Phosphorylation Regulates the Ubiquitin-independent Degradation of Yeast Pah1 Phosphatidate Phosphatase by the 20S Proteasome

• Published in: The Journal of biological chemistry Vol. 290; no. 18; pp. 11467 - 11478
• Main Authors: Hsieh, Lu-Sheng, Su, Wen-Min, Han, Gil-Soo, Carman, George M.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.05.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Diacylglycerol; Humans; Lipid; Lipids; Phosphatase; Phosphatidate; Phosphatidate Phosphatase; Phosphatidate Phosphatase - chemistry; Phosphatidate Phosphatase - genetics; Phosphatidate Phosphatase - metabolism; Phosphorylation; Proteasome; Proteasome Endopeptidase Complex - metabolism; Proteolysis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Sequence Deletion; Triacylglycerol; Ubiquitin - metabolism; Yeast; Yeast; Phosphatidate; Triacylglycerol; Diacylglycerol; Phosphatase; Lipid; Phosphatidate Phosphatase; Proteasome
• DOI: 10.1074/jbc.M115.648659
• ISSN: 0021-9258

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. Background: Yeast Pah1 phosphatidate phosphatase required for triacylglycerol synthesis is subject to proteasome-mediated degradation. Results: Pah1 is degraded by the 20S proteasome in a ubiquitin-independent manner that is governed by its phosphorylation state. Conclusion: 20S proteasomal degradation of Pah1 is regulated by phosphorylation and dephosphorylation. Significance: Pah1 function in lipid metabolism is regulated by the 20S proteasome.