Browse
Submit Data
Databases
API
Help

Dataset Information

20 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Defining the subcellular distribution and metabolic channeling of phosphatidylinositol.

ABSTRACT: Phosphatidylinositol (PI) is an essential structural component of eukaryotic membranes that also serves as the common precursor for polyphosphoinositide (PPIn) lipids. Despite the recognized importance of PPIn species for signal transduction and membrane homeostasis, there is still a limited understanding of the relationship between PI availability and the turnover of subcellular PPIn pools. To address these shortcomings, we established a molecular toolbox for investigations of PI distribution within intact cells by exploiting the properties of a bacterial enzyme, PI-specific PLC (PI-PLC). Using these tools, we find a minor presence of PI in membranes of the ER, as well as a general enrichment within the cytosolic leaflets of the Golgi complex, peroxisomes, and outer mitochondrial membrane, but only detect very low steady-state levels of PI within the plasma membrane (PM) and endosomes. Kinetic studies also demonstrate the requirement for sustained PI supply from the ER for the maintenance of monophosphorylated PPIn species within the PM, Golgi complex, and endosomal compartments.

SUBMITTER: Pemberton JG

PROVIDER: S-EPMC7054996 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Probing the subcellular distribution of phosphatidylinositol reveals a surprising lack at the plasma membrane.

Project description:The polyphosphoinositides (PPIn) are central regulatory lipids that direct membrane function in eukaryotic cells. Understanding how their synthesis is regulated is crucial to revealing these lipids' role in health and disease. PPIn are derived from the major structural lipid, phosphatidylinositol (PI). However, although the distribution of most PPIn has been characterized, the subcellular localization of PI available for PPIn synthesis is not known. Here, we used several orthogonal approaches to map the subcellular distribution of PI, including localizing exogenous fluorescent PI, as well as detecting lipid conversion products of endogenous PI after acute chemogenetic activation of PI-specific phospholipase and 4-kinase. We report that PI is broadly distributed throughout intracellular membrane compartments. However, there is a surprising lack of PI in the plasma membrane compared with the PPIn. These experiments implicate regulation of PI supply to the plasma membrane, as opposed to regulation of PPIn-kinases, as crucial to the control of PPIn synthesis and function at the PM.

| S-EPMC7054989 | biostudies-literature

Subcellular distribution of agonist-stimulated phosphatidylinositol synthesis in 1321 N1 astrocytoma cells.

Project description:In an inositol-depleted 1321 N1 astrocytoma cell line, propranolol at 0.5 mM concentration and carbachol in the presence of Li+ induce a large increase (30-60-fold) in the amount of CMP-phosphatidate, the lipid substrate of PtdIns synthase. The actions of both agents on CMP-phosphatidate accumulation were reversed by co-incubation with 1 mM inositol. In cells grown in the presence of 40 microM inositol the propranolol- and carbachol-mediated CMP-phosphatidate accumulation was much smaller (2-4-fold). Propranolol- and carbachol-mediated increases in CMP-phosphatidate accumulation were at least additive in both inositol-replete and -depleted cells. The subcellular distribution of accumulated CMP-phosphatidate was investigated by sucrose-density-gradient centrifugation of a lysate of inositol-depleted cells. There were two coincident peaks of carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, respectively. The first peak of accumulated [3H]CMP-phosphatidate and PtdIns synthase activity is characteristic of a 'light vesicle' fraction, since it sediments at sucrose densities similar to that of endocytosed 125I-transferrin. The later peak, containing both carbachol-stimulated [3H]CMP-phosphatidate and PtdIns synthase activity, has a distribution in the gradient that is similar to NADPH-cytochrome c reductase activity, an endoplasmic-reticulum marker. By contrast, propranolol-stimulated [3H]CMP-phosphatidate accumulates in membranes which sediment as a single peak corresponding to the endoplasmic-reticulum marker. These observations suggest that agonist-stimulated PtdIns synthesis occurs in the endoplasmic reticulum and in at least one additional membrane compartment which is insensitive to propranolol, an inhibitor of endoplasmic-reticulum phosphatidate phosphohydrolase.

| S-EPMC1132284 | biostudies-other

Confocal imaging of the subcellular distribution of phosphatidylinositol 3,4,5-trisphosphate in insulin- and PDGF-stimulated 3T3-L1 adipocytes.

Project description:The activation of phosphatidylinositol 3-kinase (PI 3-kinase) and production of PtdIns(3,4,5)P(3) is crucial in the actions of numerous extracellular stimuli, including insulin-stimulated glucose uptake. Platelet-derived growth factor (PDGF) also stimulates PI 3-kinase, but only weakly promotes glucose uptake when compared with insulin. Insulin and PDGF have thus been proposed to have differential effects on the subcellular targeting of PI 3-kinase. However, owing to a lack of suitable methodologies, the subcellular localization of the PtdIns(3,4,5)P(3) generated has not been examined. The pleckstrin-homology (PH) domains of the nucleotide exchange factors, ADP-ribosylation factor nucleotide-binding-site opener (ARNO) and general receptor for 3-phosphoinositides (GRP1), which have a high affinity and specificity for PtdIns(3,4,5)P(3), were fused to green fluorescent protein and used to examine the subcellular localization of PtdIns(3,4,5)P(3) generation in living 3T3-L1 adipocytes. PtdIns(3,4,5)P(3) was produced almost exclusively in the plasma membrane in response to both agonists, although the response to insulin was greater in magnitude and occurred in considerably more cells. The results suggest that the greater ability of insulin to stimulate glucose uptake may be the result of its ability to generate significantly more plasma-membrane PtdIns(3, 4,5)P(3) than PDGF. ARNO and GRP1 are nucleotide exchange factors for the small GTP-binding protein ADP-ribosylation factor 6 (ARF6). The inability of a constitutively active GTPase-deficient mutant of ARF6 (ARF6-Q67L; Gln(67)-->Leu) to cause glucose transporter GLUT4 translocation suggests that activation of this pathway is not sufficient to cause GLUT4 translocation.

| S-EPMC1220670 | biostudies-literature

Stimulation by acetylcholine of phosphatidylinositol labelling. Subcellular distribution in rat cerebral-cortex slices.

Project description:1. Rat cerebral-cortex slices were incubated with (32)P(i), acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.

| S-EPMC1178537 | biostudies-other

Subcellular distribution of RNAs in HeLa cells

Project description:HeLa cells were fractioned in nucleus and cytoplasm to investigate the subcellular distribution of lncRNAs. Two-condition experiment, nuclear fraction vs. cytoplasmatic fraction from HeLa cells. Biological replicates: 2 with dye-swap

2015-06-30 | E-GEOD-62985 | biostudies-arrayexpress

The distribution of molecular species of phosphatidylinositol in ox brain and its subcellular fractions.

Project description:1. The phosphatidylinositol content of white and grey matter of ox cerebral hemispheres did not differ. The phosphatidylinositol from grey matter was slightly enriched in palmitic acid and arachidonic acid, and that from white matter was enriched in eicosatrienoic (C(20:3)) acid. These regional differences were apparently due to the greater content of myelin in the white matter, since the same tendencies were observed when combined myelinic and non-myelinic subcellular fractions prepared from the cerebral hemispheres were compared. 2. Purified phosphatidylinositol was converted into its triacetylated methylated derivative and resolved to its molecular species by t.l.c. on AgNO(3)-impregnated silica gel. The tetraenoic molecular species was predominant in phosphatidylinositol from ox cerebral hemispheres, and this feature characterized all the phosphatidylinositol samples extracted from its regions or subcellular fractions. The grey matter was more enriched in the tetraenoic species and the white matter in the trienoic species. 3. The molecular-species composition of phosphatidylinositol from the subcellular fractions of ox cerebral hemispheres was studied. The trienoic species constituted nearly one-fifth of the phosphatidylinositol from two myelinic fractions. ;Large myelin' was more enriched in this species than was ;small myelin'. Both fractions also contained greater concentrations of the dienoic species than the non-myelinic subcellular fractions. The latter fractions, one containing nuclei and the other nerve endings plus mitochondria, were enriched in the monoenoic and tetraenoic species of phosphatidylinositol. The post-mitochondrial supernatant exhibited a pattern of distribution of phosphatidylinositol species intermediate between the myelinic and non-myelinic fractions.

| S-EPMC1173810 | biostudies-other

Enzyme clustering accelerates processing of intermediates through metabolic channeling.

Project description:We present a quantitative model to demonstrate that coclustering multiple enzymes into compact agglomerates accelerates the processing of intermediates, yielding the same efficiency benefits as direct channeling, a well-known mechanism in which enzymes are funneled between enzyme active sites through a physical tunnel. The model predicts the separation and size of coclusters that maximize metabolic efficiency, and this prediction is in agreement with previously reported spacings between coclusters in mammalian cells. For direct validation, we study a metabolic branch point in Escherichia coli and experimentally confirm the model prediction that enzyme agglomerates can accelerate the processing of a shared intermediate by one branch, and thus regulate steady-state flux division. Our studies establish a quantitative framework to understand coclustering-mediated metabolic channeling and its application to both efficiency improvement and metabolic regulation.

| S-EPMC4666537 | biostudies-literature

Phosphatidylserine subcellular distribution and functions

Project description:Develop the organelle specific phosphatidylserine sensor to observe its distribution and stady its functions

2023-10-10 | PXD046019 |

Subcellular distribution of RNAs in HeLa cells

Project description:HeLa cells were fractioned in nucleus and cytoplasm to investigate the subcellular distribution of lncRNAs.

2015-06-30 | GSE62985 | GEO

Subcellular Distribution of NAD+ between Cytosol and Mitochondria Determines the Metabolic Profile of Human Cells.

Project description:The mitochondrial NAD pool is particularly important for the maintenance of vital cellular functions. Although at least in some fungi and plants, mitochondrial NAD is imported from the cytosol by carrier proteins, in mammals, the mechanism of how this organellar pool is generated has remained obscure. A transporter mediating NAD import into mammalian mitochondria has not been identified. In contrast, human recombinant NMNAT3 localizes to the mitochondrial matrix and is able to catalyze NAD(+) biosynthesis in vitro. However, whether the endogenous NMNAT3 protein is functionally effective at generating NAD(+) in mitochondria of intact human cells still remains to be demonstrated. To modulate mitochondrial NAD(+) content, we have expressed plant and yeast mitochondrial NAD(+) carriers in human cells and observed a profound increase in mitochondrial NAD(+). None of the closest human homologs of these carriers had any detectable effect on mitochondrial NAD(+) content. Surprisingly, constitutive redistribution of NAD(+) from the cytosol to the mitochondria by stable expression of the Arabidopsis thaliana mitochondrial NAD(+) transporter NDT2 in HEK293 cells resulted in dramatic growth retardation and a metabolic shift from oxidative phosphorylation to glycolysis, despite the elevated mitochondrial NAD(+) levels. These results suggest that a mitochondrial NAD(+) transporter, similar to the known one from A. thaliana, is likely absent and could even be harmful in human cells. We provide further support for the alternative possibility, namely intramitochondrial NAD(+) synthesis, by demonstrating the presence of endogenous NMNAT3 in the mitochondria of human cells.

| S-EPMC4646015 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data