Yeast Pah1p Phosphatidate Phosphatase Is Regulated by Proteasome-mediated Degradation

Florencia Pascual,Lu-Sheng Hsieh,Aníbal Soto-Cardalda,George M Carman

doi:10.1074/jbc.m114.550103

Abstract

Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. Paradoxically, the growth phase-mediated inductions of PAH1 and phosphatidate phosphatase activity do not correlate with the amount of Pah1p; enzyme abundance declined in a growth phase-dependent manner. Pah1p from exponential phase cells was a relatively stable protein, and its abundance was not affected by incubation with an extract from stationary phase cells. Recombinant Pah1p was degraded upon incubation with the 100,000 × g pellet fraction of stationary phase cells, although the enzyme was stable when incubated with the same fraction of exponential phase cells. MG132, an inhibitor of proteasome function, prevented degradation of the recombinant enzyme. Endogenously expressed and plasmid-mediated overexpressed levels of Pah1p were more abundant in the stationary phase of cells treated with MG132. Pah1p was stabilized in mutants with impaired proteasome (rpn4Δ, blm10Δ, ump1Δ, and pre1 pre2) and ubiquitination (hrd1Δ, ubc4Δ, ubc7Δ, ubc8Δ, and doa4Δ) functions. The pre1 pre2 mutations that eliminate nearly all chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels. Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. That Pah1p abundance was stabilized in pah1Δ mutant cells expressing catalytically inactive forms of Pah1p and dgk1Δ mutant cells with induced expression of DGK1-encoded diacylglycerol kinase indicated that alteration in phosphatidate and/or diacylglycerol levels might be the signal that triggers Pah1p degradation.

Highlights

Yeast PAH1-encoded PAP2 (Pah1p) phosphatidate phosphatase is most active in stationary phase to produce diacylglycerol for triacylglycerol synthesis
We found that Pah1p is a reasonably stable protein in exponential phase cells, but it was degraded by a proteolytic activity present in stationary phase cells that was prevented by the proteasome inhibitor MG132
Pah1p Abundance Declines in a Growth Phase-dependent Manner—Recent studies have shown that PAH1 expression and Pah1p PAP activity are induced throughout growth, and this regulation is responsible for the synthesis and accumulation of TAG in the stationary phase [10]

Summary

Background

Yeast Pah1p phosphatidate phosphatase is most active in stationary phase to produce diacylglycerol for triacylglycerol synthesis. Results: Pah1p is degraded in stationary phase and stabilized by proteasome inhibitor MG132, mutations in proteasome function and ubiquitination, and elevated levels of phosphatidate. Yeast PAH1-encoded phosphatidate phosphatase is the enzyme responsible for the production of the diacylglycerol used for the synthesis of triacylglycerol that accumulates in the stationary phase of growth. The pre pre mutations that eliminate most chymotrypsin-like activity of the 20 S proteasome had the greatest stabilizing effect on enzyme levels Taken together, these results supported the conclusion that Pah1p is subject to proteasome-mediated degradation in the stationary phase. The induced expression of PAP activity in zinc-replete stationary phase cells is responsible for increased synthesis and accumulation of TAG that occurs at the expense of membrane phospholipid synthesis [10]. Pah1p abundance was stabilized in cells with elevated PA content

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION