Loss of Ypk1, the Yeast Homolog to the Human Serum- and Glucocorticoid-induced Protein Kinase, Accelerates Phospholipase B1-mediated Phosphatidylcholine Deacylation

Surlow, Beth A.; Cooley, Benjamin M.; Needham, Patrick G.; Brodsky, Jeffrey L.; Patton‐Vogt, Jana

doi:10.1074/jbc.m114.581157

Cited by 13 publications

(24 citation statements)

References 77 publications

(98 reference statements)

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“…For labeling experiments, the medium was supplemented with 5 μ m of [ 3 H]choline-GPC and grown to log phase before harvesting. A membrane fraction and TCA-extractable intracellular fraction were isolated as described previously (27). The percentages of radioactive counts in each fraction were determined using a liquid scintillation counter.…”

Section: Methodsmentioning

confidence: 99%

“…The water-soluble choline-containing metabolites were separated using anion exchange chromatography as described previously (27). Standards were used to verify the separation procedure, and label incorporated into each metabolite was quantified using liquid scintillation counting.…”

Section: Methodsmentioning

confidence: 99%

“…Standards were used to verify the separation procedure, and label incorporated into each metabolite was quantified using liquid scintillation counting. Lipids were extracted from the membrane fraction as described previously (27) and separated by TLC as described for the in vitro assays.…”

Section: Methodsmentioning

confidence: 99%

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Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants

Głąb

Beganovic

Anaokar

et al. 2016

Journal of Biological Chemistry

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Glycero-3-phosphocholine (GPC), the product of the complete deacylation of phosphatidylcholine (PC), was long thought to not be a substrate for reacylation. However, it was recently shown that cell-free extracts from yeast and plants could acylate GPC with acyl groups from acyl-CoA. By screening enzyme activities of extracts derived from a yeast knock-out collection, we were able to identify and clone the yeast gene (GPC1) encoding the enzyme, named glycerophosphocholine acyltransferase (GPCAT). By homology search, we also identified and cloned GPCAT genes from three plant species. All enzymes utilize acyl-CoA to acylate GPC, forming lyso-PC, and they show broad acyl specificities in both yeast and plants. In addition to acyl-CoA, GPCAT efficiently utilizes LPC and lysophosphatidylethanolamine as acyl donors in the acylation of GPC. GPCAT homologues were found in the major eukaryotic organism groups but not in prokaryotes or chordates. The enzyme forms its own protein family and does not contain any of the acyl binding or lipase motifs that are present in other studied acyltransferases and transacylases. In vivo labeling studies confirm a role for Gpc1p in PC biosynthesis in yeast. It is postulated that GPCATs contribute to the maintenance of PC homeostasis and also have specific functions in acyl editing of PC (e.g. in transferring acyl groups modified at the sn-2 position of PC to the sn-1 position of this molecule in plant cells).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants

Głąb

Beganovic

Anaokar

et al. 2016

Journal of Biological Chemistry

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“…Cultures were grown to logarithmic phase at 30°C in YNB in media supplemented with 100 μM choline chloride and 0.2 μCi/mL of [14C] ethanolamine. Phospholipids were isolated as described in Surlow et al (2014). Lipid extracts representing equivalent amounts of optical density units (ODUs) were spotted onto silica gel TLC plates and plates were developed in chloroform:ethanol:water:triethylamine (30:35:7:35).…”

Section: Methodsmentioning

confidence: 99%

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans

et al. 2019

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Candida albicans is an opportunistic human fungal pathogen that causes life-threatening systemic infections, as well as oral mucosal infections. Phospholipids are crucial for pathogenesis in C. albicans, as disruption of phosphatidylserine (PS) and phosphatidylethanolamine (PE) biosynthesis within the cytidine diphosphate diacylglycerol (CDP-DAG) pathway causes avirulence in a mouse model of systemic infection. The synthesis of PE by this pathway plays a crucial role in virulence, but it was unknown if downstream conversion of PE to phosphatidylcholine (PC) is required for pathogenicity. Therefore, the enzymes responsible for methylating PE to PC, Pem1 and Pem2, were disrupted. The resulting pem1Δ/Δ pem2Δ/Δ mutant was not less virulent in mice, but rather hypervirulent. Since the pem1Δ/Δ pem2Δ/Δ mutant accumulated PE, this led to the hypothesis that increased PE synthesis increases virulence. To test this, the alternative Kennedy pathway for PE/PC synthesis was exploited. This pathway makes PE and PC from exogenous ethanolamine and choline, respectively, using three enzymatic steps. In contrast to Saccharomyces cerevisiae, C. albicans was found to use one enzyme, Ept1, for the final enzymatic step (ethanolamine/cholinephosphotransferase) that generates both PE and PC. EPT1 was overexpressed, which resulted in increases in both PE and PC synthesis. Moreover, the EPT1 overexpression strain is hypervirulent in mice and causes them to succumb to system infection more rapidly than wild-type. In contrast, disruption of EPT1 causes loss of PE and PC synthesis by the Kennedy pathway, and decreased kidney fungal burden during the mouse systemic infection model, indicating a mild loss of virulence. In addition, the ept1Δ/Δ mutant exhibits decreased cytotoxicity against oral epithelial cells in vitro, whereas the EPT1 overexpression strain exhibits increased cytotoxicity. Taken altogether, our data indicate that mutations that result in increased PE synthesis cause greater virulence and mutations that decrease PE synthesis attenuate virulence.

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“…Reverse transcription was performed at 50°C for 15 min followed by 95°C for 15 min for RT inactivation and polymerase activation. The thermal profile for amplification were 40 cycles at 95°C for 15 s, 54.5°C for 30 s, and 72°C for 40 s (62). Primer set specificity was determined by melting curve analysis.…”

Section: Rna Extraction and Qrt-pcrmentioning

confidence: 99%

The glycerophosphocholine acyltransferase Gpc1 is part of a phosphatidylcholine (PC)-remodeling pathway that alters PC species in yeast

Anaokar

Kodali

Jonik

et al. 2019

Journal of Biological Chemistry

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Edited by George M. CarmanPhospholipase B-mediated hydrolysis of phosphatidylcholine (PC) results in the formation of free fatty acids and glycerophosphocholine (GPC) in the yeast Saccharomyces cerevisiae. GPC can be reacylated by the glycerophosphocholine acyltransferase Gpc1, which produces lysophosphatidylcholine (LPC), and LPC can be converted to PC by the lysophospholipid acyltransferase Ale1. Here, we further characterized the regulation and function of this distinct PC deacylation/reacylation pathway in yeast. Through in vitro and in vivo experiments, we show that Gpc1 and Ale1 are the major cellular GPC and LPC acyltransferases, respectively. Importantly, we report that Gpc1 activity affects the PC species profile. Loss of Gpc1 decreased the levels of monounsaturated PC species and increased those of diunsaturated PC species, whereas Gpc1 overexpression had the opposite effects. Of note, Gpc1 loss did not significantly affect phosphatidylethanolamine, phosphatidylinositol, and phosphatidylserine profiles. Our results indicate that Gpc1 is involved in postsynthetic PC remodeling that produces more saturated PC species. qRT-PCR analyses revealed that GPC1 mRNA abundance is regulated coordinately with PC biosynthetic pathways. Inositol availability, which regulates several phospholipid biosynthetic genes, down-regulated GPC1 expression at the mRNA and protein levels and, as expected, decreased levels of monounsaturated PC species. Finally, loss of GPC1 decreased stationary phase viability in inositol-free medium. These results indicate that Gpc1 is part of a postsynthetic PC deacylation/reacylation remodeling pathway (PC-DRP) that alters the PC species profile, is regulated in coordination with other major lipid biosynthetic pathways, and affects yeast growth.

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Loss of Ypk1, the Yeast Homolog to the Human Serum- and Glucocorticoid-induced Protein Kinase, Accelerates Phospholipase B1-mediated Phosphatidylcholine Deacylation

Cited by 13 publications

References 77 publications

Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants

Cloning of Glycerophosphocholine Acyltransferase (GPCAT) from Fungi and Plants

Overproduction of Phospholipids by the Kennedy Pathway Leads to Hypervirulence in Candida albicans

The glycerophosphocholine acyltransferase Gpc1 is part of a phosphatidylcholine (PC)-remodeling pathway that alters PC species in yeast

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