Abstract
Saccharomyces cerevisiae Pah1 phosphatidate phosphatase, which catalyzes the conversion of phosphatidate to diacylglycerol for triacylglycerol synthesis and simultaneously controls phosphatidate levels for phospholipid synthesis, is subject to the proteasome-mediated degradation in the stationary phase of growth. In this study, we examined the mechanism for its degradation using purified Pah1 and isolated proteasomes. Pah1 expressed in S. cerevisiae or Escherichia coli was not degraded by the 26S proteasome, but by its catalytic 20S core particle, indicating that its degradation is ubiquitin-independent. The degradation of Pah1 by the 20S proteasome was dependent on time and proteasome concentration at the pH optimum of 7.0. The 20S proteasomal degradation was conserved for human lipin 1 phosphatidate phosphatase. The degradation analysis using Pah1 truncations and its fusion with GFP indicated that proteolysis initiates at the N- and C-terminal unfolded regions. The folded region of Pah1, in particular the haloacid dehalogenase-like domain containing the DIDGT catalytic sequence, was resistant to the proteasomal degradation. The structural change of Pah1, as reflected by electrophoretic mobility shift, occurs through its phosphorylation by Pho85-Pho80, and the phosphorylation sites are located within its N- and C-terminal unfolded regions. Phosphorylation of Pah1 by Pho85-Pho80 inhibited its degradation, extending its half-life by ∼2-fold. The dephosphorylation of endogenously phosphorylated Pah1 by the Nem1-Spo7 protein phosphatase, which is highly specific for the sites phosphorylated by Pho85-Pho80, stimulated the 20S proteasomal degradation and reduced its half-life by 2.6-fold. These results indicate that the proteolysis of Pah1 by the 20S proteasome is controlled by its phosphorylation state.
Highlights
Yeast Pah1 phosphatidate phosphatase required for triacylglycerol synthesis is subject to proteasome-mediated degradation
By exploring the proteasomal regulation, we show here that Pah1 is degraded by the 20S proteasome in a ubiquitin-independent manner and that the proteasomal degradation is governed by its phosphorylation state
Yeast Pah1 and Human Lipin 1 Are Degraded by the 20S Proteasome but Not by the 26S Proteasome—Our previous work has shown that Pah1 expressed in E. coli, which is not subject to ubiquitination or phosphorylation, is degraded by MG132-inhibitable proteolytic activity in the 100,000 ϫ g pellet fraction of stationary phase cells [45]
Summary
Yeast Pah phosphatidate phosphatase required for triacylglycerol synthesis is subject to proteasome-mediated degradation. Pah PAP2 [1], the Saccharomyces cerevisiae ortholog of the mammalian lipin 1, 2, and 3 PAP enzymes [2, 3], catalyzes the Mg2ϩ-dependent dephosphorylation of PA to form DAG and Pi [4] (Fig. 1) It has emerged as one of the most highly regulated enzymes that controls lipid synthesis in yeast [5,6,7]. Elevated PA content causes the derepression of phospholipid synthesis genes (e.g. INO1 and OPI3) and the aberrant expansion of the nuclear/ER membrane, whereas reduced DAG and TAG contents cause the susceptibility to fatty acid-induced toxicity and defects in lipid droplet formation [1, 12, 17, 19, 20] Some of these phenotypes require expression of Dgk DAG kinase [12, 20, 21], the enzyme that converts DAG back to PA (Fig. 1A). By exploring the proteasomal regulation, we show here that Pah is degraded by the 20S proteasome in a ubiquitin-independent manner and that the proteasomal degradation is governed by its phosphorylation state
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