An essential role for phosphatidylinositol transfer protein in phospholipase C-Mediated inositol lipid signaling

Thomas, Geraint M. H.; Cunningham, Emer; Fensome, Amanda; Ball, Andrew; Totty, Nicholas F.; Truong, Oanh; Hsuan, J. Justin; Cockcroft, Shamshad

doi:10.1016/0092-8674(93)90471-2

Cited by 210 publications

(167 citation statements)

References 39 publications

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“…A salt gradient (0-1 M NaCl) was used to elute the proteins (NaCl-free buffer for 50 ml, then 0-1 M NaCl for 300 ml) ; 35 fractions, each of 10 ml, were collected and assayed for PtdIns transfer activity as described previously [15] (see Figure 1A). The active fractions (fractions 11, 12 and 13) from the three separate runs were pooled and concentrated to approx.…”

Section: Purification Of Pitp From Dictyostelium Cytoplasmmentioning

confidence: 99%

“…The samples were also transferred to nitrocellulose and blotted with the 5F12 monoclonal antibody against mammalian PITPα (see Figure 1F). Fractions 8-10, which contained the PITP activity and protein, were pooled and used for peptide sequencing as described previously [15].…”

Section: Purification Of Pitp From Dictyostelium Cytoplasmmentioning

confidence: 99%

“…The activity of PITP was measured by monitoring the transfer of $H-labelled PtdIns from donor microsomes to acceptor liposomes (PtdCho\PtdIns, 98 : 2) at 25 mC for 30 min, exactly as described previously [15].…”

Section: Assay For Pitp Activity In Vitromentioning

confidence: 99%

“…This was performed exactly as described [15]. In brief, HL60 cells were labelled for 48 h in M199 with 1 µCi\ml [$H]inositol.…”

Section: Cell Permeabilization and Plc Reconstitutionmentioning

confidence: 99%

“…The greatest demand for PtdIns comes from PLC-mediated hydrolysis of PtdIns(4,5)P # , which can occur in multiple compartments including the plasma membrane and the nucleus. Thus PLCmediated hydrolysis of PtdIns(4,5)P # is dependent on the availability of PITP, whatever the mode of activation of the PLC [15][16][17][18]. PtdIns(4,5)P # is also used as a substrate by phosphoinositide 3-kinase ; again, PITP has been identified as a requirement for this signalling pathway [19].…”

Section: Introductionmentioning

confidence: 99%

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Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae Sec14p are found in the same cell

Swigart

Insall

Cockcroft

2000

Biochemical Journal

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Soluble phosphatidylinositol transfer proteins (PITPs) have important roles in lipid-mediated signalling as well as in membrane traffic. Two PITPs (α and β) have been cloned from mammalian cells, which are unrelated in sequence to yeast PITP (the product of the SEC14 gene). However, all three PITPs can perform interchangeably to reconstitute function in mammalian cells. We have now purified the major PITP from the cytoplasm of Dictyostelium discoideum and cloned the gene. This protein, DdPITP1, is homologous with mammalian PITPα and PITPβ. We have also cloned a second gene (DdPITP2) related in sequence to DdPITP1. In addition, an independently cloned cDNA encodes a relative of the SEC14 family of yeast PITPs. DdPITP1, DdPITP2 and DdSec14 proteins were all able to mediate the transfer of PtdIns from one membrane compartment to another; they thus exhibited the hallmark of PITPs. Secondly, all three PITPs were able to rescue phospholipase C-mediated phosphoinositide hydrolysis in PITP-depleted HL60 cells, indicating that all three PITPs were capable of stimulating phosphoinositide synthesis. The identification of PITPs related to both mammalian PITPs and yeast Sec14p in a single organism will provide a unique opportunity to examine the functions of this class of protein with genetic approaches.

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Section: Purification Of Pitp From Dictyostelium Cytoplasmmentioning

confidence: 99%

Section: Purification Of Pitp From Dictyostelium Cytoplasmmentioning

confidence: 99%

Section: Assay For Pitp Activity In Vitromentioning

confidence: 99%

“…This was performed exactly as described [15]. In brief, HL60 cells were labelled for 48 h in M199 with 1 µCi\ml [$H]inositol.…”

Section: Cell Permeabilization and Plc Reconstitutionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae Sec14p are found in the same cell

Swigart

Insall

Cockcroft

2000

Biochemical Journal

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Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil

Ribbes

Cane²,

Planat

et al. 1996

J. Cell. Biochem.

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Subcellular localizations of CoA-independent transacylase and phospholipase D enzymes have been investigated in human neutrophils performing a two-step gradient system to separate plasma membranes from internal membranes and from the bulk of granules. The internal membranes were constituted by endoplasmic reticulum and by a subpopulation of specific and tertiary granules. The enzymes activities were assayed in vitro on gradient fractions using exogenous substrates. Following cell prelabelling with [3H]alkyllyso-GPC, we also analyzed the in situ localization of labelled products involving the action of both enzymes. The CoA-independent transacylase activity, together with the CoA-dependent transacylase and acyltransferase activities were only located in the internal membranes. Following 15 min cell labelling, part of the [3H]alkylacyl-GPC was recovered in plasma membranes indicating a rapid redistribution of the acylated compound. Very high contents in arachidonate containing [3H]alkylacyl-GPC were recovered both in plasma membranes and internal membranes. Phospholipase D activity being assayed in the presence of cytosol, GTP gamma S and gradient fractions, only the plasma membrane fractions from resting or stimulated cells allowed the enzyme to be active. The [3H]alkylacyl-GP and [3H]alkylacyl-GPethanol, phospholipase D breakdown products from [3H]alkylacyl-GPC, obtained after neutrophil prelabelling and activation by phorbol myristate acetate, were exclusively present in the plasma membranes. In contrast, the secondary generated [3H]alkylacylglycerols were equally distributed between plasma and internal membranes. No labelled product was recovered on azurophil granules. These data demonstrate that internal membranes are the site of action of the CoA-independent transacylase and plasma membranes are the site of action of the phospholipase D. This topographical separation between CoA-independent transacylase which generated substrate and phospholipase D which degraded it, suggested that subcellular localisation and traffic of substrates within the cell can be important to regulate the enzymes.

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An essential role for phosphatidylinositol transfer protein in phospholipase C-Mediated inositol lipid signaling

Cited by 210 publications

References 39 publications

Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae Sec14p are found in the same cell

Purification and cloning of phosphatidylinositol transfer proteins from Dictyostelium discoideum: homologues of both mammalian PITPs and Saccharomyces cerevisiae Sec14p are found in the same cell

Transacylase-mediated alkylacyl-GPC synthesis and its hydrolysis by phospholipase D occur in separate cell compartments in the human neutrophil

Phosphatidylinositol transfer proteins: a requirement in signal transduction and vesicle traffic

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