ATG8ylation facilitates an ESCRT-independent vacuolar membrane invagination in plants

ATG8-family proteins have been found to be incorporated into single-membrane structures, a process referred to as non-canonical autophagy. While several physiological roles of non-canonical conjugation of ATG8 (ATG8ylation) have been established, the specific functions of ATG8 on single membrane remain largely elusive. Here, we demonstrate that ionophores induce conjugation of ATG8 to vacuolar membrane to promote invagination in Arabidopsis. Upon monensin treatment, ATG8 is rapidly translocated to the vacuolar membrane, which is reliant on the ATG conjugation system rather than upstream autophagic regulators such as ATG1, ATG9, and phosphoinositide 3-kinase (PI3K). Moreover, inhibiting reactive oxygen species (ROS) generation or V-ATPase activity greatly impedes the targeting of ATG8 to the vacuolar membrane. Intriguingly, the recruitment of ATG8 to the tonoplast exhibits a pronounced enhancement of invagination and fosters the formation of intraluminal vesicles within the vacuoles. Further analyses elucidate that the topological remodeling of the vacuolar membrane is achieved in a ESCRT machinery-independent manner. Collectively, this study reveals a previously unrecognized role of ATG8ylation in driving the topological transformation of vacuolar membranes in plants.