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 Localization of Drosophila retinal degeneration B, a membrane-associated phosphatidylinositol transfer protein

1993 ◽  
Vol 122 (5) ◽  
pp. 1013-1022 ◽  
Author(s):  
TS Vihtelic ◽  
M Goebl ◽  
S Milligan ◽  
JE O'Tousa ◽  
DR Hyde
Keyword(s):  
Retinal Degeneration ◽  
Amino Terminal ◽  
Transfer Protein ◽  
Phosphatidylinositol Transfer Protein ◽  
Protein Functions ◽  
Drosophila Brain ◽  
Phosphatidylinositol Transfer ◽  
Acid Domain ◽  
Membrane Spanning

The Drosophila retinal degeneration B (rdgB) mutation causes abnormal photoreceptor response and light-enhanced retinal degeneration. Immunoblots using polyclonal anti-rdgB serum showed that rdgB is a 160-kD membrane protein. The antiserum localized the rdgB protein in photoreceptors, antennae, and regions of the Drosophila brain, indicating that the rdgB protein functions in many sensory and neuronal cells. In photoreceptors, the protein localized adjacent to the rhabdomeres, in the vicinity of the subrhabdomeric cisternae. The rdgB protein's amino-terminal 281 residues are > 40% identical to the rat brain phosphatidylinositol transfer protein (PI-TP). A truncated rdgB protein, which contains only this amino-terminal domain, possesses a phosphatidylinositol transfer activity in vitro. The remaining 773 carboxyl terminal amino acids have additional functional domains. Nitrocellulose overlay experiments reveal that an acidic amino acid domain, adjacent to the PI transfer domain, binds 45Ca+2. Six hydrophobic segments are found in the middle of the putative translation product and likely function as membrane spanning domains. These results suggest that the rdgB protein, unlike the small soluble PI-TPs, is a membrane-associated PI-TP, which may be directly regulated by light-induced changes in intracellular calcium.

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 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 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.

scholarly journals Isolation and Characterization of Drosophila retinal degeneration B Suppressors

Genetics ◽  
1999 ◽  
Vol 151 (2) ◽  
pp. 713-724 ◽  
Author(s):  
Don W Paetkau ◽  
Vecheslav A Elagin ◽  
Lisa M Sendi ◽  
David R Hyde
Keyword(s):  
Retinal Degeneration ◽  
Light Adaptation ◽  
Photoreceptor Cell ◽  
Light Response ◽  
Visual Transduction ◽  
Transfer Protein ◽  
Isolation And Characterization ◽  
Phosphatidylinositol Transfer ◽  
Normal Light

Abstract The Drosophila retinal degeneration B protein (RdgB) is a novel integral membrane phosphatidylinositol transfer protein required for photoreceptor cell viability and light response. We isolated one intragenic suppressor (rdgBsu100) and four autosomal suppressors of the hypomorphic rdgBKS222 retinal degeneration phenotype. The rdgBsu100 suppressor dramatically slowed rdgBKS222's photoreceptor degeneration without significantly improving the electroretinogram (ERG) light response. One autosomal recessive suppressor [su(rdgB)69] significantly slowed rdgBKS222 retinal degeneration and restored the ERG light response near to that of the wild type. Unlike all the previously characterized rdgB suppressors, the four new autosomal suppressors do not affect the ERG light response in rdgB+ flies. Only Su(rdgB)116 exhibited a mutant phenotype in a rdgB+ background, which was smaller R1-6 rhabdomeres. We also examined the extent to which two previously identified visual transduction mutations suppressed rdgB retinal degeneration. Absence of one of the light-activated calcium channels (trpCM) slowed the onset of rdgB-dependent degeneration. However, loss of protein kinase C (inaC209), which blocks photoreceptor cell deactivation, desensitization, and light adaptation, failed to suppress rdgB degeneration under normal light conditions. This demonstrates that TRP activity, but not INAC, is required for rapid rdgB-dependent degeneration.

scholarly journals Activity of phosphatidylinositol transfer protein is sensitive to ethanol and membrane curvature

2000 ◽  
Vol 348 (3) ◽  
pp. 667-673 ◽  
Author(s):  
Hiroaki KOMATSU ◽  
Barend BOUMA ◽  
Karel W. A. WIRTZ ◽  
Theodore F. TARASCHI ◽  
Nathan JANES
Keyword(s):  
Membrane Lipids ◽  
Microsomal Protein ◽  
Membrane Curvature ◽  
Unilamellar Vesicles ◽  
Transfer Protein ◽  
Drug Concentrations ◽  
Hepatic Microsomes ◽  
Phosphatidylinositol Transfer Protein ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer protein (PITP) is critical for many cellular signalling and trafficking events that are influenced by ethanol. The influence of ethanol and membrane curvature on the activity of recombinant mouse PITP-α in vitro is evaluated by monitoring the transfer of phosphatidylinositol (PtdIns) from rat hepatic microsomes to unilamellar vesicles. Acute exposure to pharmacological levels of ethanol enhanced the function of PITP. Chloroform shared a similar ability to enhance function when both drug concentrations were normalized to their respective octanol/water partition coefficients, indicating that the effect is not unique to ethanol and might be common to hydrophobic solutes. Neither the PITP activity nor its ethanol enhancement was altered by using thermally pretreated (denatured) or protease-treated microsomes, indicating that the native microsomal protein structure was unlikely to be a determinant of transfer. Kinetic analyses indicated that ethanol acted by increasing the PITP-mediated flux of PtdIns from both microsomal and liposomal surfaces. The activity of PITP was strongly dependent on the lipid structure, with a steep dependence on the expressed curvature of the membrane. Activity was greatest for small, highly curved sonicated vesicles and decreased markedly for large, locally planar unilamellar vesicles. Ethanol enhanced PITP-mediated PtdIns transfer to all vesicles, but its effect was much smaller than the enhancement due to curvature, which is consistent with ethanol's comparatively modest ability to perturb membrane lipids. The ethanol efficacy observed is as pronounced as any previously described lipid-mediated ethanol action. In addition, these observations raise the possibility that PITP specifically delivers PtdIns to metabolically active membrane domains of convex curvature and/or low surface densities of lipid.

scholarly journals Evidence that mammalian phosphatidylinositol transfer protein regulates phosphatidylcholine metabolism

1998 ◽  
Vol 335 (1) ◽  
pp. 175-179 ◽  
Author(s):  
Marie E. MONACO ◽  
Richard J. ALEXANDER ◽  
Gerry T. SNOEK ◽  
Nancy H. MOLDOVER ◽  
Karel W. A. WIRTZ ◽  
...  
Keyword(s):  
Physiological Role ◽  
Fold Increase ◽  
Transfer Protein ◽  
Antisense Mrna ◽  
Cell Clones ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer proteins (PITPs) and their yeast counterpart (SEC14p) possess the ability to bind phosphatidylinositol (PtdIns) and transfer it between membranes in vitro. However, the biochemical function of these proteins in vivo is unclear. In the present study, the physiological role of PITP was investigated by determining the biochemical consequences of lowering the cellular content of this protein. WRK-1 rat mammary tumour cells were transfected with a plasmid containing a full-length rat PITPα cDNA inserted in the antisense orientation and the resultant cell clones were analysed. Three clones expressing antisense mRNA for PITPα were compared with three clones transfected with the expression vector lacking the insert. The three antisense clones had an average of 25% less PITPα protein than control clones. Two of the three antisense clones also exhibited a decreased rate of growth. All three antisense clones exhibited a significant decrease in the incorporation of labelled precursors into PtdCho during a 90-min incubation period. Under the same conditions, however, there was no change in precursor incorporation into PtdIns. Further experimentation indicated that the decrease in precursor incorporation seen in antisense clones was not due to an increased rate of turnover. When choline metabolism was analysed more extensively in one control (2-5) and one antisense (4-B) clone using equilibrium-labelling conditions (48 h of incubation), the following were observed: (1) the decrease in radioactive labelling of PtdCho seen in short-term experiments was also observed in long-term experiments, suggesting that the total amount of PtdCho was lower in antisense-transfected clones (this was confirmed by mass measurements); (2) a similar decrease was seen in cellular sphingomyelin, lysoPtdCho and glycerophosphorylcholine; (3) an average two-fold increase in cellular phosphorylcholine was observed in the antisense-transfected clone; (4) cellular choline was, on average, decreased; and (5) cellular CDPcholine was not significantly altered.

scholarly journals Identification of a Novel Family of Nonclassic Yeast Phosphatidylinositol Transfer Proteins Whose Function Modulates Phospholipase D Activity and Sec14p-independent Cell Growth

2000 ◽  
Vol 11 (6) ◽  
pp. 1989-2005 ◽  
Author(s):  
Xinmin Li ◽  
Sheri M. Routt ◽  
Zhigang Xie ◽  
Xiaoxia Cui ◽  
Min Fang ◽  
...  
Keyword(s):  
Cell Growth ◽  
Phospholipase D ◽  
Sequence Similarity ◽  
Primary Sequence ◽  
Transfer Protein ◽  
Sequence Identity ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24–65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet,SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate thatSFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with “bypass-Sec14p” mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

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.

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