**4. V-ATPase subunit functions**

The overall molecular mechanism of V-ATPase pump activity involves the hydrolysis of ATP by the V1 domain. This step provides the necessary energy needed to pump protons across the membrane and create a pH gradient that is needed for secondary transport systems [3, 25]. However, the specific functions of each of the subunits of the V1 and V0 domains are varied. The first two subunits of the V1 domain, subunit A and subunit B, of which there are three copies of each, are encoded by *VMA1* and *VMA2* respectively. They are the ATP binding subunits that hydrolyze ATP. Additionally, both aid in the regulation of proton transport and in the disassembly of the V1 and V0 domains [26, 27]. Subunit C, which is encoded by the *VMA5* gene, is needed to help activate the pump by regulating the assembly of the V1 domain to the V0 domain. Furthermore, it plays a key role in the disassembly of the pump since it dissociates from the enzyme during glucose starvation and separates the two domains, rendering the pump inactive [28, 29]. Subunit D is encoded by *VMA8* and is also crucial for the assembly of the two domains which is needed for pump activity and proton transport [30]. Subunit E, encoded by the *VMA4* gene, helps to form the peripheral structural stalk of the pump which is needed for proper assembly and function of the pump, while subunit F which is encoded by *VMA7* is a rotor subunit and needed for proper pump assembly [31–34]. Subunit G is encoded by *VMA10* and needed for proper stalk formation, while subunit H, which is encoded by *VMA13*, is required to inhibit ATP hydrolysis when V1 and V0 domains are dissociated and does so by interacting with subunit F [32, 35].

The subunits of the V0 domain are the remaining components of the V-ATPase pump that are primarily integral proteins, with the exception of subunit d which is a peripheral protein. Subunit a is encoded by the *VPH1* gene which are specific for V-ATPases localized in the vacuolar membrane. It is needed for the appropriate assembly of the pump as well as for the transport of hydrogen ions [24, 36]. Subunits c, c' and c", which are encoded by *VMA3, VMA11,* and *VMA13* respectively, are suggested to be needed for proton translocation. All three subunits are crucial for V-ATPase function since a mutation in any of them will impair pump activity [37, 38]. Subunit d, which is encoded by *VMA6*, is the only peripheral V0 subunit and plays a key role in coupling ATP hydrolysis with proton transport [39]. Lastly, it is also important to note that the e subunit in the V0 domain, which is encoded by *VMA9*, was discovered much later than the rest of the subunits and as such has not been as well characterized [12]. Furthermore, *in vitro* studies have suggested that Vma9p is not needed for V-ATPase proton pumping activity since removal of Vma9p does not impact proton transport [40].

Importantly, the deletion of any one specific subunit in the V1 domain does not impact the stability of the remaining V1 subunits in the complex. However, it will impair the association and assembly of the entire V1 domain with the V0 domain, thus rendering the pump nonfunctional. Additionally, the deletion of any specific V1 subunit will not impact the stability of the V0 domain [13, 41]. Likewise, the loss of any one specific V0 subunit will not impact the stability of the remaining V0 subunits nor will it impact the assembly of the independent V1 domain. It will however impact the assembly of the V0 domain which is unable to produce a functional V-ATPase [13, 41]. It is important to note that if any of the thirteen genes that encode the V-ATPase pump are implicated (not including *VMA9*), the pump will not be able to work properly. Thus, if there is a mutation in any single subunit from either domain, the cell will be unable to grow in media whose pH is neutral or basic [42].

## **5. V-ATPase mechanism of disassembly and assembly**

The disassembly of the V-ATPase is a crucial step required for regulating the activity of the pump. This mechanism involves the separation of the V1 and V0 domains and will ultimately inhibit the V-ATPase pump's function *in vivo*. When undergoing disassembly, the C subunit located in the V1 domain, which acts as a bridge between the V1 and V0 domains, will depart from the complex and cause the separation of the two domains [43, 44]. Subsequently, a conformational change in the H subunit inhibits ATP hydrolysis from occurring in the newly separated V1 complex. This occurs so that energy will not be unnecessarily used without the concurrent transportation of protons across the membrane [45–48]. Furthermore, once the domains have disassembled, the passive transport of hydrogen ions across the V0 complex is also prevented [49].

This mechanism is completely reversible however once the C subunit is brought back and re-bridges the V1 and V0 domains [43]. In *Saccharomyces cerevisiae*, re-assembling the V-ATPase pump utilizes a chaperone protein that is specific for V-ATPase pumps [50]. Specifically, this process requires RAVE, or the regulator of ATPases of Vacuoles and Endosome [3]. This is a chaperone complex that includes an adaptor protein, Skp1p and the functional subunits Rav1p and Rav2p [50, 51]. This RAVE chaperone complex helps to stabilize the V1 domain and facilitates in the process of reintroducing the C subunit to bridge V1 to V0 [52, 53]. Once completed, the V-ATPase pump will be fully functional once again.

### **6. Importance of V-ATPase activity in mammalian cells**

The study of vacuolar function in *Saccharomyces cerevisiae*, and particularly the activity of the V-ATPase pump is important since yeast vacuoles are strikingly similar to the lysosomes in mammalian cells [52. 53]. The mammalian lysosome has an internal acidic pH that ranges from 4.2–5.3 and is primarily maintained by V-ATPase pumps that actively transport ions across the membrane to acidify the lysosome [54]. Similar to the acidic pH needed in the vacuole, the maintenance of lysosomal pH is required for the important hydrolytic activities and signaling roles of lysosomal homeostasis. Defects in V-ATPase machineries and functions have been associated with a number of neurodegenerative diseases, especially disorders associated with older age including some forms of Parkinson's disease and Alzheimer's disease [55–57]. In fact, the brain is one of the main organs that is the most significantly impacted by genetic diseases that interrupt normal lysosomal

#### *The Interplay of Key Phospholipid Biosynthetic Enzymes and the Yeast V-ATPase Pump… DOI: http://dx.doi.org/10.5772/intechopen.97886*

function in cells. This underlies the importance of normal lysosome function in the central nervous system [54, 58]. Additionally, impairments to proper acidification in lysosomes have been involved with cell aging and longevity. Studies conducted in yeast models have shown that longer lifespans occurred when V-ATPase components were overexpressed [59, 60]. Additionally, there have been over fifty genetic disorders that have been linked to mutated genes that encode specific proteins of the lysosome, many of which are related to the acidification function [61–63].

V-ATPases have been found in the cellular membranes of certain specialized cells in mammals. Implications in these V-ATPases cause an array of genetic disorders. For example, V-ATPases that are found in intercalated cells of the distal tubule and the collecting ducts of the kidney play a crucial role in maintaining acid–base homoeostasis. In humans, defects in the specific genes that encode V-ATPase subunits, such as a mutation in the renal isoform of subunit B or in subunit a, leads to the inherited disease of renal tubule acidosis [64, 65]. Furthermore, V-ATPase pumps have also been found to be located in the cellular membrane of osteoclast cells and are required for the process of bone resorption. As a result, genetic mutations in V-ATPase encoding genes, such as in subunit a, has been associated with osteopetrosis, which causes skeletal abnormalities caused by lack of bone degradation [64, 65]. Furthermore, V-ATPase activity has been linked to nongenetic diseases, such as cancer. The key role that these pumps play in tumor and cancer cell lines will be discussed further in a later section. Given the importance of V-ATPases with regard to diseases, research in yeast vacuoles has been crucial in helping better understand the role that these pumps play in cellular homeostasis.
