TP53INP2/DOR protein chaperones deacetylated nuclear LC3 to the cytoplasm to promote macroautophagy

LC3 (microtubule-associated protein 1 light chain 3), an essential macroautophagy (hereafter autophagy) protein, localizes in the nucleus in addition to the cytoplasm where it primarily functions. However, little is known about the role of nuclear LC3 in autophagy. In the study conducted by Huang et al., the authors characterize the molecular mechanism through which it regulates autophagosome formation. Upon nutrient deprivation, deacetylation of nuclear LC3 by the deacetylase SIRT1 promotes its association with the nuclear factor TP53INP2/DOR, resulting in the redistribution of nuclear LC3 to the cytoplasm. LC3 is then able to interact with ATG7, a step required for LC3 lipidation and subsequent autophagosome biogenesis.

The authors first confirmed that nuclear LC3 actively translocates to the cytoplasm in different cell lines under nutrient deprivation conditions.^1,2 This redistribution is dependent on intact autophagy machinery; knocking down BECN1 or ATG14 inhibits the process. Using a set of photobleaching and photoactivating experiments, through which either the nuclear or cytoplasmic pool of LC3 was highlighted, the authors demonstrated that only the nuclear LC3 can be conjugated to the phagophore membrane after starvation. Similarly, artificially retaining LC3 in the cytosol via fusing the protein with a nuclear export signal (NES) significantly inhibits the formation of LC3 puncta, suggesting failure of NES-LC3 to undergo conjugation. Moreover, electron microscopy analyses indicate that wild-type LC3, but not NES-LC3, suppress the defect in autophagosome formation observed in LC3-depleted cells. These results collectively demonstrate that nuclear LC3 is the primary source of membrane-conjugated LC3, which is indispensible for autophagosome biogenesis.

The next question the authors sought to answer was how translocation of nuclear LC3 to the cytoplasm is regulated under starvation conditions. They first observed that LC3 is deacetylated upon nutrient deprivation. Nuclear SIRT1 directly deacetylates some autophagy proteins, including LC3.² These previous findings were confirmed, and further analysis by mass spectroscopy determined that SIRT1 deacetylates LC3 at K49 and K51. An LC3^K49Q,K51Q mutant, which mimics constitutively acetylated LC3, was not able to translocate to the cytosol and be conjugated to autophagic membranes. In addition, after nutrient deprivation LC3 colocalizes with TP53INP2 in the cytoplasm. TP53INP2 functions as a scaffold, linking LC3 family proteins (including GABARAP and GABARAPL2 in addition to LC3) to VMP1;³ the recruitment of the class III PtdIns3K complex and ATG12–ATG5-ATG16L1 subsequently facilitates LC3-I lipidation.⁴ LC3 translocation to the cytosol is diminished in cells expressing a TP53INP2 mutant lacking a NES motif. Moreover, the LC3^K49Q,K51Q mutant shows weaker interaction with TP53INP2 by biochemical analysis. Taken together, these data imply that translocation of nuclear LC3 to the cytosol is dependent on deacetylation by SIRT1 and interaction with TP53INP2.

Next, the authors explored how deacetylation of nuclear LC3 promotes its conjugation to the phagophore membrane. The process of LC3 conjugation to phosphatidylethanolamine is mediated by 2 ubiquitin-like conjugation systems, and the interaction between LC3 and ATG7 is involved in this process. The authors found that knockdown of SIRT1 impairs the starvation-induced interaction between LC3 and ATG7. Furthermore, the LC3^K49Q,K51Q mutant shows significantly less binding affinity toward ATG7 than wild-type LC3. Thus, deacetylation of nuclear LC3 by SIRT1 promotes its association with ATG7 and subsequent conjugation to autophagic membranes.

By identifying the molecular mechanism governing the nucleocytosolic redistribution of LC3, this study unravels the indispensible role of nuclear LC3 and its deacetylation by SIRT1 in regulating autophagy. The temporal-spatial control of LC3 by SIRT1 provides another example of acetylation/deacetylation in auto-phagy regulation. Furthermore, the observation that interaction between Atg7 and Atg8 (the LC3 homolog in the yeast Saccharomyces cerevisiae) is also affected by acetylation of Atg8,⁵ supports the idea that this mechanism is shared among eukaryotic organisms.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by NIH grant GM053396 (to DJK).