The yeast Saccharomyces cerevisiae is a powerful experimental system to study biochemical, cell biological and molecular biological aspects of lipid synthesis. Most but not all genes encoding enzymes involved in fatty acid, phospholipid, sterol or sphingolipid biosynthesis of this unicellular eukaryote have been cloned, and many gene products have been functionally characterized. Less information is available about genes and gene products governing the transport of lipids between organelles and within membranes, turnover and degradation of complex lipids, regulation of lipid biosynthesis, and linkage of lipid metabolism to other cellular processes. Here we summarize current knowledge about lipid biosynthetic pathways in S. cerevisiae and describe the characteristic features of the gene products involved. We focus on recent discoveries in these fields and address questions on the regulation of lipid synthesis, subcellular localization of lipid biosynthetic steps, cross-talk between organelles during lipid synthesis and subcellular distribution of lipids. Finally, we discuss distinct functions of certain key lipids and their possible roles in cellular processes.

Biochemistry, Cell Biology and Molecular Biology of Lipids of Saccharomyces cerevisiae

The first committed step in the gibberellin (GA) biosynthetic pathway is the conversion of geranylgeranyl pyrophosphate (GGPP) through copalyl pyrophosphate (CPP) to ent-kaurene catalyzed by ent-kaurene synthetases A and B. The ga1 mutants of Arabidopsis are gibberellin-responsive male-sterile dwarfs. Biochemical studies indicate that biosynthesis of GAs in the ga1 mutants is blocked prior to the synthesis of ent-kaurene. The GA1 locus was cloned previously using the technique of genomic subtraction. Here, we report the isolation of a nearly full-length GA1 cDNA clone from wild-type Arabidopsis. This cDNA clone encodes an active protein and is able to complement the dwarf phenotype in ga1-3 mutants by Agrobacterium-mediated transformation. In Escherichia coli cells that express both the Arabidopsis GA1 gene and the Erwinia uredovora gene encoding GGPP synthase, CPP was accumulated. This result indicates that the GA1 gene encodes the enzyme ent-kaurene synthetase A, which catalyzes the conversion of GGPP to CPP. Subcellular localization of the GA1 protein was studied using 35S-labeled GA1 protein and isolated pea chloroplasts. The results showed that the GA1 protein is imported into and processed in pea chloroplasts in vitro.

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The Arabidopsis GA1 locus encodes the cyclase ent-kaurene synthetase A of gibberellin biosynthesis.

Multiplex metabolic pathway engineering using CRISPR/Cas9 in Saccharomyces cerevisiae

Conversion of glucose into glycogen is a major pathway that contributes to the removal of glucose from the portal vein by the liver in the postprandial state. It is regulated in part by the increase in blood-glucose concentration in the portal vein, which activates glucokinase, the first enzyme in the pathway, causing an increase in the concentration of glucose 6-P (glucose 6-phosphate), which modulates the phosphorylation state of downstream enzymes by acting synergistically with other allosteric effectors. Glucokinase is regulated by a hierarchy of transcriptional and post-transcriptional mechanisms that are only partially understood. In the fasted state, glucokinase is in part sequestered in the nucleus in an inactive state, complexed to a specific regulatory protein, GKRP (glucokinase regulatory protein). This reserve pool is rapidly mobilized to the cytoplasm in the postprandial state in response to an elevated concentration of glucose. The translocation of glucokinase between the nucleus and cytoplasm is modulated by various metabolic and hormonal conditions. The elevated glucose 6-P concentration, consequent to glucokinase activation, has a synergistic effect with glucose in promoting dephosphorylation (inactivation) of glycogen phosphorylase and inducing dephosphorylation (activation) of glycogen synthase. The latter involves both a direct ligand-induced conformational change and depletion of the phosphorylated form of glycogen phosphorylase, which is a potent allosteric inhibitor of glycogen synthase phosphatase activity associated with the glycogen-targeting protein, GL [hepatic glycogen-targeting subunit of PP-1 (protein phosphatase-1) encoded by PPP1R3B]. Defects in both the activation of glucokinase and in the dephosphorylation of glycogen phosphorylase are potential contributing factors to the dysregulation of hepatic glucose metabolism in Type 2 diabetes.

Glucokinase and molecular aspects of liver glycogen metabolism.

Genome-wide association studies have identified a number of signals for both Type 2 Diabetes and related quantitative traits. For the majority of loci, the transition from association signal to mutational mechanism has been difficult to establish. Glucokinase (GCK) regulates glucose storage and disposal in the liver where its activity is regulated by glucokinase regulatory protein (GKRP; gene name GCKR). Fructose-6 and fructose-1 phosphate (F6P and F1P) enhance or reduce GKRP-mediated inhibition, respectively. A common GCKR variant (P446L) is reproducibly associated with triglyceride and fasting plasma glucose levels in the general population. The aim of this study was to determine the mutational mechanism responsible for this genetic association. Recombinant human GCK and both human wild-type (WT) and P446L-GKRP proteins were generated. GCK kinetic activity was observed spectrophotometrically using an NADP(+)-coupled assay. WT and P446L-GKRP-mediated inhibition of GCK activity and subsequent regulation by phosphate esters were determined. Assays matched for GKRP activity demonstrated no difference in dose-dependent inhibition of GCK activity or F1P-mediated regulation. However, the response to physiologically relevant F6P levels was significantly attenuated with P446L-GKRP (n = 18; P <or= 0.03). Experiments using equimolar concentrations of both regulatory proteins confirmed these findings (n = 9; P < 0.001). In conclusion, P446L-GKRP has reduced regulation by physiological concentrations of F6P, resulting indirectly in increased GCK activity. Altered GCK regulation in liver is predicted to enhance glycolytic flux, promoting hepatic glucose metabolism and elevating concentrations of malonyl-CoA, a substrate for de novo lipogenesis, providing a mutational mechanism for the reported association of this variant with raised triglycerides and lower glucose levels.

The P446L variant in GCKR associated with fasting plasma glucose and triglyceride levels exerts its effect through increased glucokinase activity in liver

Squalene synthetase (farnesyl-diphosphate: farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is a critical branch point enzyme of isoprenoid biosynthesis that is thought to regulate the flux of isoprene intermediates through the sterol pathway. The structural gene for this enzyme was cloned from the yeast Saccharomyces cerevisiae by functional complementation of a squalene synthetase-deficient erg9 mutant. Identification of this ERG9 clone was confirmed by genetic linkage analysis in yeast and expression of enzyme activity in Escherichia coli. The predicted squalene synthetase polypeptide of 444 amino acids (Mr, 51,753) lacks significant homology to known protein sequences, except within a region that may represent a prenyl diphosphate (substrate) binding site. The ERG9-encoded protein contains a PEST consensus motif (rich in proline, glutamic acid, serine, and threonine) present in many proteins with short cellular half-lives. Modeling of the protein suggests that it contains at least one, and possibly two, membrane-spanning domains. Disruption of the chromosomal squalene synthetase coding region by insertional mutagenesis indicates that ERG9 is a single copy gene that is essential for cell growth in yeast.

https://www.pnas.org/content/pnas/88/14/6038.full.pdf

Molecular cloning and characterization of the yeast gene for squalene synthetase.

Glucokinase is a critical component of the physiological glucose sensor found in cell types that are responsive to changes in plasma glucose levels. The acute regulation of glucokinase activity has been shown to occur via a regulatory protein found in liver parenchymal cells (Van Schaftingen E, Detheux M, Da Cunha MV. Faseb J 8:414-419, 1994). The action of this protein is modulated by phosphate esters of fructose. In the presence of fructose-6-phosphate, the protein inhibits glucokinase in an allosteric competitive manner, while fructose-1-phosphate reverses this inhibition. A cDNA potentially encoding the rat liver regulatory protein has been cloned, but its identity is uncertain because of the small amounts of soluble protein obtained by expression in bacteria. We report the heterologous expression of the regulatory protein in Escherichia coli and its purification to homogeneity and high specific activity in a single chromatographic step. The properties of this recombinant protein are very similar to those of the liver protein. Direct demonstration of the binding of the recombinant protein to glucokinase has been obtained in vitro using coprecipitation experiments and in vivo, using the yeast two-hybrid system. These studies establish that the protein encoded by the cDNA is identical to the glucokinase regulatory protein and also validate tools with which to carry out structure-function studies on the interaction of the regulatory protein with glucokinase.

Yim Ha Tsay

Papers

Molecular cloning and characterization of the yeast gene for squalene synthetase.

Heterologous expression and characterization of rat liver glucokinase regulatory protein.