Rutgers University New Brunswick, New Jersey, United States
Geordan Stukey (Rutgers University)| Yeonhee Park (Rutgers University)| Gil-Soo Han (Rutgers University)| George Carman (Rutgers University)
Pah1 in Saccharomyces cerevisiae is an Mg2+-dependent phosphatidic acid (PA) phosphatase that plays a major role in lipid synthesis by controlling the levels of triacylglycerol and membrane phospholipids. The enzyme contains two conserved domains (N-lip and HAD-like) required for catalytic activity, and non-conserved regions containing multiple phosphorylation sites involved in the regulation of subcellular localization, catalytic activity, and protein stability. In a previous study, we found that Trp-637 in a putative WW-binding domain and destruction box is essential for the function of Pah1 in vivo, but not for its catalytic activity in vitro. In this work, we examined the Pah1(W637A) for its localization, abundance, and phosphorylation state. Subcellular fractionation analysis showed that the membrane translocation of Pah1(W637A), like that of the WT enzyme, depends on the Nem1-Spo7 phosphatase function. The mutant enzyme showed a higher level of membrane association than the WT enzyme, but a significantly lower cytosolic level. The phosphorylation status of Pah1(W637A), as determined by electrophoretic mobility, showed that the mutant enzyme is less phosphorylated in vivo when compared with the WT enzyme. The Nem1-Spo7-dependent membrane translocation of Pah1(W637A) was also shown in vitro by fractionation of cytosolic Pah1 incubated with the membrane containing the phosphatase complex. These results suggest that Trp-637 is not required for the translocation of Pah1, but for its catalytic activity on PA in the cellular membrane.