**8. The role of** *PAH1* **in vacuolar morphology and function**

Interestingly, studies have revealed the importance of the *PAH1* gene in vacuolar homeostasis and morphology. Research has shown that cells lacking *PAH1* have morphologically defective vacuoles that remain interminably fragmented [92, 93]. While vacuoles typically undergo fragmentation in *Saccharomyces cerevisiae* during the budding process so that the new daughter cell can obtain and inherit these organelles, the vacuoles do not remain fragmented indefinitely [94]. This normal mechanism of fragmentation begins when the budding process is initiated and the vacuole in the mother cell fragments into a series of mini vacuoles. This is followed by the development of an elongated-vesicular structure by the mini vacuoles, which guides the structures to the newly budding yeast cell [94]. Once these mini fragmented vacuoles are inherited and the newly budding cell grows, the collection of vacuoles fuse together to recreate a single large vacuole in both the mother cell and daughter cell. The two cells are separated once septa are formed and this cycle can be repeated as more daughter cells are made [95]. However, cells that lack *PAH1* are unable to keep their vacuoles unfragmented and whole [92, 93]. Research studies have found that this is due to the fusion machinery having been implicated when *PAH1* is deleted and thus prevents the vacuoles from fusing back together. Without the enzymatic phosphatase activity that exists when *PAH1* is present, the SNARES are unable to bind to Sec18p, which is the protein required to prime the SNARE complex for the fusion mechanism. Furthermore, the deletion of *PAH1* also causes a number of other key fusion machinery components to be absent. This includes Vps39p, which is a component of the homotypic fusion and vacuole protein sorting (HOPS) tethering complex. Additionally, phosphatidylinositol 3-phosphate, a lipid that is needed for SNARE function and fusion, is also absent [92]. Moreover, Pah1p is also needed to recruit, Vps34p, which is the phosphatidylinositol 3-kinase needed for vacuolar fusion [92]. As such, vacuoles in cells that lack *PAH1* remain fragmented without ever fusing into a single vacuole.

Since irregularities in vacuole morphology have been linked to implications in V-ATPase pump activity, there is reason to suspect a relationship between Pah1p and V-ATPase activity given the important role that Pah1p plays in vacuolar morphology [93, 94]. On the other hand, reports have found that vacuoles with V-ATPase pumps that have a decrease in their level of acidifying the vacuole can actually lead to increased fusion of the vacuoles. One particular study found that vacuoles that experience deacidification often have an increased level of fusion whereas mutant vacuoles with internal pHs that have maintained its acidity may have fusion impeded [96]. Thus, the uncertainty as to whether the role of Pah1p and its impact on the morphological structure of vacuoles was related to proper V-ATPase pump function remained.

Given the fact that Pah1p regulates genes in the lipid biosynthetic pathway, as well as helps play a critical role in the maintenance of proper vacuole morphology and function, additional studies were aimed to look at whether Pah1p plays a role in regulating the genes that encode for the V-ATPase pump and its impact on pump activity. Growth experiments have shown that cells lines lacking *PAH1* actually grew better in neutral environments compared to wildtype. Since neutral environments can impact cytoplasmic and vacuolar pH, a properly functioning V-ATPase pump is required to ensure that pH homeostasis is maintained in the vacuole and the entire cell, indicating that *pah1∆* cells did not negatively impact V-ATPase activity even though the morphological structure is affected. This was proven with subsequent experimentation that measured vacuolar pH and actually showed that *pah1∆* cells were even better than wildtype cells at acidifying their vacuoles, with an average internal vacuolar pH of 5.89 in their vacuoles compared to pH 6.0 in wildtype. Therefore, while Pah1p does cause morphological disturbances to the vacuole it does not adversely impact V-ATPase pump activity. This however is not a contradiction since cell lines that have mutations that cause abnormalities in vacuole morphology and fragmentation can either have fully functioning V-ATPase pumps or conversely pumps that have defects in their assembly [97, 98].

Interestingly, other research studies have shown that V-ATPase function can adversely impact the fusion of vacuoles *in vivo* [96]. Therefore, it is likely that the increased acidity in the *pah1∆* cells contribute to the fragmentation of the vacuole seen in these cell lines. This led to the discovery that perhaps Pah1p plays a regulatory role over V-ATPase genes as well, since pump activity was upregulated in the *pah1∆* strain. RNA analysis experimentation showed that 11 of the 13 vacuolar membrane ATPase genes were upregulated in the V-ATPase pump, including *VMA3, VMA6 and VMA16* which are all involved in the transport of hydrogen ions into the vacuole [93]. This indicates a potential role of Pah1p acting as a repressor for these genes since they are seemingly negatively regulated in the presence of Pah1p. Many of the 11 genes that were impacted by the deletion of PAH1p contain a UASINO, which as mentioned earlier are types of genes that Pah1p negatively regulates in the lipid biosynthetic pathway. The *VMA* genes that possess a UASINO in their promoters include *VMA1, VMA5, VMA8, VMA13* and *VMA16* [93]. There is therefore a molecular relationship between *PAH1* and the genes that encode V-ATPases since it appears that Pah1p can directly negatively impact these genes by binding to their promoters.

## **9. V-ATPases and glucose metabolism**

One important process that V-ATPase pumps have been found to be associated with is glucose metabolism. In fact, research has shown that V-ATPase pump activity is actually regulated by glucose. In both yeast and mammalian cells, a key inducer of the V-ATPase pump disassembly is glucose depletion [20, 43, 99]. When glucose is scarce, the V1 and V0 domains will disassemble and pump activity is unable to occur. Conversely, an increase in glucose levels, which triggers the activation of glycolysis, will lead to the reassembly of the V-ATPase pump and lead to an increase in pump activity. This has been found to occur in both yeast cells [8, 43] and mammalian cells [99, 100]. This process is extremely significant since during times of glucose depletion the cell aims to preserve energy. By disassembling the V-ATPase pump, it prevents unnecessary usage of ATP. Furthermore, in times of glucose abundance, the reassembling of the pump allows for a functioning V-ATPase that can lower the additional acidification of the cytosol that occurs during an uptick in glycolysis [1, 2, 25]. Research has shown that this cycle of the V-ATPase pump being disassembled and reassembled is proportional to the concentration of glucose available in the cell. This is important because it indicates that V-ATPase pumps are highly attuned to glucose metabolism and energy levels [8, 43, 101, 102].

In yeast, the highly characterized glucose sensing signaling mechanism which regulates the assembly of the V-ATPase pump is the Ras/cAMP/Protein Kinase A

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

(PKA) pathway [3, 103]. The GTP coupled protein, Ras, is inhibited by two GTPase activating proteins named Ira1p and Ira2p. When glucose is present, Ira1p and Ira2p are inhibited and Ras can stimulate the production of cAMP via adenylate cyclase. Once cAMP levels are high enough, the PKA regulatory subunit will dissociate and be free to trigger PKA's kinase activity. Additionally, studies have indicated that the assembly of the V-ATPase takes place as a result of acidification of the cytosol due to high levels of glycolysis and that this leads to changes in PKA [104, 105]. The presence of glucose, after a period of depletion, will activate PKA and thereby stimulate the assembly of the V-ATPase pump. This mechanism seemingly creates a positive feedback loop, since the heightened assembly of V-ATPase pumps aids in the maintenance of the pH in the cytosol and can stimulate PKA signaling. This in turn will cause an upregulation in glycolysis which further boosts the assembly of V-ATPase pumps and helps facilitate the switch from respiratory to fermentative growth [3, 25].
