scholarly journals Deletion of 24 amino acids from the C-terminus of phosphatidylinositol transfer protein causes loss of phospholipase C-mediated inositol lipid signalling

1997 ◽  
Vol 324 (1) ◽  
pp. 19-23 ◽  
Author(s):  
Simon PROSSER ◽  
Robert SARRA ◽  
Philip SWIGART ◽  
Andrew BALL ◽  
Shamshad COCKCROFT
Keyword(s):  
Amino Acids ◽  
Phospholipase C ◽  
Size Exclusion ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
C Terminus ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer protein α (PITPα) is a 32 kDa protein of 270 amino acids that is essential for phospholipase C-mediated phosphatidylinositol bisphosphate hydrolysis. In addition, it binds and transfers phosphatidylinositol and phosphatidylcholine between membrane compartments in vitro. Here we have used limited proteolysis of PITPα by subtilisin to identify the structural requirements for function. Digestion by subtilisin results in the generation of a number of slightly smaller peptide fragments, the major fragment being identified as a 29 kDa protein. The fragments were resolved by size-exclusion chromatography and were found to be totally inactive in both in vivo PLC reconstitution assays and in vitro phosphatidylinositol transfer assays. N-terminal sequencing and MS of the major 29 kDa fragment shows that cleavage occurs at the C-terminus of PITP at Met246, leading to a deletion of 24 amino acid residues. We conclude that the C-terminus plays an important role in mediating PLC signalling in vivo and lipid transfer in vitro, supporting the notion that lipid transfer may be a facet of PITP function in vivo.

scholarly journals Differential expression of a C-terminal splice variant of phosphatidylinositol transfer protein β lacking the constitutive-phosphorylated Ser262 that localizes to the Golgi compartment

2006 ◽  
Vol 398 (3) ◽  
pp. 411-421 ◽  
Author(s):  
Clive P. Morgan ◽  
Victoria Allen-Baume ◽  
Marko Radulovic ◽  
Michelle Li ◽  
Alison Skippen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Rat Liver ◽  
Splice Variant ◽  
Single Gene ◽  
Hl60 Cells ◽  
Transfer Protein ◽  
Golgi Localization ◽  
C Terminus ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

Mammalian PITPβ (phosphatidylinositol transfer protein β) is a 272-amino-acid polypeptide capable of transferring PtdIns, PtdCho and SM (sphingomyelin) between membrane bilayers. It has been reported that Ser262 present in the C-terminus of PITPβ is constitutively phosphorylated and determines Golgi localization. We provide evidence for the expression of an sp (splice) variant of PITPβ (PITPβ-sp2) where the C-terminal 15 amino acids of PITPβ-sp1 are replaced by an alternative C-terminus of 16 amino acids. PITPβ-sp1 is the product of the first 11 exons, whereas PITPβ-sp2 is a product of the first 10 exons followed by the twelfth exon – exon 11 being ‘skipped’. Both splice variants are capable of PtdIns and PtdCho transfer, with PITPβ-sp2 being unable to transport SM. PITPβ is ubiquitously expressed, with the highest amounts of PITPβ found in HL60 cells and in rat liver; HL60 cells express only PITPβ-sp1, whereas rat liver expresses both sp variants in similar amounts. In both cell types, PITPβ-sp1 is constitutively phosphorylated and both the PtdIns and PtdCho forms of PITPβ-sp1 are present. In contrast, PITPβ-sp2 lacks the constitutively phosphorylated Ser262 (replaced with glutamine). Nonetheless, both PITPβ variants localize to the Golgi and, moreover, dephosphorylation of Ser262 of PITPβ-sp1 does not affect its Golgi localization. The presence of PITPβ sp variants adds an extra level of proteome complexity and, in rat liver, the single gene for PITPβ gives rise to seven distinct protein species that can be resolved on the basis of their charge differences.

scholarly journals The Phosphatidylinositol Transfer Protein Domain of Drosophila Retinal Degeneration B Protein Is Essential for Photoreceptor Cell Survival and Recovery from Light Stimulation

1997 ◽  
Vol 139 (2) ◽  
pp. 351-363 ◽  
Author(s):  
Scott C. Milligan ◽  
James G. Alb ◽  
Raya B. Elagina ◽  
Vytas A. Bankaitis ◽  
David R. Hyde
Keyword(s):  
Retinal Degeneration ◽  
Light Response ◽  
Protein Domain ◽  
Inherited Disease ◽  
Light Stimulation ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

The Drosophila retinal degeneration B (rdgB) gene encodes an integral membrane protein involved in phototransduction and prevention of retinal degeneration. RdgB represents a nonclassical phosphatidylinositol transfer protein (PITP) as all other known PITPs are soluble polypeptides. Our data demonstrate roles for RdgB in proper termination of the phototransduction light response and dark recovery of the photoreceptor cells. Expression of RdgB's PITP domain as a soluble protein (RdgB-PITP) in rdgB2 mutant flies is sufficient to completely restore the wild-type electrophysiological light response and prevent the degeneration. However, introduction of the T59E mutation, which does not affect RdgB-PITP's phosphatidylinositol (PI) and phosphatidycholine (PC) transfer in vitro, into the soluble (RdgB-PITP-T59E) or full-length (RdgB-T59E) proteins eliminated rescue of retinal degeneration in rdgB2 flies, while the light response was partially maintained. Substitution of the rat brain PITPα, a classical PI transfer protein, for RdgB's PITP domain (PITPα or PITPα-RdgB chimeric protein) neither restored the light response nor maintained retinal integrity when expressed in rdgB2 flies. Therefore, the complete repertoire of essential RdgB functions resides in RdgB's PITP domain, but other PITPs possessing PI and/or PC transfer activity in vitro cannot supplant RdgB function in vivo. Expression of either RdgB-T59E or PITPα-RdgB in rdgB+ flies produced a dominant retinal degeneration phenotype. Whereas RdgB-T59E functioned in a dominant manner to significantly reduce steady-state levels of rhodopsin, PITPα-RdgB was defective in the ability to recover from prolonged light stimulation and caused photoreceptor degeneration through an unknown mechanism. This in vivo analysis of PITP function in a metazoan system provides further insights into the links between PITP dysfunction and an inherited disease in a higher eukaryote.

scholarly journals Noncanonical regulation of phosphatidylserine metabolism by a Sec14-like protein and a lipid kinase

2020 ◽  
Vol 219 (5) ◽  
Author(s):  
Yaxi Wang ◽  
Peihua Yuan ◽  
Aby Grabon ◽  
Ashutosh Tripathi ◽  
Dongju Lee ◽  
...  
Keyword(s):  
Lipid Transfer Protein ◽  
Physical Interaction ◽  
Lipid Transfer ◽  
Uncharacterized Protein ◽  
Lipid Kinase ◽  
Transfer Protein ◽  
Transfer Activity ◽  
Phosphatidylinositol Transfer

The yeast phosphatidylserine (PtdSer) decarboxylase Psd2 is proposed to engage in a membrane contact site (MCS) for PtdSer decarboxylation to phosphatidylethanolamine (PtdEtn). This proposed MCS harbors Psd2, the Sec14-like phosphatidylinositol transfer protein (PITP) Sfh4, the Stt4 phosphatidylinositol (PtdIns) 4-OH kinase, the Scs2 tether, and an uncharacterized protein. We report that, of these components, only Sfh4 and Stt4 regulate Psd2 activity in vivo. They do so via distinct mechanisms. Sfh4 operates via a mechanism for which its PtdIns-transfer activity is dispensable but requires an Sfh4-Psd2 physical interaction. The other requires Stt4-mediated production of PtdIns-4-phosphate (PtdIns4P), where Stt4 (along with the Sac1 PtdIns4P phosphatase and endoplasmic reticulum–plasma membrane tethers) indirectly modulate Psd2 activity via a PtdIns4P homeostatic mechanism that influences PtdSer accessibility to Psd2. These results identify an example in which the biological function of a Sec14-like PITP is cleanly uncoupled from its canonical in vitro PtdIns-transfer activity and challenge popular functional assumptions regarding lipid-transfer protein involvements in MCS function.

Phosphatidylinositol-transfer protein and its homologues in yeast

2006 ◽  
Vol 34 (3) ◽  
pp. 377-380 ◽  
Author(s):  
P. Griac ◽  
R. Holic ◽  
D. Tahotna
Keyword(s):  
Membrane Trafficking ◽  
Diverse Group ◽  
Secretory Function ◽  
Lipid Transfer ◽  
Lipid Transfer Proteins ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylcholine Transfer Protein

Yeast Sec14p acts as a phosphatidylinositol/phosphatidylcholine-transfer protein in vitro. In vivo, it is essential in promoting Golgi secretory function. Products of five genes named SFH1–SFH5 (Sec Fourteen Homologues 1–5) exhibit significant sequence homology to Sec14p and together they form the Sec14p family of lipid-transfer proteins. It is a diverse group of proteins with distinct subcellular localizations and varied physiological functions related to lipid metabolism and membrane trafficking.

Rapid replenishment of sphingomyelin in the plasma membrane upon degradation by sphingomyelinase in NIH3T3 cells overexpressing the phosphatidylinositol transfer protein β

2000 ◽  
Vol 346 (2) ◽  
pp. 537-543 ◽  
Author(s):  
Claudia M. VAN TIEL ◽  
Chiara LUBERTO ◽  
Gerry T. SNOEK ◽  
Yusuf A. HANNUN ◽  
Karel W. A. WIRTZ
Keyword(s):  
Plasma Membrane ◽  
De Novo ◽  
Choline Chloride ◽  
Recovery Period ◽  
Nih3t3 Cells ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

In order to study the in vivo function of the phosphatidylinositol transfer protein β (PI-TPβ), mouse NIH3T3 fibroblasts were transfected with cDNA encoding mouse PI-TPβ. Two stable cell lines were isolated (SPIβ2 and SPIβ8) in which the levels of PI-TPβ were increased 16- and 11-fold respectively. The doubling time of the SPIβ cells was about 1.7 times that of the wild-type (wt) cells. Because PI-TPβ expresses transfer activity towards sphingomyelin (SM) in vitro, the SM metabolism of the overexpressors was investigated. By measuring the incorporation of [methyl-3H]choline chloride in SM and phosphatidylcholine (PtdCho), it was shown that the rate of de novo SM and PtdCho synthesis was similar in transfected and wt cells. We also determined the ability of the cells to resynthesize SM from ceramide produced in the plasma membrane by the action of bacterial sphingomyelinase (bSMase). In these experiments the cells were labelled to equilibrium (60 h) with [3H]choline. At relatively low bSMase concentrations (50 munits/ml), 50% of [3H]SM in wt NIH3T3 cells was degraded, whereas the levels of [3H]SM in SPIβ cells appeared to be unaffected. Since the release of [3H]choline phosphate into the medium was comparable for both wt NIH3T3 and SPIβ cells, these results strongly suggest that breakdown of SM in SPIβ cells was masked by rapid resynthesis of SM from the ceramide formed. By increasing the bSMase concentrations to 200 munits/ml, a 50% decrease in the level of [3H]SM in SPIβ cells was attained. During a recovery period of 6 h (in the absence of bSMase) the resynthesis of SM was found to be much more pronounced in these SPIβ cells than in 50% [3H]SM-depleted wt NIH3T3 cells. After 6 h of recovery about 50% of the resynthesized SM in the SPIβ cells was available for a second hydrolysis by bSMase. When monensin was present during the recovery period, the resynthesis of SM in bSMase-treated SPIβ cells was not affected. However, under these conditions 100% of the resynthesized SM was available for hydrolysis. On the basis of these results we propose that, under conditions where ceramide is formed in the plasma membrane, PI-TPβ plays an important role in restoring the steady-state levels of SM.

scholarly journals A Sec14-like phosphatidylinositol transfer protein paralog defines a novel class of heme-binding proteins

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Danish Khan ◽  
Dongju Lee ◽  
Gulcin Gulten ◽  
Anup Aggarwal ◽  
Joshua Wofford ◽  
...  
Keyword(s):  
Open Shell ◽  
Heme Binding ◽  
Phosphoinositide Signaling ◽  
Transfer Protein ◽  
Collective Data ◽  
Redox Active ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIII hemoprotein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.

scholarly journals A Sec14-like Phosphatidylinositol Transfer Protein Paralog Defines a Novel Class of Heme-binding Proteins With An Unusual Heme Coordination Mechanism

2020 ◽  
Author(s):  
Danish Khan ◽  
Dongju Lee ◽  
Gulcin Gulten ◽  
Anup Aggarwal ◽  
Joshua Wofford ◽  
...  
Keyword(s):  
Open Shell ◽  
Coordination Mechanism ◽  
Heme Binding ◽  
Phosphoinositide Signaling ◽  
Transfer Protein ◽  
Redox Active ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

AbstractYeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin FeIII heme-binding protein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities.

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

Cell ◽  
1993 ◽  
Vol 74 (5) ◽  
pp. 919-928 ◽  
Author(s):  
Geraint M.H. Thomas ◽  
Emer Cunningham ◽  
Amanda Fensome ◽  
Andrew Ball ◽  
Nicholas F. Totty ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Essential Role ◽  
Lipid Signaling ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer
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