Fluorescence spectroscopy measures yeast PAH1-encoded phosphatidate phosphatase interaction with liposome membranes

Zhi Xu,George M. Carman,Wen-Min Su

doi:10.1194/jlr.m022798

Zhi Xu, George M. Carman + Show 1 more

Open Access

https://doi.org/10.1194/jlr.m022798

Copy DOI

Abstract

Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. Fluorescence spectroscopy was used to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes. PA phosphatase contains five tryptophan residues and exhibited inherit fluorescence that increased upon interaction with phosphatidylcholine liposomes. The interaction was enhanced by inclusion of other phospholipids and especially the substrate PA. Interaction was dependent on both the concentration of phosphatidylcholine-PA liposomes as well as the surface concentration of PA in liposomes. Mg(2+) ions, which were required for catalysis, did not affect PA phosphatase interaction with phosphatidylcholine-PA liposomes. PA phosphatase was a substrate for protein kinase A, protein kinase C, and casein kinase II, and these phosphorylations decreased PA phosphatase interaction with phosphatidylcholine-PA liposome membranes.

Highlights

Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides
Tryptophan fluorescence of PA phosphatase increases with liposome interaction We sought a convenient assay to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes
At the highest concentration (0.4 mM) of phospholipid used in this experiment, about 50% of the PA phosphatase was associated with liposome membranes

Summary

Introduction

Phosphatidate (PA) phosphatase, the enzyme that catalyzes the penultimate step in triacylglycerol synthesis, is a cytosolic enzyme that must associate with the membrane where its substrate PA resides. Fluorescence spectroscopy was used to measure the interaction of yeast PAH1-encoded PA phosphatase with model liposome membranes. Fluorescence spectroscopy measures yeast PAH1-encoded phosphatidate phosphatase interaction with liposome membranes. PA phosphatase plays a major role in controlling the cellular content of its substrate PA [3], the precursor of phospholipids that are synthesized via the liponucleotide intermediate CDP-DAG [1, 8]. The elevated PA content causes the induction of phospholipid synthesis gene expression and the aberrant expansion of the nuclear/ER membrane [3, 7, 10], whereas the reduced capacity to synthesize DAG and TAG causes a defect in lipid droplet formation [11] and an acute sensitivity to fatty acid-induced toxicity [12], respectively. Mutations in mammalian lipins (counterpart of yeast PAH1-encoded PA phosphatase) cause defects in lipid metabolism and cell physiology.

Methods

Results

Conclusion