Architecture and function of yeast phosphatidate phosphatase Pah1 domains/regions

Abstract

Phosphatidate (PA) phosphatase, which catalyzes the Mg²⁺-dependent dephosphorylation of PA to produce diacylglycerol, provides a direct precursor for the synthesis of the storage lipid triacylglycerol and the membrane phospholipids phosphatidylcholine and phosphatidylethanolamine. The enzyme controlling the key phospholipid PA also plays a crucial role in diverse aspects of lipid metabolism and cell physiology. PA phosphatase is a peripheral membrane enzyme that is composed of multiple domains/regions required for its catalytic function and subcellular localization. In this review, we discuss the domains/regions of PA phosphatase from the yeast Saccharomyces cerevisiae with reference to the homologous enzyme from mammalian cells.

Keywords: Diacylglycerol; Lipin; Pah1; Phosphatidic acid; Phospholipid; Phosphorylation; Protein kinase; Protein phosphatase; Triacylglycerol; Yeast.

Fig. 1.

Roles of PA phosphatase Pah1 in lipid synthesis. The structures of CDP-DAG, PA, DAG, and TAG are shown with fatty acyl groups of 16 and 18 carbons with and without a single double bond where indicated. Pah1 plays a key role in the production of DAG for TAG synthesis and thereby controls the use of PA for the synthesis of membrane phospholipids via CDP-DAG. The PA phosphatase reaction is counterbalanced by the CTP-dependent conversion of DAG to PA by Dgk1. In addition to its role as a precursor in lipid synthesis, PA signals the transcriptional regulation of phospholipid synthesis genes via the Henry (Opi1/Ino2-Ino4) regulatory circuit. Under certain conditions (i.e., choline and/or ethanolamine supplementation), the DAG generated by the PA phosphatase reaction is utilized for the synthesis of phosphatidylcholine and/or phosphatidylethanolamine via the Kennedy pathway (not shown). More comprehensive pathways of lipid synthesis, along with details of the Henry regulatory circuit may be found in Refs. [3,4]. PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; PI, phosphatidylinositol, PG, phosphatidylglycerol; CL, cardiolipin.

Fig. 2.

Model for the phosphorylation/dephosphorylation-mediated regulation of Pah1 function. The PA phosphatase activity of Pah1 is primarily controlled by the localization of the enzyme. Following expression, Pah1 is present in the cytosol where it is phosphorylated by many protein kinases (A). The multiple phosphorylations mask the amphipathic helix and protect the enzyme against degradation by the 20S proteasome. Phosphorylated Pah1 is recruited (B) and dephosphorylated (C) by Nem1-Spo7 present at the nuclear/ER membrane. The dephosphorylation exposes the amphipathic helix to permit Pah1 to associate with the membrane surface (D). The membrane-associated Pah1 recognizes PA for its catalytic reaction to generate DAG, which is acylated to form TAG that is stored in lipid droplets (E). Following rounds of reaction, Pah1 dissociates from the nuclear/ER membrane for proteasomal degradation (indicated by dark shading) (F). AlphaFold2 structures of Pah1 and Nem1-Spo7 are depicted. For simplicity, some domains/regions (e.g., acidic tail and IDRs) of Pah1 are not shown. Nem1 is colored pink and Spo7 is colored blue. Green dots on Pah1 represent a phosphate group.

Fig. 3.

Linear schematics of yeast Pah1 and human lipins. The domains and regions of Pah1 and human lipin isoforms. AH, amphipathic helix; W, conserved tryptophan residue; AT, acidic tail; NLS, nuclear localization signal; M-Lip, middle lipin domain; β, lipin 1β specific sequence; γ, lipin 1γ specific sequence.

Fig. 4.

Predicted structures of S. cerevisiae Pah1 and human lipin 1α. The structures of Pah1 (A) and lipin 1α (B) are predicted by AlphaFold2 and visualized using the PyMol program. The crystal structure of T. thermophila Pah2 (C) is shown for comparison.

Fig. 5.

Predicted structures of the catalytic cores of S. cerevisiae Pah1 and human lipin 1α. The diagram generated from the AlphaFold2 structures displays the co-folding of the N-LIP and HAD-like domains of Pah1 (A) and lipin 1α (B). Experimental evidence for the co-folding of the N-LIP and HAD-like domains is provided by the crystal structure of T. thermophila Pah2 (C). The residues from the DXDXT catalytic motif are indicated and shown as a stick diagram to see the orientation of the residues in the active site. The conserved glycine residue in the N-LIP domain is also indicated. The Ig-like domain, originally identified in T. thermophila Pah2, is shown along with the suggested location of the squiggle motif. The β strands that form the central β sheet of the Rossman-like fold are numbered, and their positions are indicated.

Fig. 6.

Phosphorylation sites in Pah1. The serine and threonine residues known to be phosphorylated [–,,,–150] are grouped at their approximate regions in the Pah1 protein (A). The sites phosphorylated by casein kinase I (CKI) [66], casein kinase II (CKII) [65], Cdc28 [62], Hsl1 [117], Pho85 [61], protein kinase A [63], protein kinase C [64], and Rim11 [67] are indicated. AlphaFold3 predictions of the unphosphorylated and phosphorylated forms of Pah1 (B). The green dots on unphosphorylated Pah1 represent all of the serine/threonine residues that are targets of phosphorylation. The phosphorylated structure represents Pah1 with all serine/threonine residues in their phosphorylated state. S, serine; T, threonine; CKI, casein kinase; PKA, protein kinase A; PKC, protein kinase C.