Mutant phosphatidate phosphatase Pah1-W637A exhibits altered phosphorylation, membrane association, and enzyme function in yeast

Yeonhee Park,Geordan J Stukey,Ruta Jog,Joanna M Kwiatek,Gil-Soo Han,George M Carman

doi:10.1016/j.jbc.2022.101578

Abstract

The Saccharomyces cerevisiae PAH1-encoded phosphatidate (PA) phosphatase, which catalyzes the dephosphorylation of PA to produce diacylglycerol, controls the bifurcation of PA into triacylglycerol synthesis and phospholipid synthesis. Pah1 is inactive in the cytosol as a phosphorylated form and becomes active on the membrane as a dephosphorylated form by the Nem1–Spo7 protein phosphatase. We show that the conserved Trp-637 residue of Pah1, located in the intrinsically disordered region, is required for normal synthesis of membrane phospholipids, sterols, triacylglycerol, and the formation of lipid droplets. Analysis of mutant Pah1-W637A showed that the tryptophan residue is involved in the phosphorylation-mediated/dephosphorylation-mediated membrane association of the enzyme and its catalytic activity. The endogenous phosphorylation of Pah1-W637A was increased at the sites of the N-terminal region but was decreased at the sites of the C-terminal region. The altered phosphorylation correlated with an increase in its membrane association. In addition, membrane-associated PA phosphatase activity in vitro was elevated in cells expressing Pah1-W637A as a result of the increased membrane association of the mutant enzyme. However, the inherent catalytic function of Pah1 was not affected by the W637A mutation. Prediction of Pah1 structure by AlphaFold shows that Trp-637 and the catalytic residues Asp-398 and Asp-400 in the haloacid dehalogenase-like domain almost lie in the same plane, suggesting that these residues are important to properly position the enzyme for substrate recognition at the membrane surface. These findings underscore the importance of Trp-637 in Pah1 regulation by phosphorylation, membrane association of the enzyme, and its function in lipid synthesis.

Highlights

The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) enzyme [1], which catalyzes the Mg2+-dependent dephosphorylation of phosphatidate (PA) to produce diacylglycerol (DAG) [2], plays a major role in governing whether cells utilize PA to synthesize the storage lipid triacylglycerol (TAG) or synthesize membrane phospholipids [1, 3, 4] (Fig. 1)
Because the function of Pah1 depends on its PAP activity, the functional loss of catalytically competent Pah1-W637A is an enigma that we addressed in this work
We considered that the loss of Pah1 function by the W637A mutation in TAG synthesis and lipid droplet formation is rooted in a defective membrane association of the mutant protein in vivo

Summary

Introduction

The Saccharomyces cerevisiae PAH1-encoded phosphatidate phosphatase (PAP) enzyme [1], which catalyzes the Mg2+-dependent dephosphorylation of phosphatidate (PA) to produce diacylglycerol (DAG) [2], plays a major role in governing whether cells utilize PA to synthesize the storage lipid triacylglycerol (TAG) or synthesize membrane phospholipids [1, 3, 4] (Fig. 1). The Nem1–Spo7 complex [38] is a sole protein phosphatase that is responsible for the recruitment and dephosphorylation of Pah1 at the nuclear/ ER membrane; this process is required for Pah1 to associate with the membrane for PAP activity [10, 19, 32,33,34, 38,39,40,41,42] (Fig. 1).

Results

Conclusion