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

Abstract

The Nem1–Spo7 complex in the yeast Saccharomyces cerevisiae is a protein phosphatase that catalyzes the dephosphorylation 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 a 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 the Nem1–Spo7 complex formation and its role in the phosphatase cascade that regulates the function of Pah1 phosphatidate phosphatase.

Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex

“…However, deletion of ICE2 slightly destabilized Nem1 and Spo7, and ICE2 overexpression had essentially no effect (Figure 7A, B). In contrast, deletion of SPO7 strongly reduced the levels of Nem1, as reported (Mirheydari et al, 2020). Thus, it is likely that Ice2 negatively regulates the Nem1-Spo7 complex by inhibiting its phosphatase activity.…”

“…Ice2 co-precipitated with both Nem1 and Spo7 (Figure 7C, D). We were unable to test whether the association of Ice2 and Nem1 depends on Spo7 because Nem1 is unstable in the absence of Spo7 (Figure 7A; Mirheydari et al, 2020). However, Ice2 still co-precipitated with Spo7 in the absence of Nem1 (Figure 7E).…”

Ice2 promotes ER membrane biogenesis in yeast by inhibiting the conserved lipin phosphatase complex

“…Ice2 coprecipitated with both Spo7 and Nem1, but not with the abundant ER transmembrane protein Dpm1 (Fig 7A and B). We were unable to test whether the association of Ice2 and Nem1 depends on Spo7 because Nem1 is unstable in the absence of Spo7 (Fig EV4A; Mirheydari et al, 2020). However, Ice2 still co-precipitated with Spo7 in the absence of Nem1, albeit less efficiently (Fig 7C).…”

Mutant phosphatidate phosphatase Pah1-W637A exhibits altered phosphorylation, membrane association, and enzyme function in yeast

“…Lipid droplets in stationary phase cells were stained with the fluorescent dye BODIPY 493/503 (4,46). The green fluorescence signal of the lipid droplets was observed under a Nikon Eclipse Ni-U microscope with the EGFP/FITC/Cy2/AlexaFluor 488 filter, recorded by the DS-Qi2 camera, and subjected to imaging analysis with the NIS-Elements BR software.…”

“…1). In turn, the regulation of the Nem1-Spo7 complex impacts on the function of Pah1 (10,(44)(45)(46)(47)(48)(49). Phosphorylation has a protective effect on Pah1 against its degradation by the 20S proteasome, whereas its dephosphorylation makes it susceptible to the proteolytic degradation (50,51).…”