SNX18 promotes autophagosome formation by remodeling membranes and providing membrane to forming autophagosomes.
Trafficking of mammalian ATG9A between the Golgi apparatus, endosomes and peripheral ATG9A compartments is important for autophagosome biogenesis. Here, we show that the membrane remodelling protein SNX18, previously identified as a positive regulator of autophagy, regulates ATG9A trafficking from recycling endosomes. ATG9A is recruited to SNX18‐induced tubules generated from recycling endosomes and accumulates in juxtanuclear recycling endosomes in cells lacking SNX18. Binding of SNX18 to Dynamin‐2 is important for ATG9A trafficking from recycling endosomes and for formation of ATG16L1‐ and WIPI2‐positive autophagosome precursor membranes. We propose a model where upon autophagy induction, SNX18 recruits Dynamin‐2 to induce budding of ATG9A and ATG16L1 containing membranes from recycling endosomes that traffic to sites of autophagosome formation.
A fundamental question is how autophagosome formation is regulated. Here we show that the PX domain protein HS1BP3 is a negative regulator of autophagosome formation. HS1BP3 depletion increased the formation of LC3-positive autophagosomes and degradation of cargo both in human cell culture and in zebrafish. HS1BP3 is localized to ATG16L1- and ATG9-positive autophagosome precursors and we show that HS1BP3 binds phosphatidic acid (PA) through its PX domain. Furthermore, we find the total PA content of cells to be significantly upregulated in the absence of HS1BP3, as a result of increased activity of the PA-producing enzyme phospholipase D (PLD) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. Our findings provide key insights into how autophagosome formation is regulated by a novel negative-feedback mechanism on membrane lipids.
Macroautophagy/autophagy is a membrane trafficking and intracellular degradation process involving the formation of double-membrane autophagosomes and their ultimate fusion with lysosomes. Much is yet to be learned about the regulation of this process, especially at the level of the membranes and lipids involved. We have recently found that the PX domain protein HS1BP3 (HCLS1 binding protein 3) is a negative regulator of autophagosome formation. HS1BP3 depletion increases the formation of LC3-positive autophagosomes both in human cells and zebrafish. HS1BP3 localizes to ATG16L1-and ATG9-positive autophagosome precursors deriving from recycling endosomes, which appear to fuse with LC3-positive phagophores. The HS1BP3 PX domain interacts with phosphatidic acid (PA) and 3'-phosphorylated phosphoinositides. When HS1BP3 is depleted, the total cellular PA content is upregulated stemming from increased activity of the PA-producing enzyme PLD (phospholipase D) and increased localization of PLD1 to ATG16L1-positive membranes. We propose that HS1BP3 negatively regulates autophagy by decreasing the PA content of the ATG16L1-positive autophagosome precursor membranes through inhibition of PLD1 activity and localization. Several aspects of the role of specific lipids in autophagy are poorly understood, such as the lipid composition of the autophagosomal membrane and the involvement of lipid-binding proteins and lipid-modifying enzymes in altering the properties of forming autophagosomes. To approach some of these questions we have focused on PX domain-containing proteins, which are known to bind phosphoinositides (PIs), but little is known about their function in autophagy.To search for PX proteins in autophagy, we performed an imaging-based siRNA screen, where HS1BP3 was identified as a putative negative regulator of autophagy. Knockdown of HS1BP3 increases LC3 puncta formation and LC3 lipidation, both under basal and starved conditions. Autophagic flux assays indicate that HS1BP3 functions during autophagosome formation. Accordingly, depletion of HS1BP3 increases the degradation of the autophagic cargo SQSTM1/p62 and longlived proteins. For in vivo analysis, HS1BP3 was silenced in transgenic GFP-LC3 zebrafish and as observed in cells, the number of GFP-LC3 puncta increases in embryos lacking HS1BP3. This increase was partly rescued by co-injecting human HS1BP3-mRNA, indicating that HS1BP3 negatively regulates autophagy also in zebrafish.HS1BP3 is generally cytosolic, but also enriched on structures containing autophagy proteins ATG9 and ATG16L1 as well as recycling endosome markers TF (transferrin) and TFRC (transferrin receptor) (Fig. 1). Live-cell imaging revealed that these structures fuse with LC3-positive vesicles, indicating that they represent recycling endosome-derived membranes providing input to forming autophagosomes. This is in line with recent findings showing that ATG9-and ATG16L1-positive vesicles traffic via the plasma membrane through recycling endosomes and that such trafficking is important for a...
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