**2. V-ATPase pump function**

V-ATPase pumps have been shown to be crucial for proper cell function and survival. In fact, research experiments involving the deletion of genes that cause the ubiquitous loss of V-ATPase subunits show that it is lethal in almost all organisms, including *Drosophila* fruit flies and mice [10, 11]. Unlike most organisms however, yeast cells are still viable when V-ATPase subunit encoding genes are deleted. As a result, *Saccharomyces cerevisiae* has been an ideal model organism for studying V-ATPase function [12].

Deletion mutation studies in *S. cerevisiae* have shown that the vacuolar acidification process is impaired when genes that encode V-ATPase subunit are removed. Furthermore, growth phenotypes have indicated that cells with V-ATPase subunit deletions are sensitive to alkaline extracellular pH. While these mutants can grow in an extracellular environment of pH 5, they are unable to grow at pH 7.5. They are also sensitive to high levels of calcium in their growth medium, as well as when they are in the presence of heavy metals and oxidants. They also cannot grow on nonfermentable carbon sources [4, 12].

While V-ATPases are necessary for the vacuoles to maintain their internal acidic pH, they actually play a much more widespread role in the homeostasis of the cell by regulating cytoplasmic pH. Research has shown that cells with defective V-ATPase pumps are not only unable to maintain the acidity in their vacuoles but cannot uphold the necessary pH of the cell's cytoplasm [5, 13]. This is due to the fact that other pumps are dependent on V-ATPase activity which utilizes ATP to transport hydrogen ions into the vacuole. For instance, the Ca+2/H+ antiporter requires a

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

functional V-ATPase to transport calcium into the vacuole. If the V-ATPase is defective, calcium will enter the vacuole at a much slower rate and thus these mutants cannot grow well in presence of excess calcium as mentioned above [14–16].

Additionally, the vacuole plays a large role in detoxifying the cell of heavy metals via antiporters in the vacuole membrane. These pumps are also dependent on functional ATPases and rely on their activity to function properly. Cells with nonfunctional V-ATPases are unable to thrive in the presence of heavy metals, such as cadmium which causes oxidative stress in the cell [17, 18]. Furthermore, V-ATPases play an important part in promoting hydrolytic enzymes that degrade and recycle biomolecules [9, 19]. When a cell undergoes nitrogen starvation, nearly 4/5ths of the cell's protein degradation process takes place in the vacuole. Additionally, the vacuoles recycle organelles as well, which also helps to supply the cells with an abundance of amino acids and other biomolecule building blocks that can help the cell survive during times of stress [20–22]. V-ATPases therefore play a critical role in protecting the cell when exposed to various adverse environmental conditions and stress.

## **3. Structural composition of the vacuolar V-ATPase pump**

Given the importance of V-ATPase pumps in maintaining intravacuolar acidity, the structural components of the pump have been well characterized in *Saccharomyces cerevisiae*. V-ATPase pumps are comprised of two distinctive domains that include the V1 domain and the V0 domain. The V1 domain is the catalytic component in the pump that is linked to the ATP binding sites and is located on the cytosolic part of the membrane. The V0 domain is the proton translocating component and consists of integral membrane proteins as well as peripheral ones. Both the V1 and V0 domains are linked together by a stalk-like structure within the V1 domain [13, 23]. These domains each consist of multiple subunits that come together to form the overall pump. V1 contains eight subunits, labeled A through H (A, B, C, D, E, F, G, and H) while the V0 domain contains 6 subunits labeled a, c, c', c", d and e (**Figure 1**).

The 14 subunits of the V-ATPase pump are each encoded by their own gene, except for subunit a of the V0 domain which has two isoforms. 13 of these subunits are encoded by vacuolar membrane ATPase (VMA) genes [4]. These genes include *VMA1, VMA2, VMA3, VMA4, VMA5, VMA6, VMA7, VMA8, VMA9, VMA10, VMA11, VMA13, VMA16* (**Table 1**)*.* The final subunit in the vacuolar V-ATPase is encoded by a gene named *VPH1* and encodes for the largest subunit in the pump, subunit a [15]. However, this subunit has two isoforms, with the second isoform being encoded by *STV1*. The Stv1p isoform is not located in the vacuolar membrane but is instead found in the V-ATPases located in the Golgi complex and endosome membranes. Research has shown that vacuolar V-ATPases that contain the Vph1p subunit are better at coupling the hydrolysis of ATP to the transport of hydrogen ions across the membrane. They also have increased assembly levels of the V-ATPase pump, and are more responsive to extracellular glucose levels compared to V-ATPases that contain Stv1p [4, 24]. For the purposes of this review, we will be focusing on vacuolar V-ATPases and will be looking at the Vph1p subunit.

Thus, eight of the V-ATPase subunits in the vacuole are found in the V1 domain and are encoded by *VMA1, VMA2, VMA4, VMA5, VMA7, VMA8, VMA10, VMA13.* The remaining six subunits are encoded by *VPH1, VMA3, VMA6, VMA9, VMA11*, and *VMA16* and are found in the V0 domain. A table outlining the various genes of the V-ATPase pump and the subunits encoded by them has been included (**Table 1**).

#### **Figure 1.**

*Model of the* Saccharomyces cerevisiae *vacuolar V-ATPase. The V1 domain contains subunits A, B, C, D, E, F, G, and H. Subunits A and B each have three copies (not all depicted) and alternate in an A, B, A, B, A, B fashion in the pump. The V0 domain consists of subunits a, c, c', c", d, and e.*


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


**Table 1.**

*The genes of the* Saccharomyces cerevisiae *vacuolar V-ATPase and their corresponding subunits.*
