The insufficiency of ATG4A in macroautophagy
2020; Elsevier BV; Volume: 295; Issue: 39 Linguagem: Inglês
10.1074/jbc.ra120.013897
ISSN1083-351X
AutoresNathan Nguyen, Taryn J. Olivas, Antonio Mires, Jiaxin Jin, Shenliang Yu, Lin Luan, Shanta Nag, Karlina J. Kauffman, Thomas J. Melia,
Tópico(s)Mosquito-borne diseases and control
ResumoDuring autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. These results suggest that to support efficient production and consumption of autophagosomes, additional factors are essential including possibly ATG4B itself or one of its proteolytic products in the LC3 family. During autophagy, LC3 and GABARAP proteins become covalently attached to phosphatidylethanolamine on the growing autophagosome. This attachment is also reversible. Deconjugation (or delipidation) involves the proteolytic cleavage of an isopeptide bond between LC3 or GABARAP and the phosphatidylethanolamine headgroup. This cleavage is carried about by the ATG4 family of proteases (ATG4A, B, C, and D). Many studies have established that ATG4B is the most active of these proteases and is sufficient for autophagy progression in simple cells. Here we examined the second most active protease, ATG4A, to map out key regulatory motifs on the protein and to establish its activity in cells. We utilized fully in vitro reconstitution systems in which we controlled the attachment of LC3/GABARAP members and discovered a role for a C-terminal LC3-interacting region on ATG4A in regulating its access to LC3/GABARAP. We then used a gene-edited cell line in which all four ATG4 proteases have been knocked out to establish that ATG4A is insufficient to support autophagy and is unable to support GABARAP proteins removal from the membrane. As a result, GABARAP proteins accumulate on membranes other than mature autophagosomes. 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Thus, ATG4 family proteins act constitutively to prime newly expressed Atg8/LC3B/GABARAP proteins but also must function under temporal and spatial regulation to release lipid-conjugated forms of Atg8/LC3/GABARAP from fully formed mature autophagosomes. We and others have begun to study how priming and delipidation are independently regulated to achieve these drastically different kinetics in the cell (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar, 30Agrotis A. Pengo N. Burden J.J. Ketteler R. Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells.Autophagy. 2019; 15 (30661429): 976-99710.1080/15548627.2019.1569925Crossref PubMed Scopus (86) Google Scholar). ATG4B is an extremely fast priming enzyme in vitro (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar, 31Li M. Hou Y. Wang J. Chen X. Shao Z.-M. Yin X.-M. Kinetics comparisons of mammalian Atg4 homologues indicate selective preferences toward diverse Atg8 substrates.J. Biol. Chem. 2011; 286 (21177865): 7327-733810.1074/jbc.M110.199059Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar) and is also the major driver of priming for both the LC3 and GABARAP subfamilies in cells. KO of ATG4B, but not of the other three mammalian ATG4 proteases, eliminates priming of all LC3 proteins and dramatically impairs priming of the GABARAP family proteins (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar, 30Agrotis A. Pengo N. Burden J.J. Ketteler R. Redundancy of human ATG4 protease isoforms in autophagy and LC3/GABARAP processing revealed in cells.Autophagy. 2019; 15 (30661429): 976-99710.1080/15548627.2019.1569925Crossref PubMed Scopus (86) Google Scholar). Delipidation is more complicated. Our laboratory has established that although ATG4A, ATG4C, and ATG4D are very poor at priming, consistent with many previous studies, they are surprisingly just as efficient as ATG4B in vitro at delipidation (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar), suggesting the possibility that they play a key role in deciding when and which LC3 or GABARAP proteins are released from the membrane. Thus far, however, the study of ATG4A, ATG4C, and ATG4D function in cells has largely been confounded by the redundancy of the family. In particular, how and whether ATG4A-mediated priming and delipidation are independently controlled has not been known. Here, we explore the limits of ATG4A-mediated delipidation in a gene-edited cell line we created in which all four mammalian ATG4 genes are missing, which allows us to specifically rescue with ATG4A and various engineered mutants. We discover that like ATG4B, the C-terminal LC3-interaction region (LIR) of ATG4A is essential to its function, but unlike ATG4B, this motif controls both priming and delipidation on ATG4A. Furthermore, we show that ATG4A is not sufficient to support autophagy in our cell lines and, more surprisingly, does not appear to support any delipidation of its substrate, GABARAPL1 (from now on referred to as GL1), in this context. We show that instead, GL1 accumulates on a variety of membranes and remains membrane-associated even if the normal cues for activating the lipidation pathway are shut off for as long as 24 h. This strongly implies that ATG4A does not have access to GL1–PE (also known as GL1-II) under these conditions, suggesting more complex intracellular regulation of its delipidation activity. The redundancy of function across the ATG4 family limits the useful interpretation of single knockouts, and thus we have created a gene-edited HEK293 cell line that we call our ATG4 quadruple knockout (ATG4QKO). In this cell line, there is no detectable expression of any of the ATG4 proteins by Western blotting (Fig. S1A and Ref. 29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar), and we have confirmed genetic alteration at each locus (Fig. S1B). Without any ATG4 expression, these cells are completely incapable of priming the ATG8 protein family, and thus all lipidation of the ATG8 proteins is also inhibited (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar). We previously showed that re-expression of ATG4B in this background is sufficient to rescue LC3 and GABARAP protein priming and lipidation and even to support flux of these proteins in a bafilomycin A1 (Baf A1)–dependent way, suggesting restoration of autophagy (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar). In contrast, re-expression of ATG4A is unable to rescue LC3 processing, consistent with many previous publications, and also leads to anomalous processing of GABARAP proteins. In particular, these cells continue to have a large pool of unprimed pro-GL1 and accumulate fully lipidated GL1-II to high levels but do not show any significant levels of free-floating processed GL1 (GL1-I) (Ref. 29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar and Fig. S2). This result suggests that ATG4A can prime a portion of the GABARAP family protein pool, and this entire population of primed GL1-I is utilized for lipidation. Should any delipidation take place, newly released protein is again rapidly lipidated. Thus, we have an opportunity to consider how autophagy progresses when only the GABARAP family is able to become lipid-attached and to assess how ATG4A contributes to this process. Recent papers have suggested that the GABARAPs can be fully sufficient for at least some forms of macroautophagy and that even in the absence of all LC3 and GABARAP proteins, some macroautophagy persists (32Nguyen T.N. Padman B.S. Usher J. Oorschot V. Ramm G. Lazarou M. Atg8 family LC3/GABARAP proteins are crucial for autophagosome–lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.J. Cell Biol. 2016; 215 (27864321): 857-87410.1083/jcb.201607039Crossref PubMed Scopus (348) Google Scholar, 33Nishida Y. Arakawa S. Fujitani K. 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Critically, when these cells were treated with the PI3K inhibitor 3-MA to block initiation of the macroautophagy pathway, starvation-dependent LLPD is mostly prevented. In contrast to WT cells, ATG4QKO cells were incapable of starvation-dependent LLPD. Thus, starvation-dependent flux depends on ATG4 proteins in this cell line. Lentiviral-mediated rescue of individual ATG4A, B, C, or D genes in the ATG4QKO restored ATG8 protein family processing to varying degrees (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar). We observed in these rescues that high expression of ATG4A drove significant lipidation of GL1, whereas re-expression of ATG4C or ATG4D resulted in more modest levels of lipidated GABARAP family proteins, and only ATG4B re-expression restored any LC3 processing (Fig. S2). To establish whether the ATG4A-, ATG4C-, or ATG4D-mediated processing of GABARAP proteins alone can drive full autophagy, we looked at LLPD in these lines. Surprisingly, only ATG4B re-expression was sufficient to support a starvation-mediated LLPD and did so at essentially WT levels. Cells expressing only ATG4A, ATG4C, or ATG4D behaved exactly like the ATG4QKO (Fig. 1A). Thus, bulk cargo delivery depends absolutely on the presence of ATG4B, consistent with other reports (36Lystad A.H. Carlsson S.R. Ballina L.R. De Kauffman K.J. Nag S. Yoshimori T. Melia T.J. Simonsen A. Distinct functions of ATG16L1 isoforms in membrane binding and LC3B lipidation in autophagy-related processes.Nat. 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Interestingly, however, overall p62 levels were elevated ∼2-fold higher in ATG4QKO cells relative to WT cells (Fig. 1B), indicating that loss of the ATG4 family alters the overall homeostasis of p62 (29Kauffman K.J. Yu S. Jin J. Mugo B. Nguyen N. O'Brien A. Nag S. Lystad A.H. Melia T.J. Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases.Autophagy. 2018; 14 (29458288): 992-101010.1080/15548627.2018.1437341PubMed Google Scholar). Re-expression of ATG4B in the ATG4QKO but not re-expression of ATG4A, C, or D restored p62 to levels similar to WT (Fig. 1C and Figs. S2 and S4). Thus, ATG4B is able to restore essentially normal processing of the LC3 and GABARAP families, fully support LLPD, and maintain normal p62 homeostasis, whereas expression of any other ATG4 alone can prime GABARAP protein families (to varying levels) but cannot otherwise support autophagy. Some studies have observed that in cells depleted of LC3, the GABARAP family is sufficient to drive autophagosome formation and even cargo delivery (32Nguyen T.N. Padman B.S. Usher J. Oorschot V. Ramm G. Lazarou M. Atg8 family LC3/GABARAP proteins are crucial for autophagosome–lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation.J. Cell Biol. 2016; 215 (27864321): 857-87410.1083/jcb.201607039Crossref PubMed Scopus (348) Google Scholar, 41Szalai P. Hagen L.K. Sætre F. Luhr M. Sponheim M. Øverbye A. Mills I.G. Seglen P.O. Engedal N. Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs.Exp. Cell Res. 2015; 333 (25684710): 21-3810.1016/j.yexcr.2015.02.003Crossref PubMed Scopus (54) Google Scholar). Thus, in principle, the ATG4QKO-ATG4A cell line that primes GABARAP, GL1, and GL2 and accumulates lipidated forms of all three proteins might also support macroautophagy. However, the absence of both starvation-mediated LLPD and normal p62 homeostasis indicates that instead there are unexpected detriments to autophagy. To that end, we suspected that the strong accumulation of GL1-II in cells expressing only ATG4A (Fig. S2) reflected a breakdown in the normal GL1 cycle in cells that might underlie a larger problem with autophagosome progression (Fig. 2A, cartoon). In particular, the complete absence of GL1-I could arise because the total pool of primed LC3 and GABARAP proteins is very small, and thus all free GL1-I formed from either priming or from delipidation becomes immediately relipidated. Alternatively, GL1-I depletion could indicate a general failure of ATG4A to support delipidation in these cells. To distinguish between these two possibilities, we reasoned that by blocking autophagosome biogenesis signals that generally drive lipidation, we might uncouple the two processes and measure delipidation rates. We treated cells with wortmannin, a potent phosphatidylinositol 3-kinase inhibitor that blocks autophagy initiation. In the absence of PI3P, the activation of autophagy in general and of the lipidation machinery in particular is largely inhibited. For example, although WT cells incubated in Baf A1 accumulate significant LC3-II and less but detect
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