A conserved ATG2-GABARAP interaction is critical for phagophore closure
Preprint posted on 3 May 2019 https://www.biorxiv.org/content/10.1101/624627v1
ATG2- autophagosome closing cue. Great insight into the function of ATG2A/B proteins in cells!
Selected by Mila Basic
Categories: biochemistry, cell biology, molecular biology
Background
Autophagy is a process where cellular components are recycled with the aim of preserving homeostasis under basal conditions, as well as a process specifically initiated to overcome stress induced by damaged organelles, protein aggregates, pathogens, etc. (reviewed in Dikic and Elazar, 2018). In more detail, this well orchestrated process results in freeing amino acids and lipids for essential processes to preserve, or reestablish physiological conditions. In short, autophagy can be defined, or illustrated the following way: The formation of a double layer membrane surrounding and enclosing random cellular components, or specific cargo. Once closed, the autophagosome fuses with the lysosome leading to degradation of engulfed material by lysosomal proteases, and its subsequent release. The importance of autophagy as a process, and efforts to understand how it is so precisely executed and regulated have been made clear by awarding the Nobel prize in Physiology or Medicine to Yoshinori Ohsumi for his discoveries of mechanisms for autophagy.
A lot is known about the initiation steps, but exact complexes responsible for autophagosome closure and subsequent fusion with the lysosome remain elusive. Mammalian ATG2A and ATG2B are ≈212kDa and ≈232kDa large proteins, respectively, that share approximately 40% amino acid sequence homology but are only 13% similar to the single isoform of S. cerevisiae Atg2 and 24-26% to the D. melanogaster Atg2, indicating a potential divergence of function (this preprint). In yeast, Atg2 constitutively interacts with Atg18 at phosphatidylinositol-3-phosphate- rich membrane regions, and tethers pre-autophagosomal membranes to the endoplasmic reticulum for autophagosome formation (Obara et al., 2008, Kotani et al. 2018). Similarly, in mammals, ATG2A/B have been previously shown to interact with WIPI4 (member of the WIPI family of proteins, homologue of yeast Atg18) (Zheng et al., 2017, Chowdhury et al., 2018). Depletion of these proteins leads to formation of immature, open autophagosome-like structures, suggesting their role in late stages of autophagy (Velikkakath et al., 2012, Kishi-Itakura et al., 2014, Bakula et.al 2017), but the exact role of ATG2A/B proteins, as well as the role of the interaction with WIPI4, is not well understood, hence, this study aimed to address these questions.
Key Findings
In this well designed and thorough study, the authors set out to elucidate the molecular mechanism of autophagosome closure, dissecting the roles of ATG2 isoforms and WIPI4 in this process. The authors created an endogenously tagged GFP-ATG2A U2OS cell line using CRISPR/Cas9 knock-in strategy. Characterizing the behavior of the cell line, they observed that under physiological conditions ATG2A appears to be dispersed throughout the cell. However, upon starvation, distinct ring and punctae structures were formed. These structures were in proximity to LC3B, an ATG8 family autophagosomal marker, and co-localized with known markers of early autophagosomes: ATG2B, WIPI2, and ATG16L1. Interestingly, GABARAP-L1, another member of the ATG8 family, was also observed in close proximity to ATG2A. Following the co-localization data, the authors demonstrate that ATG2A co-immunoprecipitates with ATG2B, WIPI4, and GABARAP, but not with LC3B, thus suggesting a specific and distinct role of the ATG2-GABARAP interaction in autophagy. The authors identified LIR motifs (LC3-interaction region) in both ATG2A and ATG2B, and utilizing mutagenesis approaches, they conclude that LIR#5 in ATG2A, and LIR#6 in ATG2B are necessary for the interaction with ATG8 family of proteins. Interestingly, these LIR motifs seem to be conserved across vertebrates and invertebrates, but not in yeast, suggesting a functional divergence in context of evolution. Furthermore, LIR motifs in both of the ATG2 isoforms are in close proximity to WIPI4 interaction region (Y/HFS) (Bakula et al., 2017, Zheng et al., 2017, Chowdhury et al., 2018). Mutating LIR or Y/HFS abolished ATG2 binding to GABARAP, or WIPI4, respectively, but not vice versa, suggesting these interactions are independent. To address the functional consequence of these interactions, through biochemical, and autophagy flux assays, the authors nicely show that depletion of both ATG2A/B, using double knockout cell lines DKO, leads to impaired autophagy, and that this can be rescued by reintroducing WT ATG2A, or WIPI4 interacting mutant (ATG2A-mYFS), but not the LC3-interacting mutant ATG2A-mLIR. This finding clearly suggests that ATG2-WIPI4 interaction is dispensable for autophagy; however, the interaction with GABARAP is absolutely required. The authors observe that expression of ATG2A-mLIR mutant in DKO results in large LC3B/p62 positive structures and accumulation of ATG9A, WIPI2, and ATG16L. The presence of these proteins, albeit at non-autophagosome-like structures, suggests that the absence of a functional, LIR containing ATG2A affects only late stages of autophagy, namely autophagosome maturation or closure. Utilizing two elegant approaches to address the question of autophagosome closure, the authors demonstrate that in DKO cells, or DKO cells expressing ATGA-mLIR, autophagosomes are not sealed, as compared to WT ATG2A expressing cells, or those expressing ATG2A-mYFS. This was shown using a Proteinase K protection assay, exploiting the vulnerability of proteins that are not protected by membrane compartmentalisation (e.g. in autophagosomes). Similarly, failure of STX17 recruitment to the LC3-positive vesicles in DKO cells, or cells reconstituted with ATG2A-mLIR, suggested that these structures are not matured autophagosomes (Itakura et al., 2012). Taken together, the authors show that ATG2 isoforms play an important role in autophagosome maturation, and that in the absence of ATG2 the autophagosomes fail to close. The function of ATG2 depends on its LIR motif, and surprisingly, the interaction with WIPI4 is dispensable for autophagy.
What I like about this preprint
I chose to highlight this preprint due to several scientific, but also technical reasons. The preprint demonstrates the power of having adequate, simple, yet sophisticated tools (e.g. DKO cells complemented with various ATG2 mutants, or endogenously tagged ATG2) in combination with different methods allows you to accurately address, and dissect, molecular mechanisms, that would otherwise require a plethora of additional complementary approaches.
In context of autophagy, this study on the function of ATG2 contributes to our general understanding of the process, but also raises interesting questions for future research in the field, such as the need to further understand distinct function of WIPI proteins, exact mechanism of the ATG2 dependent autophagosome closure, and the dynamics and the importance of the conserved interaction region between ATG2 and WIPI4 (in yeast, Atg2-Atg18).
Open questions
In autophagy, in higher eukaryotes, we generally observe occurrence of multiple isoforms of proteins, examples are ATG8 family of proteins, WIPI, or ATG2 explored in this work, to which degree can we talk about cell-line specificity, process specificity (as in cargo specificity for example), functional complementation, or redundancy? What is the function of ATG2A and ATG2B isoforms?
If LIR mutation in ATG2A leads to loss of interaction with GABARAP, but not with WIPI4, and loss of WIPI4 interaction alone leads to no particular autophagy phenotype, could the interface simply suggest their coexistence at the autophagosome within the same complex, without direct recruitment by ATG2A? Is WIPI4 co-localizing at the autophagosome in DKO ATG2A/B complemented with ATG2A-mYFS?
If mutation of WIPI4 interaction region leads to increase of ATG2A interaction with lipidated GABARAP, could ATG2A-WIPI4 interaction be a regulatory mechanism (inhibitory)? Can GABARAP titrate WIPI4 out of interaction with ATG2A, meaning beyond a certain threshold (e.g.: cytoplasmic versus autophagosomal localisation) they compete for the binding? In the absence of WIPI4 in context of ATG2 dependent autophagy, could another WIPI isoform complement the required role in DKO cells with stably expressing ATG2A-mYFS (adaptation effect)?
References
- Dikic I., and Elazar Z., (2018) Mechanism and medical implications of mammalian autophagy Nature Reviews Molecular Cell Biology volume 19, pages349–364
- Obara K., Sekito T., Niimi K., Ohsumi Y., (2008) The Atg18-Atg2 complex is recruited to autophagic membranes via phosphatidylinositol 3-phosphate and exerts an essential function. The Journal of biological chemistry 283: 23972-80
- Kotani T., Kirisako H., Koizumi M., Ohsumi Y., Nakatogawa H., (2018) The Atg2-Atg18 complex tethers pre-autophagosomal membranes to the endoplasmic reticulum for autophagosome formation. Proceedings of the National Academy of Sciences of the United States of America 115: 10363-10368
- Zheng J.X., Li Y., Ding Y.H., Liu J.J., Zhang M.J., Dong M.Q., Wang H.W., Yu L., (2017) Architecture of the ATG2B-WDR45 complex and an aromatic Y/HF motif crucial for complex formation. Autophagy 13: 1870- 1883
- Chowdhury S., Otomo C., Leitner A., Ohashi K., Aebersold R., Lander G.C., Otomo T., (2018) Insights into autophagosome biogenesis from structural and biochemical analyses of the ATG2A-WIPI4 complex. Proceedings of the National Academy of Sciences of the United States of America 115: E9792-E9801
- Velikkakath A.K., Nishimura T., Oita E., Ishihara N., Mizushima N., (2012) Mammalian Atg2 proteins are essential for autophagosome formation and important for regulation of size and distribution of lipid droplets. Mol Biol Cell 23: 896-909
- Kishi-Itakura C., Koyama-Honda I., Itakura E., Mizushima N., (2014) Ultrastructural analysis of autophagosome organization using mammalian autophagy-deficient cells. J Cell Sci 127: 4089-102
- Bakula D., Muller A.J., Zuleger T., Takacs Z., Franz-Wachtel M., Thost A.K., Brigger D., Tschan M.P., Frickey T., Robenek H., et al., (2017) WIPI3 and WIPI4 beta-propellers are scaffolds for LKB1-AMPK-TSC signalling circuits in the control of autophagy. Nat Commun 8: 15637
- Itakura E., Kishi-Itakura C., Mizushima N., (2012) The hairpin-type tail- anchored SNARE syntaxin 17 targets to autophagosomes for fusion with endosomes/lysosomes. Cell 151: 1256-69
Posted on: 12 September 2019
doi: https://doi.org/10.1242/prelights.13762
Read preprint
(No Ratings Yet)Sign up to customise the site to your preferences and to receive alerts
Register hereAlso in the biochemistry category:
Structural basis of respiratory complexes adaptation to cold temperatures
Lens Placode Modulates Extracellular Matrix Formation During Early Eye Development
Generalized Biomolecular Modeling and Design with RoseTTAFold All-Atom
Also in the cell biology category:
Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments
Structural basis of respiratory complexes adaptation to cold temperatures
RIPK3 coordinates RHIM domain-dependent inflammatory transcription in neurons
Also in the molecular biology category:
Clusters of lineage-specific genes are anchored by ZNF274 in repressive perinucleolar compartments
Nanos2+ cells give rise to germline and somatic lineages in the sea anemone Nematostella vectensis
Plant plasmodesmata bridges form through ER-driven incomplete cytokinesis
AND
Plasmodesmata act as unconventional membrane contact sites regulating inter-cellular molecular exchange in plants
preLists in the biochemistry category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
Preprint Peer Review – Biochemistry Course at UFRJ, Brazil
Communication of scientific knowledge has changed dramatically in recent decades and the public perception of scientific discoveries depends on the peer review process of articles published in scientific journals. Preprints are key vehicles for the dissemination of scientific discoveries, but they are still not properly recognized by the scientific community since peer review is very limited. On the other hand, peer review is very heterogeneous and a fundamental aspect to improve it is to train young scientists on how to think critically and how to evaluate scientific knowledge in a professional way. Thus, this course aims to: i) train students on how to perform peer review of scientific manuscripts in a professional manner; ii) develop students' critical thinking; iii) contribute to the appreciation of preprints as important vehicles for the dissemination of scientific knowledge without restrictions; iv) contribute to the development of students' curricula, as their opinions will be published and indexed on the preLights platform. The evaluations will be based on qualitative analyses of the oral presentations of preprints in the field of biochemistry deposited in the bioRxiv server, of the critical reports written by the students, as well as of the participation of the students during the preprints discussions.
| List by | Marcus Oliveira |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
20th “Genetics Workshops in Hungary”, Szeged (25th, September)
In this annual conference, Hungarian geneticists, biochemists and biotechnologists presented their works. Link: http://group.szbk.u-szeged.hu/minikonf/archive/prg2021.pdf
| List by | Nándor Lipták |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |
Also in the cell biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
preLights peer support – preprints of interest
This is a preprint repository to organise the preprints and preLights covered through the 'preLights peer support' initiative.
| List by | preLights peer support |
The Society for Developmental Biology 82nd Annual Meeting
This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.
| List by | Joyce Yu, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
Journal of Cell Science meeting ‘Imaging Cell Dynamics’
This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.
| List by | Helen Zenner |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
Fibroblasts
The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!
| List by | Osvaldo Contreras |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
Planar Cell Polarity – PCP
This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.
| List by | Ana Dorrego-Rivas |
BioMalPar XVI: Biology and Pathology of the Malaria Parasite
[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria
| List by | Dey Lab, Samantha Seah |
1
Cell Polarity
Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.
| List by | Yamini Ravichandran |
TAGC 2020
Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20
| List by | Maiko Kitaoka et al. |
3D Gastruloids
A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.
| List by | Paul Gerald L. Sanchez and Stefano Vianello |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
EMBL Seeing is Believing – Imaging the Molecular Processes of Life
Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019
| List by | Dey Lab |
Autophagy
Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.
| List by | Sandra Malmgren Hill |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
Cellular metabolism
A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.
| List by | Pablo Ranea Robles |
BSCB/BSDB Annual Meeting 2019
Preprints presented at the BSCB/BSDB Annual Meeting 2019
| List by | Dey Lab |
Biophysical Society Annual Meeting 2019
Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA
| List by | Joseph Jose Thottacherry |
ASCB/EMBO Annual Meeting 2018
This list relates to preprints that were discussed at the recent ASCB conference.
| List by | Dey Lab, Amanda Haage |
Also in the molecular biology category:
BSCB-Biochemical Society 2024 Cell Migration meeting
This preList features preprints that were discussed and presented during the BSCB-Biochemical Society 2024 Cell Migration meeting in Birmingham, UK in April 2024. Kindly put together by Sara Morais da Silva, Reviews Editor at Journal of Cell Science.
| List by | Reinier Prosee |
‘In preprints’ from Development 2022-2023
A list of the preprints featured in Development's 'In preprints' articles between 2022-2023
| List by | Alex Eve, Katherine Brown |
CSHL 87th Symposium: Stem Cells
Preprints mentioned by speakers at the #CSHLsymp23
| List by | Alex Eve |
9th International Symposium on the Biology of Vertebrate Sex Determination
This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.
| List by | Martin Estermann |
Alumni picks – preLights 5th Birthday
This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.
| List by | Sergio Menchero et al. |
CellBio 2022 – An ASCB/EMBO Meeting
This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.
| List by | Nadja Hümpfer et al. |
EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)
A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.
| List by | Alex Eve |
FENS 2020
A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020
| List by | Ana Dorrego-Rivas |
ECFG15 – Fungal biology
Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome
| List by | Hiral Shah |
ASCB EMBO Annual Meeting 2019
A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)
| List by | Madhuja Samaddar et al. |
Lung Disease and Regeneration
This preprint list compiles highlights from the field of lung biology.
| List by | Rob Hynds |
MitoList
This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.
| List by | Sandra Franco Iborra |