Molecular dissection of the human autophagy signaling pathway
Final Report Abstract
Autophagy is an evolutionarily conserved cellular process that enables cells to engulf and digest portions of their cytoplasm in a regulated manner, thereby accomplishing quality and quantity control of organelles, pathogens, proteins and other macromolecules. These homeostatic and adaptive functions intricately link autophagy to diverse health and disease states including suppression of tumor development, prevention of neurodegeneration and innate immunity. Central to autophagy are dynamic membrane rearrangements to sequester cytosolic constituents in autophagosomes and deliver this cargo load for lysosomal degradation. Our work builds on a systematic proteomic analysis of the human autophagy system that provided a glimpse into the global interaction architecture of this pathway. However, for many of the candidate autophagy pathway proteins we do not fully understand their role in autophagy. We set out to functionally dissect selected components of this system with a focus on the family of human ubiquitin-like ATG8 proteins and their binding partners. Human cells contain six ATG8 members that can be grouped into LC3 and GABARAP subfamilies and are conjugated to the lipid phosphatidylethanolamine (PE). The function of these proteins is best understood in the case of LC3B. LC3B-PE conjugates are incorporated into both layers of incipient autophagosomes where LC3B provides a docking site for cargo receptors and regulatory adaptor proteins. Though, we do not fully understand the role of each of the six ATG8 family members. Given the fact that yeast only harbors one Atg8 isoform, it is unclear whether LC3 and GABARAP proteins are functionally redundant or have unique properties. While analyzing three novel ATG8-binding proteins, namely KBTBD6, KBTBD7 and TECPR2, we uncovered that LC3 and GABARAP proteins exhibit an unexpected function beyond autophagy as membrane localized binding/signaling scaffolds. Briefly, we identified a novel CUL3 RING ubiquitin ligase complex containing the substrate adaptors KBTBD6/7 that mediates ubiquitylation and proteasomal degradation of TIAM1, a RAC1 specific GEF. Increasing the abundance of TIAM1 by depletion of KBTBD6 and/or KBTBD7 led to elevated RAC1 activity, changes in actin morphology, loss of focal adhesions, reduced proliferation and enhanced invasion (Genau et al. Mol Cell 2015). KBTBD6/7 employed ATG8 family-interacting motifs to bind preferentially to GABARAP proteins. Surprisingly, ubiquitylation and degradation of TIAM1 by CUL3KBTBD6/7 depended on its binding to GABARAP proteins. Our efforts revealed that recruitment of CUL3KBTBD6/7 to GABARAP-containing vesicles regulates the abundance of membrane-associated TIAM1 and subsequently spatially restricted RAC1 signaling. In addition, we found that the hereditary spastic paraplegias (HSP)- associated disease protein TECPR2 serves as a scaffold to bind and stabilize two distinct endosomal fusion and sorting complexes as well as the COPII coat protein SEC24D. Depletion of TECPR2 caused decreased ER exit sites (ERES), impaired ER export and altered ER morphology. Intriguingly, regulation of ER export by TECPR2 required its binding to ERES-localized LC3C and can be recapitulated in skin fibroblasts from a HSP patient. Thus, we proposed that TECPR2 is a novel regulator of COPII-dependent ER export (Stadel et al. Mol Cell 2015). Intriguingly, autophagosomal degradation or localization to autophagosomes does not seem to be involved in both processes. Collectively, we provide strong evidence that LC3 and GABARAP proteins act as reversible, membrane-bound scaffolds that help organizing protein and protein complexes spatially to control cellular pathways other than autophagy. Thus, our work has the potential to drive research on human ATG8 proteins into new directions.
Publications
- Rab GTPase-activating proteins in autophagy: regulation of endocytic and autophagy pathways by direct binding to human ATG8 modifiers. MCB 2012 May; 32(9): 1733-44
Popovic D, Akutsu M, Novak I, Harper JW, Behrends C & Dikic I
(See online at https://doi.org/10.1128/MCB.06717-11) - Cellular mechanotransduction relies on tension-induced and chaperone-assisted autophagy. Current Biology 2013 Mar 4; 23(5): 430-5
Ulbricht A, Eppler FJ, Tapia VE, van der Ven PF, Hampe N, Hersch N, Vakeel P, Stadel D, Haas A, Saftig P, Behrends C, Furst DO, Volkmer R, Hoffmann B, Kolanus W & Hohfeld J
(See online at https://doi.org/10.1016/j.cub.2013.01.064) - NRBF2 regulates autophagy and prevents liver injury by modulating Atg14L- linked phosphatidylinositol-3 kinase III activity. Nature Comm 2014 May 22; 5: 3920
Lu J, He L, Behrends C, Araki M, Araki K, Jun Wang Q, Catanzaro JM, Friedman SL, Zong WX, Fiel MI, Li M & Yue Z
(See online at https://doi.org/10.1038/ncomms4920) - RAB3GAP1 and RAB3GAP2 modulate basal and rapamycin-induced autophagy. Autophagy 2014; 10(12): 2297-309
Spang N, Feldmann A, Huesmann H, Bekbulat F, Schmitt V, Hiebel C, Koziollek-Drechsler I, Clement AM, Moosmann B, Jung J, Behrends C, Dikic I, Kern A & Behl C
(See online at https://doi.org/10.4161/15548627.2014.994359) - Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9. MCB 2015 Jul 15; 35(14): 2479-94
Jung J, Genau HM & Behrends C
(See online at https://doi.org/10.1128/MCB.00125-15) - CUL3-KBTBD6/KBTBD7 ubiquitin E3 ligase cooperates with ubiquitin-like GABARAP proteins to spatially restrict TIAM1-RAC1 signaling. Mol Cell 2015 Mar 19; 57(6): 995-1010
Genau HM, Huber J, Baschieri F, Akutsu M, Dötsch V, Farhan H, Rogov V & Behrends C
(See online at https://doi.org/10.1016/j.molcel.2014.12.040) - PLEKHM1 Regulates Autophagosome-Lysosome Fusion through HOPS Complex and LC3/GABARAP Proteins. Mol Cell 2015 Jan 8; 57(1): 39-54
McEwan DG, Popovic D, Gubas A, Terawaki S, Suzuki H, Stadel D, Coxon FP, Miranda de Stegmann D, Bhogaraju S, Maddi K, Kirchof A, Gatti E, Helfrich MH, Wakatsuki S, Behrends C, Pierre P & Dikic I
(See online at https://doi.org/10.1016/j.molcel.2014.11.006) - TBC1D14 regulates autophagy via the TRAPP complex and ATG9 traffic. EMBO J 2015 Dec 28
Lamb CA, Nuhlen S, Judith D, Frith D, Snijders B, Behrends C & Tooze SA
(See online at https://doi.org/10.15252/embj.201592695) - TECPR2 Cooperates with LC3C to Regulate COPII-Dependent ER Export. Mol Cell 2015 Oct 1; 60(1): 89-104
Stadel D, Millarte V, Tillmann KD, Huber J, Tamin-Yecheskel B-C, Akutsu M, Demishtein A, Ben- Zeev B, Anikster Y, Perez F, Dötsch V, Elazar Z, Rogov V, Farhan H & Behrends C
(See online at https://doi.org/10.1016/j.molcel.2015.09.010) - An siRNA screen for ATG protein depletion reveals the extent of the unconventional functions of the autophagy proteome in virus replication. JCB 29 August 2016, 214 (5): 619
Mauthe M, Langereis M, Jung J, Zhou X, Jones A, Omta W, Tooze SA, Stork B, Paludan SR, Ahola T, Egan D, Behrends C, Mokry M, de Haan C, van Kuppeveld F & Reggiori F
(See online at https://doi.org/10.1083/jcb.201602046)
