Elucidating the Role of Lipid Binding Domains in V-ATPase a-subunits

V-ATPases are H+ pumps essential for housekeeping functions such as acidifying Golgi, lysosomes, and endosomes. V-ATPases on the plasma membrane (PM) of osteoclasts pump acid onto the surface of bone to demineralize bone, while V-ATPases on the PM of tumour cells acidify the extracellular space required for metastasis. A drug design towards inhibiting V-ATPase activity to prevent bone loss or metastasis must develop a strategy for targeting only V-ATPases on the PM of tumour cells or osteoclasts without affecting V-ATPases involved in housekeeping roles. In mammalian cells, the V-ATPase a subunit has four isoforms, a1-4, that are responsible for targeting V-ATPases to specific cellular locations. Human mutations in the four isoforms have been linked to different diseases. Among the four isoforms, a2 is found in the Golgi membrane while a3 and a4 are primarily expressed on the PM of osteoclast, and kidney cells, respectively. In invasive cancer, both a3 and a4 at the PM of tumour cells are required for tissue invasion and metastasis. Protein primary structures reveal a putative phosphoinositide (PIP) binding domains, containing a highly conserved basic motifs within all a isoforms. Using site-directed mutagenesis, I showed that the basic motifs to affect PIPs interaction and that disruption of the interaction impaired membrane targeting of the N-terminal domains of both a2 and a4 isoforms. In addition, altered levels of membrane PIPs mislocalized both proteins. Mutations in a1, a2, a3 and a4 are associated with epileptic encephalopathy, cutis laxa, osteopetrosis and renal tubular acidosis (dRTA), respectively. To understand the role of a subunit-PIPs interaction in human diseases, disease-causing mutations occurring within the putative binding domains, specifically: a2.K237_V238del (cutis laxa), and a4.K237del (dRTA) demonstrated defective PIP binding in vitro and impaired membrane retention/targeting in HEK293 cells. This data suggests that the a2-PI(4)P interaction is required for Golgi retention/targeting, and a4¬-PI(4,5)P2 interaction is required plasma membrane retention/targeting. This work enhances our understanding of a subunit functional domains, and informs potential targeted therapeutic treatment for metastatic cancer and bone loss pathologies.

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