E3 autophagy complex and LC3/GABARAP proteins in autophagosome generation

Póster presentado al 8th International Iberian Biophysics Congress celebrado en Bilbao los días 20 y 21 de junio de 2022.In macroautophagy, autophagosome (AP) formation in humans requires the concerted action of the LC3/GABARAP and ATG12 ubiquitin-like conjugation systems. In the present study, the conjugation to the membrane of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of the product of the ATG12 system (the E3 complex). In addition, the mechanisms by which the different LC3/GABARAP proteins and E3 could participate in vesicle tethering and fusion during AP expansion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulation should exist to inhibit the formationof an E3 immobile scaffold

Effect of ATG12-ATG5-ATG16L1 autophagy E3 complex on the ability of LC3/GABARAP proteins to induce

Trabajo presentado en el 44º Congreso Nacional de la Sociedad Española de Bioquímica y Biología Molecular (SEBBM), celebrado en Málaga entre el 6 y el 9 de septiembre de 2022.In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12–ATG5-ATG16L1 (E3) complex acts as a ubiquitin-like E3 ligase enzyme, promoting LC3/GABARAP protein anchoring to the AP membrane. The role of the various proteins in the AP expansion process is still unclear, in part because there are no studies comparing LC3/GABARAP-family member roles under the same conditions, and also because the full human E3 complex was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However E3 hampers LC3/GABARAP capacity to induce intervesicular lipid mixing or subsequent fusion, presumably through formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulation should exist to inhibit the formation of an E3 immobile scaffold

Effect of ATG12–ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12–ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grants No. PGC2018-099857-B-I00 and PID2021-124461NB-I00), by the Basque Government (grants No. IT1625-22 and IT1270-19), by Fundación Biofísica Bizkaia, by Fundación Ramón Areces (grant No. CIVP20A6619), and by the Basque Excellence Research Centre (BERC) program of the Basque Government. MI and YV were recipients of predoctoral FPU fellowships from the Spanish Ministry of Science, Innovation and Universities (FPU16/05873, FPU18/00799), UB thanks the University of the Basque Country for a predoctoral contract. This work was supported by Human Frontiers Science Program RGP0026/2017 (S.M.)

Effect of ATG12-ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

In macroautophagy, the autophagosome (AP) engulfs portions of cytoplasm to allow their lysosomal degradation. AP formation in humans requires the concerted action of the ATG12 and LC3/GABARAP conjugation systems. The ATG12-ATG5-ATG16L1 or E3-like complex (E3 for short) acts as a ubiquitin-like E3 enzyme, promoting LC3/GABARAP proteins anchoring to the AP membrane. Their role in the AP expansion process is still unclear, in part because there are no studies comparing six LC3/GABARAP family member roles under the same conditions, and also because the full human E3 was only recently available. In the present study, the lipidation of six members of the LC3/GABARAP family has been reconstituted in the presence and absence of E3, and the mechanisms by which E3 and LC3/GABARAP proteins participate in vesicle tethering and fusion have been investigated. In the absence of E3, GABARAP and GABARAPL1 showed the highest activities. Differences found within LC3/GABARAP proteins suggest the existence of a lipidation threshold, lower for the GABARAP subfamily, as a requisite for tethering and inter-vesicular lipid mixing. E3 increases and speeds up lipidation and LC3/GABARAP-promoted tethering. However, E3 hampers LC3/GABARAP capacity to induce inter-vesicular lipid mixing or subsequent fusion, presumably through the formation of a rigid scaffold on the vesicle surface. Our results suggest a model of AP expansion in which the growing regions would be areas where the LC3/GABARAP proteins involved should be susceptible to lipidation in the absence of E3, or else a regulatory mechanism would allow vesicle incorporation and phagophore growth when E3 is present.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported in part by the Spanish Ministerio de Ciencia e Innovación (MCI), Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (FEDER) (grants No. PGC2018-099857-B-I00 and PID2021-124461NB-I00), by the Basque Government (grants No. IT1625-22 and IT1270-19), by Fundación Biofísica Bizkaia, by Fundación Ramón Areces (grant No. CIVP20A6619), and by the Basque Excellence Research Centre (BERC) program of the Basque Government. MI and YV were recipients of predoctoral FPU fellowships from the Spanish Ministry of Science, Innovation and Universities (FPU16/05873, FPU18/00799), UB thanks the University of the Basque Country for a predoctoral contract. This work was supported by Human Frontiers Science Program RGP0026/2017 (S.M.).Peer reviewe

Supplementary Information for Effect of ATG12–ATG5-ATG16L1 autophagy E3-like complex on the ability of LC3/GABARAP proteins to induce vesicle tethering and fusion

15 pages. -- This file includes: Supp. Fig. 1-13. -- Supp. Table 1. List of protein constructs used in this work: vector, expression system, detailed protein encoded, and reference study.Peer reviewe

A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy.

Autophagy degrades cytoplasmic cargo by its delivery to lysosomes within double membrane autophagosomes. Synthesis of the phosphoinositide PI(3)P by the autophagic class III phosphatidylinositol-3 kinase complex I (PI3KC3-C1) and conjugation of ATG8/LC3 proteins to phagophore membranes by the ATG12-ATG5-ATG16L1 (E3) complex are two critical steps in autophagosome biogenesis, connected by WIPI2. Here, we present a complete reconstitution of these events. On giant unilamellar vesicles (GUVs), LC3 lipidation is strictly dependent on the recruitment of WIPI2 that in turn depends on PI(3)P. Ectopically targeting E3 to membranes in the absence of WIPI2 is insufficient to support LC3 lipidation, demonstrating that WIPI2 allosterically activates the E3 complex. PI3KC3-C1 and WIPI2 mutually promote the recruitment of each other in a positive feedback loop. When both PI 3-kinase and LC3 lipidation reactions were performed simultaneously, positive feedback between PI3KC3-C1 and WIPI2 led to rapid LC3 lipidation with kinetics similar to that seen in cellular autophagosome formation

Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy.

Autophagy is a major pathway for the clearance of harmful material from the cytoplasm. During autophagy, cytoplasmic material is delivered into the lysosomal system by organelles called autophagosomes. Autophagosomes form in a de novo manner and, in the course of their formation, isolate cargo material from the rest of the cytoplasm. Cargo specificity is conferred by autophagic cargo receptors that selectively link the cargo to the autophagosomal membrane decorated with ATG8 family proteins such as LC3B. Here we show that the human cargo receptor p62/SQSTM-1 employs oligomerization to stabilize its interaction with LC3B and linear ubiquitin when they are clustered on surfaces. Thus, oligomerization enables p62 to simultaneously select for the isolation membrane and the ubiquitinated cargo. We further show in a fully reconstituted system that the interaction of p62 with ubiquitin and LC3B is sufficient to bend the membrane around the cargo