Structure‐function Analysis of Yeast Pah1 Phosphatidate Phosphatase

Abstract

Mg2+‐dependent phosphatidate phosphatase (PAP), encoded by PAH1 in Saccharomyces cerevisiae, catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol. The diacylglycerol produced in the reaction is utilized for the synthesis of triacylglycerol or the major membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. The PAP enzyme from yeast contains two conserved domains; the NLIP domain is found at the N‐terminus and the haloacid dehalogenase (HAD)‐like domain is found in the middle region of Pah1. The G80R mutation in the NLIP domain of Pah1 causes a 98% reduction in PAP activity along with other phenotypes characteristic of the pah1Δ mutation. The HAD‐like domain of Pah1 contains the DXDX (T/V) motif that is essential for catalytic activity. In this study, we carried out a structure‐function analysis of the conserved and non‐conserved regions of yeast Pah1. For constructs expressed and purified from Escherichia coli, G80R and G80A mutations, as well as deletion of the NLIP domain caused > 99% loss of PAP activity; the deletion of the non‐conserved region at the N‐terminus had little effect on PAP activity; and the deletion of the non‐conserved region at the C‐terminus caused a 3‐fold increase in PAP activity. The effects of the mutations on the in vivo function of Pah1 were examined by the ability to complement cold or warm sensitivities exhibited by the pah1Δ mutant. Cells expressing the deletion of the non‐conserved region at the N‐terminus complemented both the cold and warm sensitive phenotypes of the pah1Δ mutant, whereas the mutations in the conserved NLIP domain and the deletion of the non‐conserved region at the C‐terminus did not rescue the temperature sensitive phenotypes. The effects of the mutations on Pah1 PAP functions such as the synthesis of triacylglycerol and lipid droplet formation are being investigated.Support or Funding InformationSupported by NIH grant GM028140