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  • br Conflicts of interest br Introduction Phosphatidic


    Conflicts of interest
    Introduction Phosphatidic Alarelin Acetate australia phosphatase (PAP) enzymes are responsible for catalyzing the reaction that dephosphorylates phosphatidic acid (PA), which in turn produces diacylglycerol (DAG) and a phosphate group during phospholipid regulation (Fig. 1A) [1]. Subsequently, DAG can be used to either generate triacylglycerol (TAG), which stores energy and fatty acids in lipid droplets, or can be used to generate phosphatidylethanolamine (PE) and phosphotidylcholine (PC) via the Kennedy pathway [2]. Therefore, concentrations of PA and DAG are critical in maintaining the production of phospholipid levels, as well as determining the types of phospholipids that are synthesized [3]. Because of their role in regulating PA and DAG levels, PAP enzymes play a critical role in regulating the lipid biosynthetic pathway. Although four PAP enzymes have been identified, only PAH1 is responsible for de novo TAG and phospholipid synthesis [4], [5]. PAH1 encodes for the PA phosphatase, Pah1p, which is part of a subfamily of PA phosphatases. Pah1p's activity depends on the presence of Mg2+ and is not only found in yeast, but also in higher organisms as well [4], [6], [7], [8]. The physiological relevance of Pah1p has been studied intensively [4], [9], [10], [11], [12], [13]. Since Pah1p is necessary for catalyzing the key step of dephosphorylating PA to form DAG, the absence of Pah1p leads to elevated levels of PA. The abnormal elevation of PA not only causes the hyperproliferation of the nuclear ER membrane, but also leads to reduced amounts of DAG and TAG [5], [14], [15]. Furthermore, the reduced levels of DAG observed in pah1Δ cells hampers TAG synthesis and the ability to cope with excess exogenous fatty acids, thus inducing lipotoxicity Alarelin Acetate australia [1], [4], [10], [11]. It has been shown that Pah1p is responsible for the recruitment of the phosphatidylinositol (PI) 3-kinase, Vps34p, to the vacuoles, which then produces PI3P there (Fig. 1A) [12]. The lack of Pah1p results in the absence of Vps34p at the vacuole and thus causes the defect of vacuolar fusion, suggesting that V-ATPase pump activity is implicated in pah1Δ cells. This is because irregular vacuolar morphology has been associated with dysfunctional V-ATPase pump activity [16]. However, recent studies have shown that de-acidification of the vacuole actually induces vacuolar fusion and that mutated vacuoles that had retained their internal acidic pH might have a blockage of this fusion [17]. These results suggest that the lack of pump activity might actually favor vacuolar fusion in vivo. As such, it is uncertain whether irregular vacuolar morphology, caused by the lack of Pah1p, can be associated with the V-ATPase activity. Although Pah1p plays an important role in lipid synthesis and in vacuolar activity and morphology, it is still unclear how Pah1p regulates V-ATPase activity. Previous research has shown that PAH1 negatively regulates UAS containing genes, including INO1, INO2 and OPI3[9]. Although many V-ATPase genes are also UAS containing genes, it is still unknown whether Pah1p regulates the expression of V-ATPase genes. Here, we performed biochemical analysis to understand how Pah1p affects vacuolar morphology and its activity. We first examined the inducing conditions for maximum PAH1 expression through qRT-PCR analysis. An electron microscopy (EM) study was performed to examine whether the lack of Pah1p influences vacuolar morphology. Subsequently, a growth sensitivity assay and a vacuolar pH assay were employed to study how Pah1p affects vacuolar function. We also examined how Pah1p regulates the expression of V-ATPase genes through qRT-PCR analysis.
    Materials and methods
    Discussion In yeast, PAH1 is important for cell homeostasis and lipid biosynthesis. Here, we have demonstrated that PAH1 transcript levels are at a minimum at the exponential phase, and that even in the presence of inositol, PAH1 expression is still low at the exponential phase. However, PAH1 is greatly induced at the stationary phase and reaches the maximum expression level in the presence of 100 μM inositol compared to other inducing conditions (Fig. 1). The maximum induction of PAH1 at the stationary phase is more than two folds of other inducing conditions and four folds of repressing conditions. Therefore, the expression of PAH1 was induced as cells progressed from the exponential to the stationary phases of growth. Furthermore, the growth phase-mediated induction of PAH1 was further stimulated by inositol supplementation in stationary phase cells.