Einar Trosdal

The mechanisms governing mammalian proton pump V-ATPase function are of fundamental and medical interest. The assembly and disassembly of cytoplasmic V1 domain with the membrane-embedded V0 domain of V-ATPase is a key aspect of V-ATPase localization and function. Here, we show that the mammalian protein ATG16L1, primarily appreciated for its role in canonical autophagy and in noncanonical membrane atg8ylation processes, controls V-ATPase. ATG16L1 knockout elevated V-ATPase activity, increased V1 presence on endomembranes, and increased the number of acidified intracellular compartments. ATG16L1’s ability to efficiently bind V-ATPase was required for its inhibitory role in endolysosomal acidification and for control of Mycobacterium tuberculosis infection in mice. These findings uncover a hitherto unappreciated role of ATG16L1 in regulating V-ATPase, a key pump governing acidification and functionality of the endolysosomal system along with its physiological roles.

Canonical autophagy is an intracellular pathway that degrades and recycles cellular components. A key step of this pathway is the formation of double-membraned organelles, known as autophagosomes, an emblematic feature of macroautophagy. For convenience, the formation of autophagosomes can be categorized into sequential steps, initiation (X), expansion (Y) and closure (Z). ATG9A is an integral membrane protein known for its role in the X and Y steps. whereby it organizes phagophore membrane assembly and its growth. Here, we report a previously unappreciated function of mammalian ATG9A in directing the last step Z. In particular, ATG9A partners with the key ESCRT-III component CHMP2A through IQGAP1 to facilitate autophagosome closure. Thus, ATG9A orchestrates all stages of autophagosome membrane biogenesis, from phagophore initiation to its closure. This makes ATG9A a unique ATG factor that works as a central hub in autophagosome biogenesis.

Canonical autophagy captures within specialized double-membrane organelles, termed autophagosomes, an array of cytoplasmic components destined for lysosomal degradation. An autophagosome is completed when the growing phagophore undergoes ESCRT-dependent membrane closure, a prerequisite for its subsequent fusion with endolysosomal organelles and degradation of the sequestered cargo. ATG9A, a key integral membrane protein of the autophagy pathway, is best known for its role in the formation and expansion of phagophores. Here, we report a hitherto unappreciated function of mammalian ATG9A in directing autophagosome closure. ATG9A partners with IQGAP1 and key ESCRT-III component CHMP2A to facilitate this final stage in autophagosome formation. Thus, ATG9A is a central hub governing all major aspects of autophagosome membrane biogenesis, from phagophore formation to its closure, and is a unique ATG factor with progressive functionalities affecting the physiological outputs of autophagy.

ATG5 plays a pivotal role in membrane Atg8ylation, influencing downstream processes encompassing canonical autophagy and noncanonical processes. Remarkably, genetic ablation of ATG5 in myeloid cells leads to an exacerbated pathological state in murine models of tuberculosis, characterized by an early surge in mortality much more severe when compared to the depletion of other components involved in Atg8ylation or canonical autophagy. This study shows that in the absence of ATG5, but not other core canonical autophagy factors, endolysosomal organelles display a lysosomal hypersensitivity phenotype when subjected to damage. This is in part due to a compromised recruitment of ESCRT proteins to lysosomes in need of repair. Mechanistically, in the absence of ATG5, the ESCRT protein PDCD6IP/ALIX is sequestered by the alternative conjugate ATG12-ATG3, contributing to excessive exocytic processes while not being available for lysosomal repair. Specifically, this condition increases secretion of extracellular vesicles and particles, and leads to excessive degranulation in neutrophils. Our findings uncover unique functions of ATG5 outside of the autophagy and Atg8ylation paradigm. This finding is of in vivo relevance for tuberculosis pathogenesis as modeled in mice.