAP1LC3 and GABARAP isoforms (solid squares). b, Human ATG8 protein phylogenetic tree. c, Overlap of interacting proteins found between and among MAP1LC3 and GABARAP subfamilies by LC-MS/MS. d, Effect of autophagy activation by Torin1 on HCIP-ATG8 association. BN-NSAF, bait normalized-normalized spectral abundance factor.

Figure 4. Specificity within the ATG8 sub-network

a, In vitro validation. S-tagged proteins were tested for GST-ATG8 binding (Supplementary Fig. S10). Green: binding. Red: no binding. b, LDS dependence. As in panel a using wild-type, GABARAP^Y49A/L50A or MAP1LC3B^F52A/L53A proteins (Supplementary Fig. S11a,b). Blue edges: loss of binding. Green edges: binding maintained. Red edges: no binding with WT. c, ATG8^ΔGly dependence. Wild-type and ATG8^ΔGly proteins from 293T cells were subjected to LC-MS/MS. Blue edge: decreased binding. Red edge: increased binding. (Supplementary Fig. S12a, Table S3).

Figure 5. RNAi analysis of the autophagy interaction network

a, GFP-MAP1LC3B foci after RNAi and/or Rapamycin in U2OS cells. b, Integrated spot signal/cell (ISSC) for 344 siRNAs targeting 86 genes (n = 4). Control siRNA (siCK) indicated by black vertical bars. c, Normalized ISSC (N-ISSC) for GFP-MAP1LC3B/U2OS with or without Rapamycin (6 h) (4 siRNAs/gene with each bar representing one of four siRNAs). Unless noted otherwise, p < 0.01 using Students T-test; *, p<0.05; white rectangles, p>0.05. d, α-LC3 blots of U2OS cell extracts after depletion of the indicated proteins in the presence or absence of BafA1 (100 nM, 3 h), and re-probed with α-PCNA. e, Normalized-mean fluorescence intensity (N-MFI) of GFP-MAP1LC3B cells after depletion with the indicated siRNAs in the presence or absence of BafA1. Error bars, Standard Deviation; n = 2.

Figure 6. Functional integration of the Autophagy Interaction Network

Proteins in yellow boxes are HCIPs and proteins labeled with an asterisk were sub-threshold (WD^N-score <1.0) for HCIP identification. Dotted lines, cross-module interaction. Solid lines, this study. Dotted arrows, potential functional interactions. Red or green full circle, three-quarter circle, and half-circle represent a reduction or increase in autophagosomes with 4, 3, and 2 siRNAs, respectively, in GFP-MAP1LC3B expressing U2OS cells without rapamycin. Proteins in white boxes were not found by proteomics. The six ATG8 family members are represented by ATG8.