Summary Vesicular- or vacuolar-type adenosine triphosphatases (V-ATPases) are multi-component, ATP-driven proton pumps consisting of a V1 complex that possesses ATPase activity and a Vo complex for proton transfer across the membrane. V-ATPases play important roles in the acidification of intracellular vesicles, organelles, and the extracellular milieu, and are essential for maintaining the pH homeostasis of endosomes, lysosomes, and the Golgi apparatus in all eukaryotic cells. V-ATPase deficiency in mammals is embryonic lethal, and malfunction of V-ATPases is associated with numerous diseases, including microbial infection, renal tubular acidosis, osteoporosis, sensorineural deafness, neurodegenerative diseases, and cancer. Despite the critical functions of V-ATPases, we have limited understanding on the biogenesis, assembly, regulation, and signaling of mammalian V-ATPases. A major challenge in studying mammalian V-ATPases is that the pure complexes are difficult to obtain for biochemical and biophysical experiments. We developed an innovative method to purify large amounts of human V-ATPase to homogeneity directly from cells. Our preliminary cryo-electron microscopy (cryo-EM) structures of human V-ATPases show three functional states at up to 3.1 Å resolution and with all known subunits, which together represent the most complete mechanistic model of V-ATPase to date. Our study revealed that mammalian V-ATPases are composed of proteins, glycans, glycolipids, and lipids. Therefore, we defined the V- ATPase as a glycoproteolipid complex. Our study opened up the field for comprehensively understanding the biogenesis, assembly, regulation, and signaling of V-ATPases. Based on our prior work, we will complement cryo-EM structure determination with biochemical and functional assays, yeast genetics, and mass spectrometry analysis to address fundamental questions in the field, including the roles of glycolipids in the V-ATPase assembly and function, the regulation of V-ATPases by reversible assembly, the detailed mechanism of proton transfer, and the mechanisms of V-ATPase mediated cell signaling. The completion of this project will not only provide conceptual innovations regarding the V-ATPases assembly, regulation, and signaling, but also inspire new therapeutic strategies for treating V-ATPase-related diseases.