Abstract
The acetylation of autophagy protein 9 A (ATG9A) in the lumen of the endoplasmic reticulum (ER) by ATase1 and ATase2 regulates the induction of reticulophagy. Analysis of the ER-specific ATG9A interactome identified calreticulin (CALR), an ER luminal Ca+2-binding chaperone, as key for ATG9A activity. Specifically, if acetylated, ATG9A is sequestered by CALR and prevented from engaging FAM134B and SEC62. Under this condition, ATG9A is unable to activate the autophagy core machinery. In contrast, when non-acetylated, ATG9A is released by CALR and able to engage FAM134B and SEC62. In this study, we report that Ca+2 dynamics across the ER membrane regulate the ATG9A-CALR interaction as well as the ability of ATG9A to trigger reticulophagy. We show that the Ca+2-binding sites situated on the C-domain of CALR are essential for the ATG9A-CALR interaction. Finally, we show that K359 and K363 on ATG9A can influence the ATG9A-CALR interaction. Collectively, our results disclose a previously unidentified aspect of the complex mechanisms that regulate ATG9A activity. They also offer a possible area of intersection between Ca+2 metabolism, acetyl-CoA metabolism, and ER proteostasis.