**7. Regulation of the lipid biosynthetic pathway**

The relationship between V-ATPases and the lipid biosynthetic pathway is only recently becoming better understood. The lipid biosynthetic pathway is a highly controlled pathway that synthesizes the crucial membrane phospholipids, in addition to other various fats and lipids that are necessary for cell survival [66–68]. The transcription of genes in this pathway is tightly governed by a number of key regulators. One of these primary regulators is inositol, a crucial phospholipid precursor that plays a key role in regulating phospholipid metabolism based on its availability [69–72]. When present in the growth medium, inositol is not required to be synthesized by the cell and thus the genes involved in its production are turned off. If inositol is absent in the growth medium, then the cell will need to produce the crucial phospholipid precursor. Multiple genes in the phospholipid biosynthetic pathway have a UASINO, or an inositol responsive cis-acting element, that is located in their promoter regions [73, 74]. This region is the binding site for the Ino2p/Ino4p activator which activates the transcription of genes needed to produce inositol. Conversely, when inositol is richly present in the media, the Opi1p repressor will bind to the Ino2p/Ino4p activator and thus repress transcription from occurring [74, 75].

Another crucial regulator of the lipid biosynthetic pathway is the phosphatidate (PA) phosphatase enzyme, Pah1p. Pah1p, which is encoded by the *PAH1* gene, catalyzes the crucial reaction that dephosphorylates PA and leads to the production of diacylglycerol (DAG) and a phosphate group in the lipid biosynthetic pathway [76]. Both PA and DAG are two central players in this pathway and their levels are highly regulated, which in turn regulates the synthesis of triacylglycerides and membrane phospholipids [77]. Depending on the pathway taken, DAG can either be the precursor of the phospholipids phosphotidylcholine and phosphatidylethanolamine or can be converted into triacylglycerol (TAG). PA on the other hand can generate all

four major phospholipids that are found in the membrane of *Saccharomyces cerevisiae*. If it is not dephosphorylated and converted into DAG, then PA can be used to manufacture phosphatidylinositol and phosphotidylserine and can also be used to produce phosphotidylcholine and phosphatidylethanolamine [77].

Since PA and DAG play such critical roles in the lipid biosynthetic pathway, they are both highly regulated by the key PA phosphatase enzyme, Pah1p. While there are other PA phosphatase enzymes, Pah1p is considered to participate more directly in the synthesis of phospholipids and plays a pivotal regulatory role in the pathway [78, 79] and is essential for the *de novo* synthesis of membrane phospholipids and TAG [80, 81]. Pah1p is mainly located in the cytosol where it can be easily translocated onto the endoplasmic reticulum to catalyze the conversion of PA into DAG [82]. However, Pah1p's regulatory role in the lipid biosynthetic pathway extends even further due to the fact that it exerts transcriptional regulation over other genes involved in the manufacturing of phospholipids. As explained earlier, numerous genes in the lipid biosynthetic pathway contain a UASINO element in their promoter regions which acts as a binding site for the transcriptional activator, Ino2p/Ino4p [83, 84]. The levels of PA actually help control the transcription of UASINO containing genes, since Opi1p, which is a negative regulator of transcription, is physically connected to PA on the endoplasmic reticulum and nuclear membrane [85]. Thus, when there are greater levels of PA, the Opi1p repressor will continue to remain tethered to PA on the ER/nuclear membrane which will prevent it from being able to cross into the nucleus and prevent the transcription of UASINO containing genes [86]. Conversely, when PA levels are decreased, Opi1p is free to cross the nuclear membrane and bind to the Ino2p/Ino4p activator complex on the UASINO and block transcription. Since Pah1p regulates PA levels by catalyzing the reaction to turn PA into DAG, it indirectly impacts the transcription of other lipid biosynthetic genes. Therefore, higher concentrations of Pah1p will lead to less PA which represses transcription of genes with a UASINO, while lower concentrations of Pah1p leads to increased amounts of PA and activates transcription of genes with a UASINO [86]. However, additional research has uncovered that Pah1p actually plays a more direct role in the regulation of lipid biosynthetic genes as well. Studies have found that Pah1p is located in the nucleus as well so that it can directly act as a repressor for UASINO genes in the pathway [83]. In fact, it can directly bind to the promoter of UASINO containing genes and physically block gene expression. It is therefore not surprising that studies looking into the impact of deleting the *PAH1* gene show that there is an upregulation of gene expression of UASINO containing genes [87].

In addition to its regulatory role, deletion experiments have shown just how pivotal Pah1p is to overall cell homeostasis. Mutants that lack the *PAH1* gene have much higher levels of PA present in the cell. Additionally, these cells contain much lower levels of TAG due to the loss of the Pah1p phosphatase activity and the conversion of PA into DAG, which is the precursor of TAG. Furthermore, there is an abnormal expansion of the nuclear and endoplasmic reticulum membrane as well as increased levels of phospholipids, sterol esters and fatty acids in cell lines lacking *PAH1* [88, 89]. This is because, the gene expression of UASINO genes in the lipid biosynthetic pathway is upregulated. This is due to the lack of Pah1p which causes a derepression of genes that are typically repressed in the presence of Pah1p in the CDP-DAG pathway and Kennedy pathways, both of which lead to the synthesis of membrane phospholipids [87]. These mutant strains also experience fatty acid toxicity due to the higher levels of lipids present [89]. *PAH1* has also been shown to be needed for lipid droplet formation, which is dependent on the presence of DAG. Thus, in the absence of *PAH1* and the resulting lower concentrations of DAG, there is a decrease in the concentration of lipid droplets in these cell [90]. Furthermore,

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

cells that are missing *PAH1* are not able to grow in the presence of non-fermentable carbon and are also temperature sensitive [91]. Most notably for this review article, *PAH1* has been shown to be important for vacuole morphology and function. Given the importance of V-ATPase activity in overall cell homeostasis, the link between Pah1p and vacuole function is important to understand and will now be explored in the upcoming sections.
