Abstract
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.
Highlights
A fundamental question is how autophagosome formation is regulated
We propose that HS1BP3, through its binding to phosphatidic acid (PA) and inhibition of PLD1 activity, provides a novel negative-feedback mechanism to ensure the proper regulation of autophagosome biogenesis
To identify PX domain proteins involved in autophagy, we recently performed an imaging-based short interfering RNA screen in HEK GFP-LC3 cells[13] and one of the candidate proteins was HS1BP3
Summary
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. We propose that HS1BP3 regulates autophagy by modulating the PA content of the ATG16L1-positive autophagosome precursor membranes through PLD1 activity and localization. PI(3)P, the lipid product of the class III PI3K complex, has a central role in autophagy and several PI(3)P-binding proteins in autophagy have been identified[17,24], including the FYVE domain proteins DFCP1, a marker for omegasomes[6], the scaffold protein ALFY that links cargo to the autophagic machinery for selective autophagy[25,26] and FYCO1, which is involved in trafficking of autophagosomes on microtubuli[27]. We propose that HS1BP3, through its binding to PA and inhibition of PLD1 activity, provides a novel negative-feedback mechanism to ensure the proper regulation of autophagosome biogenesis
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