Autophagosome targeting and membrane curvature sensing by Barkor/Atg14(L)

Abstract

The class III phosphatidylinositol 3-kinase (PI3KC3) is crucial for autophagosome biogenesis. It has been long speculated to nucleate the autophagosome membrane, but the biochemical mechanism of such nucleation activity remains unsolved. We recently identified Barkor/Atg14(L) as the targeting factor for PI3KC3 to autophagosome membrane. Here, we show that we have characterized the region of Barkor/Atg14(L) required for autophagosome targeting and identified the BATS [Barkor/Atg14(L) autophagosome targeting sequence] domain at the carboxyl terminus of Barkor. Bioinformatics and mutagenesis analyses revealed that the BATS domain binds to autophagosome membrane via the hydrophobic surface of an intrinsic amphipathic alpha helix. BATS puncta overlap with Atg16 and LC3, and partially with DFCP1, in a stress-inducible manner. Ectopically expressed BATS accumulates on highly curved tubules that likely represent intermediate autophagic structures. PI3KC3 recruitment and autophagy stimulation by Barkor/Atg14(L) require the BATS domain. Furthermore, our biochemical analyses indicate that the BATS domain directly binds to the membrane, and it favors membrane composed of phosphatidylinositol 3-phosphate [PtdIns(3)P] and phosphatidylinositol 4,5-biphosphate [PtdIns(4,5)P2]. By binding preferentially to curved membranes incorporated with PtdIns(3)P but not PtdIns(4,5)P2, the BATS domain is capable of sensing membrane curvature. Thus, we propose a novel model of PI3KC3 autophagosome membrane nucleation in which its autophagosome-specific adaptor, Barkor, accumulates on highly curved PtdIns(3)P enriched autophagic membrane via its BATS domain to sense and maintain membrane curvature.omegasome | phagophore | lysosome | endocytosis | membrane trafficking

Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection

“…Similarly, Atg14L was not reported to localize to any intracellular pathogenassociated compartment, although Atg14L is required for completion of the intracellular life cycle of Brucella (6). Atg14L accumulates on highly curved PI3P-enriched autophagic membrane through its C-terminal Beclin 1-associated autophagy-related key regulator/Atg14L autophagosome targeting sequence domain (37). It was suggested that, when autophagosomes expand by membrane fusion, PI3P is dephosphorylated, which leads to the dissociation of the Atg14L-PI3KC3 complex from autophagosomes (37).…”

“…Atg14L accumulates on highly curved PI3P-enriched autophagic membrane through its C-terminal Beclin 1-associated autophagy-related key regulator/Atg14L autophagosome targeting sequence domain (37). It was suggested that, when autophagosomes expand by membrane fusion, PI3P is dephosphorylated, which leads to the dissociation of the Atg14L-PI3KC3 complex from autophagosomes (37). Furthermore, a study proposed that Atg14L and PI3KC3 dissociation from autophagosome might cause membrane curvature instability and prime the membrane for fusion with endosomes-lysosomes (37).…”

A current perspective of autophagosome biogenesis

“…Since the mutation of the cysteine repeat abrogated the autophagy-inducing ability of Atg14L, and the addition of the ER localization sequence from DFCP1 rescued the deficiency, it was concluded that the ER localization of Atg14L is crucial for autophagosome biogenesis [66]. In addition, the C-terminus of mammalian Atg14L, which is also required for its ER localization [66], has been shown to preferentially bind to liposomes containing PI3P or PI(4,5)P2 [67].…”

Differential regulatory functions of three classes of phosphatidylinositol and phosphoinositide 3-kinases in autophagy

“…142,144 Indeed, the Barkor/Atg14(L) autophagosome targeting sequence/BATS domain of ATG14 has been confirmed to be required for targeting the PIK3C3 complex to the phagophore and/or omegasome membrane and for the initiation of autophagosome formation. 145 Exogenously expressed ATG14 colocalizes with LC3, ATG12 and ATG5, indicating that ATG14 may engage in the recruitment of LC3, ATG12, and ATG5 at an early step of autophagy. 75,77,78 More interestingly, ATG14 likely mediates the differential regulation of PIK3C3 in different class III PtdIns3K complexes.…”