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How Eukaryotes Add Head Groups In Phospholipid Synthesis

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. 2002 Sep;thirteen(ix):3148-61.

doi: x.1091/mbc.01-11-0540.

The major sites of cellular phospholipid synthesis and molecular determinants of Fatty Acid and lipid caput group specificity

Affiliations

  • PMID: 12221122
  • PMCID: PMC124149
  • DOI: x.1091/mbc.01-eleven-0540

Free PMC article

The major sites of cellular phospholipid synthesis and molecular determinants of Fatty Acrid and lipid head grouping specificity

Annette 50 Henneberry  et al. Mol Biol Prison cell. 2002 Sep .

Free PMC article

Abstract

Phosphatidylcholine and phosphatidylethanolamine are the 2 main phospholipids in eukaryotic cells comprising ~50 and 25% of phospholipid mass, respectively. Phosphatidylcholine is synthesized nigh exclusively through the CDP-choline pathway in essentially all mammalian cells. Phosphatidylethanolamine is synthesized through either the CDP-ethanolamine pathway or by the decarboxylation of phosphatidylserine, with the contribution of each pathway beingness cell type dependent. Two man genes, CEPT1 and CPT1, code for the total compliment of activities that direct synthesize phosphatidylcholine and phosphatidylethanolamine through the CDP-booze pathways. CEPT1 transfers a phosphobase from either CDP-choline or CDP-ethanolamine to diacylglycerol to synthesize both phosphatidylcholine and phosphatidylethanolamine, whereas CPT1 synthesizes phosphatidylcholine exclusively. Nosotros show through immunofluorescence that brefeldin A treatment relocalizes CPT1, but not CEPT1, implying CPT1 is establish in the Golgi. A combination of coimmunofluorescence and subcellular fractionation experiments with various endoplasmic reticulum, Golgi, and nuclear markers confirmed that CPT1 was establish in the Golgi and CEPT1 was establish in both the endoplasmic reticulum and nuclear membranes. The charge per unit-limiting stride for phosphatidylcholine synthesis is catalyzed by the amphitropic CTP:phosphocholine cytidylyltransferase alpha, which is establish in the nucleus in near cell types. CTP:phosphocholine cytidylyltransferase alpha is found immediately upstream cholinephosphotransferase, and it translocates from a soluble nuclear location to the nuclear membrane in response to activators of the CDP-choline pathway. Thus, substrate channeling of the CDP-choline produced by CTP:phosphocholine cytidylyltransferase blastoff to nuclear located CEPT1 is the mechanism past which upregulation of the CDP-choline pathway increases de novo phosphatidylcholine biosynthesis. In add-on, a serial of CEPT1 site-directed mutants was generated that allowed for the assignment of specific amino acrid residues every bit structural requirements that directly alter either phospholipid head group or fatty acyl composition. This pinpointed glycine 156 within the catalytic motif as being responsible for the dual CDP-alcohol specificity of CEPT1, whereas mutations within helix 214-228 immune for the orientation of transmembrane helices surrounding the catalytic site to be definitively positioned.

Figures

Figure 1
Figure one

Synthesis of PtdCho and PtdEtn by the CDP-alcohol pathways. The cistron names of the enzymes that catalyze each step are indicated. EKI, ethanolamine kinase; ET CTP:phosphocholine cytidylyltransferase; CEPT1, choline/ethanolaminephosphotransferase; CKI, choline kinase; CT, CTP:phosphocholine cytidylyltransferase; CPT1, cholinephosphotransferase.

Figure 2
Figure 2

Effect of brefeldin A on CEPT1 and CPT1 subcellular localization. (A) CHO-K1 cells transiently transfected with T7-CEPT1-GFP or the outset 81 amino of the Golgi resident β-1,four-galactosyltransferase fused to yellow fluorescent protein were treated with 2 μg/ml brefeldin A for 30 min. Alive and stock-still cells resulted in identical images. (B) CHO-K1 cells stably expressing T7-CPT1 were treated with brefeldin A and T7 monoclonal antibodies followed by Texas Red–conjugated secondary antibodies were used to make up one's mind the location of CPT1. The location of the Golgi was determined past the addition of FITC-coupled 50. culinaris lectin.

Figure 3
Figure three

Intracellular localization of CPT1. The intracellular location of CPT1 was adamant in CHO-K1 cells stably expressing T7-CPT1 using T7 monoclonal principal antibodies. The location of the endoplasmic reticulum was assessed using chief antibodies to calnexin. Golgi location was adamant by the addition of FITC-coupled L. culinaris lectin. MitoTracker dye was used to determine the location of the mitochondria. Secondary antibodies were coupled to either FITC or Texas Crimson. The yellow colour in merged images indicates overlap between CPT1 and the organelle marker.

Figure 4
Effigy 4

Intracellular localization of CEPT1. The intracellular location of CEPT1 was determined in CHO-K1 cells stably expressing T7-CEPT1 using T7 monoclonal primary antibodies. The location of the endoplasmic reticulum was assessed using primary antibodies to calnexin. Golgi location was adamant by the improver of FITC coupled L. culinaris lectin. MitoTracker dye was used to determine the location of the mitochondria. Secondary antibodies were coupled to either FITC or Texas Red. The xanthous color in merged images indicates overlap between CEPT1 and the organelle mark.

Figure 5
Figure five

PtdCho synthesis pathway reconstitution at the nuclear membrane. (A) CHO-K1 stably expressing T7-CEPT1 were treated with oleic acid (500 μM in 0.5% bovine serum albumin) for 24 h to translocate CTα from its inactive soluble intranuclear location to its active nuclear membrane location before immunofluorescence. (B) Nuclei (N) were separated from extranuclear structures including the endoplasmic reticulum (E) by subcellular fractionation as described in MATERIALS AND METHODS, and the distribution of nuclear and endoplasmic reticulum markers are compared with CEPT1.

Figure 6
Effigy 6

Predicted secondary structures for human CEPT1. Seven or 8 membrane spans are strongly predicted with helix 181–199 positioned either in (A) or exterior (B) of the membrane using the SMART or TmPred algorithms. The numbers denote amino acid residues with the black filled circles representing those mutated in the electric current study. Based on comparisons to studies on the yeast Cpt1p and Ept1p enzymes, the night gray circles stand for the maximum region required for diacylglycerol binding, and the light gray the maximum region required for CDP-alcohol binding. The conserved residues within the CDP-alcohol phosphotransferase motif, DG(x)iiAR(x)8Thousand(x)3D(x)3D, are also indicated.

Figure 7
Figure seven

CEPT1 CDP-alcohol phosphotransferase motif mutants. (A) The site-directed mutations fabricated in CEPT1 are indicated. The numbers announce CEPT1 amino acid residues. (B) Western blot of wild-type and mutant versions of CEPT1.

Figure 8
Figure eight

Enzyme activeness of CEPT1 CDP-alcohol phosphotransferase motif mutants. Enzyme activities were determined from microsomal membrane preparations of S. cerevisiae cells (cpt1:: LEU2 ept1 − ) constitutively expressing CEPT1 or the indicated site-directed mutants. The power to use either CDP-choline (blackness) or CDP-ethanolamine (white) as a substrate is indicated.

Figure 9
Figure 9

Metabolic reconstitution of PtdCho and PtdEtn synthesis by CEPT1 CDP-booze phosphotransferase mutants. Exponentially growing South. cerevisiae cells (cpt1:: LEU2 ept1 − ) constitutively expressing CEPT1 or the indicated site-directed mutants were radiolabeled with (A) [xivC]choline to label PtdCho or (B) [14C]ethanolamine to label PtdEtn, for 1 h. Radiolabel incorporated into each phospholipid was determined by scintillation counting.

Figure 10
Figure 10

CEPT1 diacylglycerol specificity mutants. (A) The site-directed mutations made in CEPT1 are indicated. The same region in human being CPT1 is provided for comparison. The numbers denote CEPT1 amino acid residues. (B) Western blot of wild-type and mutant versions of CEPT1.

Figure 11
Effigy 11

Enzyme activity of CEPT1 diacylglycerol specificity motif mutants. Enzyme activities were determined from microsomal membrane preparations of S. cerevisiae cells (cpt1:: LEU2 ept1 − ) constitutively expressing CEPT1 or the indicated site-directed mutants.

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How Eukaryotes Add Head Groups In Phospholipid Synthesis,

Source: https://pubmed.ncbi.nlm.nih.gov/12221122/

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