Rebellious autophagy proteins bypass ATG8 lipidation, taking their own path to autophagic degradation

LC3/GABARAP (hereafter ATG8) conjugation machineries have long been thought to play an essential role in autophagy by driving ATG8 lipidation on autophagosomal membranes. In this issue, Ohnstad et al (2020) describe an ATG8 lipidation bypass pathway which governs autophagy‐dependent turnover of NBR1, highlighting that there is more than one road to autophagic degradation.

A new study characterises a pathway that drives autophagy‐dependent degradation of NBR1‐positive cargos in the absence of LC3/GABARAP lipidation.

Despite the importance of ATG8 lipidation systems in many forms of autophagy, there is an emerging body of evidence showing that autophagosome formation does not strictly rely on ATG8s or their conjugation machineries (Nishida et al, 2009; Nguyen et al, 2016; Tsuboyama et al, 2016; Vaites et al, 2018). In a CRISPR/Cas9‐based genetic screen utilizing a tandem fluorescent reporter (tf, RFP‐GFP) fused to autophagy receptors, it was noted that the autophagic turnover of tf‐NBR1 was largely independent of ATG8 lipidation machineries (Shoemaker et al, 2019). Following confirmation that tf‐NBR1 was indeed loaded into autophagosomes in cells lacking ATG8 conjugation, Ohnstad et al (2020) used their screening platform to identify factors required for ATG8 lipidation‐independent turnover of tf‐NBR1. The tf‐NBR1 screen was conducted using three different cell lines incapable of lipidating ATG8s: ATG7^KO, ATG3^KO, and ATG10^KO. The screen led to the identification of TAX1BP1 and TBK1 as key players, along with canonical autophagy factors (e.g., FIP200 and ATG9A) which lie outside the ATG8 lipidation axis. Knockout of TAX1BP1 and TBK1 blocked the degradation of tf‐NBR1 in lipidation‐deficient cells but had no effect on tf‐NBR1 turnover in wild‐type (WT) cells. This demonstrates that TAX1BP1 and TBK1 drive autophagic turnover of NBR1 only during lipidation‐independent autophagy. The finding opened the door to an important question: When might lipidation‐independent autophagy occur under physiological conditions? The bacterial pathogen Legionella pneumophila attacks the ATG8 lipidation system in order to subvert its turnover by autophagy. It does so via an effector protein called RavZ, which irreversibly cleaves lipidated ATG8s (Choy et al, 2012). Along this line of query, the authors asked whether tf‐NBR1 turnover can persist in cells expressing BFP‐RavZ. Indeed, TAX1BP1‐dependent turnover of tf‐NBR1 was resistant to BFP‐RavZ inhibition. Therefore, by engaging the TAX1BP1‐TBK1‐mediated lipidation bypass pathway, NBR1 turnover can be resistant to pathogens which target autophagy via ATG8 lipidation inhibition. Although the role of NBR1 and its cargo turnover during pathogen infection such as Legionella remains unclear, it is an interesting avenue to explore further to better understand host‐mediated defense. It would also be intriguing to explore NBR1 lipidation bypass under cellular stress conditions in which ATG8 lipidation‐mediated autophagy is overwhelmed, perhaps during prolonged starvation or high levels of sustained selective autophagy.

To gain a mechanistic understanding of how TAX1BP1 and TBK1 mediate the lipidation‐independent turnover of NBR1, the authors took a closer look at protein–protein interactions and autophagy factor recruitment. It was found that NBR1, TAX1BP1, and p62 form a heterotypic complex, although p62 was shown to be dispensable for NBR1 turnover. Despite being dispensable, it is likely that p62 may contribute to important cargo substrates that are degraded together with NBR1 cargoes. By making various truncation constructs, the authors also discovered that TAX1BP1 recognizes NBR1 not via ubiquitin binding (as is typical for this class of protein) but via direct binding. The second half of TAX1BP1’s coiled‐coil 2 domain, named the N‐domain (Fig 1), was found to mediate binding to NBR1 and was required for lipidation‐independent turnover of tf‐NBR1. TAX1BP1 also contains an N‐terminal SKICH domain which has been previously shown to interact with FIP200 and TBK1 (Fu et al, 2018; Ravenhill et al, 2019). Given that the SKICH domain within NDP52 was recently shown to be important for selective autophagy through FIP200 and TBK1 binding (Fu et al, 2018; Ravenhill et al, 2019; Vargas et al, 2019), the authors asked whether TAX1BP1's SKICH domain functions by binding FIP200 and TBK1 to drive tf‐NBR1 turnover. Mutation of Ala¹¹⁴ to Gln within TAX1BP1's SKICH domain abrogated TBK1 and FIP200 binding and subsequently resulted in a loss of tf‐NBR1 turnover in lipidation‐deficient cells.

Figure 1. ATG8 lipidation bypass turnover of NBR1.

In lipidation‐deficient cells, NBR1 binds to the N‐domain (located within coiled‐coil 2 (CC2) of TAX1BP1) which recruits TAX1BP1 to NBR1 foci. TAX1BP1 (SKICH domain) and TBK1 function together to recruit FIP200 and drive local autophagosome formation independently of lipidated ATG8s. p62 is also part of the NBR1‐TAX1BP1 complex but dispensable for autophagic turnover of NBR1.

Analysis of autophagy factor recruitment to tf‐NBR1 foci revealed that both TBK1 and TAX1BP1 are necessary for FIP200 recruitment. Thus, TAX1BP1 alone is not sufficient to recruit FIP200, indicating that TBK1 plays a key role in promoting or stabilizing FIP200‐TAX1BP1‐TBK1 complexes. It is also noteworthy that TAX1BP1 binding to FIP200 and TBK1 was found to be dispensable for canonical autophagy turnover of tf‐TAX1BP1, demonstrating that the lipidation‐independent activity of TAX1BP1 is distinct from canonical autophagy. Interestingly, TAX1BP1, TBK1, and FIP200 have also been reported to drive lysosomal turnover of ferritin independently of lipidated ATG8s (Goodwin et al, 2017). Therefore, it appears that TAX1BP1, TBK1, and FIP200 constitute a crucial part of the core machinery that is responsible for targeting various cargos for lysosomal degradation independently of ATG8 lipidation systems.

Putting all the pieces of the puzzle together, the authors show that TAX1BP1 directly binds NBR1 and functions together with its interaction partner TBK1 to recruit FIP200 and drive local autophagosome formation around NBR1 foci (Fig 1). Key areas to follow up would be to clarify which NBR1 cargoes are turned over by this ATG8 lipidation bypass pathway, and whether the cargo profile might change depending on cellular stress, for example, during pathogen invasion or during times of extreme autophagic stress. Whether p62 also delivers its own set of cargoes during lipidation‐independent autophagy via its association with NBR1 is also unclear. One thing is certain; this study reveals important insights into selective autophagy and the many roads to autophagosome formation, opening multiple new avenues of research inquiry along the way.

The EMBO Journal (2020) 39: e106990.