A new role of RAB21 and VARP in autophagy and autophagic exocytosis of ATP.

Autophagy has been implicated in various cellular processes, including non-conventional secretion. Our previous findings suggest that ATP is loaded into amphisomes and secreted upon autophagy stimulation at focal adhesion sites in a VAMP7-dependent manner. Here, we demonstrate that the knockout (KO) of VAMP7, along with its partners RAB21 and its guanine nucleotide exchange factor (GEF) VARP, inhibits ATP release, indicating a key role for this pathway in amphisome secretion. Constitutively inactive RAB21 also inhibited ATP secretion. RAB21 overexpression rescued starvation-induced ATP secretion in RAB21 KO, but not in VAMP7 or VARP KO cells. RAB21-LC3-positive vesicles redistributed to the cell periphery upon starvation. KO cells and overexpression experiments showed that RAB21 plays a positive role in autophagosome biogenesis, particularly in controlling the number of LC3-II- and DFCP1-positive structures upon starvation, suggesting a role in the early steps of autophagosome formation. Accordingly, VARP partially colocalized with LC3 upon starvation. Together, these findings identify a novel role for RAB21 in regulating autophagic ATP secretion likely in amphisome biogenesis and their localization in the cell periphery.