The Spo7 sequence LLI is required for Nem1-Spo7/Pah1 phosphatase cascade function in yeast lipid metabolism

Mona Mirheydari,Prabuddha Dey,Geordan J Stukey,Yeonhee Park,Gil-Soo Han,George M Carman

doi:10.1074/jbc.ra120.014129

Abstract

The Nem1-Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphory-lation of Pah1 phosphatidate phosphatase, required for its translocation to the nuclear/endoplasmic reticulum membrane. The Nem1-Spo7/Pah1 phosphatase cascade plays a major role in triacylglycerol synthesis and in the regulation of phospholipid synthesis. In this work, we examined Spo7, a regulatory subunit required for Nem1 catalytic function, to identify residues that govern formation of the Nem1-Spo7 complex. By deletion analysis of Spo7, we identified a hydrophobic Leu-Leu-Ile (LLI) sequence comprising residues 54-56 as being required for the protein to complement the temperature-sensitive phenotype of an spo7Δ mutant strain. Mutational analysis of the LLI sequence with alanine and arginine substitutions showed that its overall hydrophobicity is crucial for the formation of the Nem1-Spo7 complex as well as for the Nem1 catalytic function on its substrate, Pah1, in vivo Consistent with the role of the Nem1-Spo7 complex in activating the function of Pah1, we found that the mutational effects of the Spo7 LLI sequence were on the Nem1-Spo7/Pah1 axis that controls lipid synthesis and related cellular processes (e.g. triacylglycerol/phospholipid synthesis, lipid droplet formation, nuclear/endoplasmic reticulum membrane morphology, vacuole fusion, and growth on glycerol medium). These findings advance the understanding of Nem1-Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

Highlights

In the model eukaryote Saccharomyces cerevisiae, the lipid intermediate phosphatidic acid (PA) is used for the synthesis of phospholipids and the neutral lipid triacylglycerol (TAG) [1,2,3,4] (Fig. 1)
To address the hypothesis that the LLI sequence is required for the protein–protein interaction of Spo7 and Nem1, we examined the mutational effects of the hydrophobic sequence on Nem1-Spo7 complex formation
The role of Spo7 in sporulation is unclear, but its role in the nuclear morphology is based on the formation of a protein phosphatase complex with Nem1 to regulate Pah1 PA phosphatase [20, 43]

Summary

Introduction

In the model eukaryote Saccharomyces cerevisiae (in this paper, “yeast” is used interchangeably with Saccharomyces cerevisiae), the lipid intermediate phosphatidic acid (PA) is used for the synthesis of phospholipids and the neutral lipid triacylglycerol (TAG) [1,2,3,4] (Fig. 1). Deletion analysis of Spo7 indicated that its amino acid region 46–65 is required for Nem1-Spo7/Pah1 axis function, and the N-terminal region contains the hydrophobic sequence Leu-Leu-Ile (LLI) (residues 54–56), conserved in fungi. Mutational analysis showed that the overall hydrophobicity of the LLI sequence is required for the complex formation of Spo7 with Nem1, and its defect causes a significant loss of the phosphatase complex-controlled Pah1 function (e.g. TAG synthesis and lipid droplet formation, the nuclear/ER membrane morphology, vacuole fusion, and growth at elevated temperature and on glycerol as a carbon source).

Results

Conclusion