Abstract 1714 Leveraging the V0 a-isoform knockout in defining phosphoinositide-dependent regulation of mammalian V-ATPase function

V-ATPase, an ATP-dependent proton translocating pump, is responsible for organelle acidification and its regulation is multifaceted. The multi-subunit V-ATPase has a membrane-embedded V0 sub-complex, where the V0 a-subunit dictates organelle-specific distribution and assembly of V-ATPases. The V0 a-subunit has four isoforms (a1-a4) in mammals. However, the a-isoform-specific regulation of V-ATPase function is not completely understood. Here, we used individual a-isoform knockouts (KO) in mouse 4T1 breast cancer cell lines generated in the Forgac lab (McGuire, Collins et al. 2019) to unveil a-isoform-specific regulation and function of V-ATPases in lysosomes and Golgi. The a1 and a3 isoforms both localize to endolysosomal compartments in many cells. Pulse-chase endocytosis of FITC-Dextran revealed that V-ATPase-mediated lysosomal acidification still occurs both in a1KO and a3KO cell lines as well as wild-type (WT) cells. Interestingly, lysosomal fractions from a1KO 4T1 cells fail to re-establish the pH gradient in vitro, indicating a central role for a1-containing V-ATPases in lysosomal acidification. The a2 isoform has been localized to Golgi. Expression of Golgi sialyltransferase St6gal1, associated with breast cancer invasiveness and metastasis, is significantly increased in a2KO cells compared to WT. This may suggest that increasing Golgi pH can alter glycosylation patterns. We recently showed that N-terminal cytosolic domains (NT) of endolysosomal V0 a1 and Golgi V0 a2 bind specifically to organelle-enriched phosphoinositides (PIPs); potential PIP binding sites were identified in each isoform. Mutations in these sites abolish the interaction with these PIPs in vitro. Using a CRISPR-Cas9-based homology-directed repair approach, we are introducing these mutations into genomic V0 a1 and a2 of 4T1 cells. Using these mutants, we will assess the PIP-dependent regulation of a1- and a2-containing V-ATPases at their resident organelles. Altogether, the findings of our study will elucidate unresolved mechanisms of V-ATPase regulation, and provide new insight for target-based development of therapeutics to treat disorders caused by V-ATPase misregulation. McGuire, C. M., M. P. Collins, G. Sun-Wada, Y. Wada and M. Forgac (2019). "Isoform-specific gene disruptions reveal a role for the V-ATPase subunit a4 isoform in the invasiveness of 4T1-12B breast cancer cells." Journal of Biological Chemistry 294(29): 11248-11258. This research work has been supported by NIH R35GM145256