Ligand and membrane-binding behavior of the phosphatidylinositol transfer proteins PITPα and PITPβ

2016 ◽  
Vol 94 (6) ◽  
pp. 528-533 ◽  
Author(s):  
Matilda Baptist ◽  
Candace Panagabko ◽  
Shamshad Cockcroft ◽  
Jeffrey Atkinson
Keyword(s):  
Transfer Rate ◽  
Resonance Energy ◽  
Lipid Vesicles ◽  
Lipid Transfer ◽  
Binding Behavior ◽  
Dual Polarization Interferometry ◽  
Phosphatidylinositol Transfer ◽  
Ligand Transfer ◽  
Phosphatidylinositol Transfer Proteins

Phosphatidylinositol transfer proteins (PITPs) are believed to be lipid transfer proteins because of their ability to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments, in vitro. However, the detailed mechanism of this transfer process is not fully established. To further understand the transfer mechanism of PITPs we examined the interaction of PITPs with membranes using dual polarization interferometry (DPI), which measures protein binding affinity on a flat immobilized lipid surface. In addition, a fluorescence resonance energy transfer (FRET)-based assay was also employed to monitor how quickly PITPs transfer their ligands to lipid vesicles. DPI analysis revealed that PITPβ had a higher affinity to membranes compared with PITPα. Furthermore, the FRET-based transfer assay revealed that PITPβ has a higher ligand transfer rate compared with PITPα. However, both PITPα and PITPβ demonstrated a preference for highly curved membrane surfaces during ligand transfer. In other words, ligand transfer rate was higher when the accepting vesicles were highly curved.

scholarly journals Phosphatidylinositol transfer protein β displays minimal sphingomyelin transfer activity and is not required for biosynthesis and trafficking of sphingomyelin

2002 ◽  
Vol 366 (1) ◽  
pp. 23-34 ◽  
Author(s):  
Bruno SÉGUI ◽  
Victoria ALLEN-BAUME ◽  
Shamshad COCKCROFT
Keyword(s):  
Intracellular Trafficking ◽  
Lipid Transfer ◽  
Transfer Protein ◽  
Golgi Membranes ◽  
Bound Lipids ◽  
Cellular Lipids ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Mammalian phosphatidylinositol transfer proteins (PITPs) α and β, which share 77% identity, have been shown to exhibit distinct lipid-transfer activities. In addition to transferring phosphatidylinositol (PI) and phosphatidylcholine (PC), PITPβ has been shown to transfer sphingomyelin (SM), and this has led to the suggestion that PITPβ is important for the regulation of SM metabolism. In the present study, we have analysed the ability of human PITPβ to transfer and regulate the metabolism of cellular SM. We report that, in vitro, the two PITP isoforms were comparable in mediating PI, PC or SM transfer. Using permeabilized HL-60 cells as the donor compartment, both PITP isoforms efficiently transferred PI and PC, and were slightly active towards SM, with the activity of PITPβ being slightly greater. To identify which cellular lipids were selected by PITPs, PITPα and PITPβ were exposed to permeabilized HL-60 cells, and subsequently repurified and analysed for their bound lipids. Both PITPs were able to select only PI and PC, but not SM. SM synthesis takes place at the Golgi, and PITPβ was shown to localize in that compartment. To examine the role of PITPβ in SM biosynthesis, Golgi membranes were used. Purified Golgi membranes had lost their endogenous PITPβ, but were able to recruit PITPβ when added exogenously. However, PITPβ did not enhance the activities of either SM synthase or glucosylceramide synthase. Further analysis in COS-7 cells overexpressing PITPβ showed no effects on (a) SM and glucosylceramide biosynthesis, (b) diacylglycerol or ceramide levels, (c) SM transport from the Golgi to the plasma membrane, or (d) resynthesis of SM after exogenous sphingomyelinase treatment. Altogether, these observations do not support the suggestion that PITPβ participates in the transfer of SM, the regulation of SM biosynthesis or its intracellular trafficking.

scholarly journals Phospholipid transfer proteins revisited

1997 ◽  
Vol 324 (2) ◽  
pp. 353-360 ◽  
Author(s):  
Karel. W. A WIRTZ
Keyword(s):  
Mammalian Cells ◽  
Lipid Transfer Protein ◽  
Fusion Reaction ◽  
Lipid Binding ◽  
Lipid Transfer ◽  
Cell Functions ◽  
Transfer Protein ◽  
Phospholipid Transfer ◽  
Phosphatidylinositol Transfer

Phosphatidylinositol transfer protein (PI-TP) and the non-specific lipid transfer protein (nsL-TP) (identical with sterol carrier protein 2) belong to the large and diverse family of intracellular lipid-binding proteins. Although these two proteins may express a comparable phospholipid transfer activity in vitro, recent studies in yeast and mammalian cells have indicated that they serve completely different functions. PI-TP (identical with yeast SEC14p) plays an important role in vesicle flow both in the budding reaction from the trans-Golgi network and in the fusion reaction with the plasma membrane. In yeast, vesicle budding is linked to PI-TP regulating Golgi phosphatidylcholine (PC) biosynthesis with the apparent purpose of maintaining an optimal PI/PC ratio of the Golgi complex. In mammalian cells, vesicle flow appears to be dependent on PI-TP stimulating phosphatidylinositol 4,5-bisphosphate (PIP2) synthesis. This latter process may also be linked to the ability of PI-TP to reconstitute the receptor-controlled PIP2-specific phospholipase C activity. The nsL-TP is a peroxisomal protein which, by its ability to bind fatty acyl-CoAs, is most likely involved in the β-oxidation of fatty acids in this organelle. This protein constitutes the N-terminus of the 58 kDa protein which is one of the peroxisomal 3-oxo-acyl-CoA thiolases. Further studies on these and other known phospholipid transfer proteins are bound to reveal new insights in their important role as mediators between lipid metabolism and cell functions.

The role of phosphatidylinositol-transfer proteins at membrane contact sites

2016 ◽  
Vol 44 (2) ◽  
pp. 419-424 ◽  
Author(s):  
Michael Selitrennik ◽  
Sima Lev
Keyword(s):  
Underlying Mechanism ◽  
Cellular Processes ◽  
Membrane Contact Sites ◽  
Contact Sites ◽  
Membrane Compartments ◽  
Potential Mode ◽  
Membrane Contact ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Phosphatidylinositol-transfer proteins (PITPs) have been initially identified as soluble factors that accelerate the monomeric exchange of either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane bilayers in vitro. They are highly conserved in eukaryotes and have been implicated in different cellular processes, including vesicular trafficking, signal transduction, and lipid metabolism. Recent studies suggest that PITPs function at membrane contact sites (MCSs) to facilitate the transport of PI from its synthesis site at the endoplasmic reticulum (ER) to various membrane compartments. In this review, we describe the underlying mechanism of PITPs targeting to MCSs, discuss their cellular roles and potential mode of action.

Role for Megakaryocyte Phosphatidylinositol Transfer Proteins-Alpha and -Beta in TGF Beta-Mediated Regulation of Hematopoietic Homeostasis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 780-780
Author(s):  
Hal E. Broxmeyer ◽  
Maegan L. Capitano ◽  
Liang Zhao ◽  
Scott Cooper ◽  
Charles S. Abrams
Keyword(s):  
Colony Formation ◽  
Mammalian Cells ◽  
Conflicts Of Interest ◽  
Specific Activity ◽  
High Specific Activity ◽  
Homeostatic Regulation ◽  
Hematopoietic Stem ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Abstract There are still unknowns regarding homeostatic regulation of hematopoietic stem (HSC) and progenitor (HPC) cells. Deciphering these processes are important for understanding and treating hematopoietic diseases. Phosphatidylinositol is a rare membrane structure lipid, but is critical for cellular signaling upon phosphorylation by lipid kinases to generate phosphoinositide. While phosphoinositide pathways contribute to events linked to the cytoskeleton, little is known of these pathways in regulating hematopoiesis. Critical to this pathway are phosphatidylinositol transfer proteins (PITPs) that in vitro enhance transfer of aqueous insoluble phosphatidylinositol from one membrane to another. Class I PITP proteins PITP α and β are highly conserved, small, and ubiquitously expressed in mammalian cells. To test the hypothesis that phosphatidylinositol signaling contributes to hematopoiesis, we generated conditional knock out mice that lack either PITPα single isoform (PITPαfl/fl PF4Cre+) or both PITPα and PITPβ (PITPαfl/fl βfl/fl PF4Cre+) specifically in their platelets and megakaryocytes, and observed a bone marrow (BM) HSC/HPC phenotype. BM from these mice and their littermate controls were evaluated for absolute numbers of nucleated cells, HSC, and HPC. Cells were analyzed by rigorous phenotyping for long-term (LT)-HSC, short-term (ST)-HSC, multipotential (MPP), common myeloid (CMP), and granulocyte macrophage (GMP) progenitors. They were also assessed for functional HPC by colony assays in vitro for multi-cytokine (Epo, GM-CSF, IL-3, SCF, hemin) stimulated granulocyte macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitors, and for their cycling status using a high specific activity tritiated thymidine kill assay. PITPα-/-, and to a greater extent PITPα/β-/-, progenitor cells demonstrated significant decreases in LT-HSC and ST-HSC per femur. While there were no significant changes in numbers of MPP, CMP, and GMP in the PITPα and PITPα/β-/- BM compared to controls, there were significant decreases of approximately 50% in numbers of CFU-GM, BFU-E, and CFU-GEMM per femur. PITC-/- HPC were in a slow or non-cycling state compared to the rapid cell cycle (40-57% in S-phase) of control HPC. Thus PITPα-/- and PITPα/β -/- BM cells were associated with decreased HSC and functional HPC numbers. To evaluate mechanisms for this phenotype, we focused on BM megakaryocytes, as they have been implicated in microenvironmental regulation of hematopoiesis, and PITPα and PITPα/β activities are associated with megakaryocyte/platelet function. BM derived TPO-culture expanded megakaryocytes were allowed to condition medium for 48 hours, and conditioned medium (CM) from PITPα-/-, PITPα/β-/-, and control BM megakaryocytes were assayed for effects on colony formation by multicytokine stimulated BM cells derived from normal mice. CM from PITPα-/- and PITPα/β-/- megakaryocytes, but not from control mice, significantly suppressed colony formation by CFU-GM, BFU-E and CFU-GEMM (by ~50%). Limiting dilution analysis of the CM demonstrated that PITPα/β-/- cells had more potent suppressor activity than PITPα-/- cells. Bioplex analysis of the CM from PITPα -/- and PITPα/β -/- megakaryocytes demonstrated significantly higher levels of cytokines/chemokines with known myelosuppressive activities (including: TNF-α, VEGF, LIF, IP-10, ENA-78, MDC, MIG, and MIP-1α). However, ELISA analysis of TGF-β1, demonstrated minimal protein in BM flushes from control mice, but large amounts of TGF-β (>350 pg/ml) in BM flushes from the PITPα/β -/- mice. CM from PITPα and α/β-/- megakaryocytes also contained highly elevated TGF-β protein. Thus, we hypothesized that the effect of PITP -/- on the suppression of HPC colony formation was mediated by TGF-β. The myelosuppressive CM derived from PITPα and PITPα/β -/- megakaryocytes was completely neutralized by a monoclonal TGF-β antibody. This demonstrates that PITPα and PITP α/β-/- megakaryocytes produce elevated TGF-β that at least in part, and possibly in synergy with other myelosuppressive cytokines/chemokines, decreases numbers of HSC and functional HPC. Our studies demonstrate a link between PITPα and α/β and TGF-β levels with significant effects on HSCs and HPCs, thus demonstrating involvement of the phosphoinositide pathway in homeostatic regulation of hematopoiesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phylogenetic analysis of plant multi-domain SEC14-like phosphatidylinositol transfer proteins and structure–function properties of PATELLIN2

2020 ◽  
Vol 104 (6) ◽  
pp. 665-678
Author(s):  
Karolin Montag ◽  
Jannik Hornbergs ◽  
Rumen Ivanov ◽  
Petra Bauer
Keyword(s):  
Structure Function ◽  
Green Algae ◽  
Stress Responses ◽  
Land Plants ◽  
Modular Structure ◽  
Flowering Plant ◽  
Cellular Interactions ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Abstract Key message SEC14L-PITPs guide membrane recognition and signaling. An increasingly complex modular structure of SEC14L-PITPs evolved in land plants compared to green algae. SEC14/CRAL-TRIO and GOLD domains govern membrane binding specificity. Abstract SEC14-like phosphatidylinositol transfer proteins (SEC14L-PITPs) provide cues for membrane identity by exchanging lipophilic substrates, ultimately governing membrane signaling. Flowering plant SEC14L-PITPs often have modular structure and are associated with cell division, development, and stress responses. Yet, structure–function relationships for biochemical–cellular interactions of SEC14L-PITPs are rather enigmatic. Here, we evaluate the phylogenetic relationships of the SEC14L-PITP superfamily in the green lineage. Compared to green algae, land plants have an extended set of SEC14L-PITPs with increasingly complex modular structure. SEC14-GOLD PITPs, present in land plants but not Chara, diverged to three functional subgroups, represented by the six PATELLIN (PATL) proteins in Arabidopsis. Based on the example of Arabidopsis PATL2, we dissect the functional domains for in vitro binding to phosphoinositides and liposomes and for plant cell membrane association. While the SEC14 domain and its CRAL-TRIO-N-terminal extension serve general membrane attachment of the protein, the C-terminal GOLD domain directs it to the plasma membrane by recognizing specific phosphoinositides. We discuss that the different domains of SEC14L-PITPs integrate developmental and environmental signals to control SEC14L-PITP-mediated membrane identity, important to initiate dynamic membrane events.

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.

Class I Phosphatidylinositol Transfer Proteins (PITPs) Promote Platelet Activation by Functioning As Essential Cofactors for Phospholipid Kinases and Hydrolases

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 91-91
Author(s):  
Liang Zhao ◽  
Lurong Lian ◽  
Aae Suzuki ◽  
Charles S. Abrams
Keyword(s):  
Platelet Activation ◽  
Kinase Activity ◽  
Ex Vivo ◽  
Class I ◽  
Null Mutation ◽  
Exogenous Substrate ◽  
Phosphatidylinositol Transfer ◽  
Phosphatidylinositol Transfer Proteins

Abstract Abstract 91 In contrast to the more abundant phospholipids within the platelet membrane bilayer, phosphatidylinositol (PI) can be phosphorylated by PI kinases to generate seven distinct phosphoinositides that function as signaling molecules in series of cellular events including platelet activation. The synthesis of individual phosphoinositides in different cellular compartments is tightly regulated both in time and in space, and the relative amount of various phosphoinositides change within a few seconds after agonist stimulation of platelets. Thus, demonstrating how the synthesis of specific phosphoinositides is regulated in platelet activation would be critical to understanding their role in platelet biology. Class I PhosphatidylInositol Transfer Proteins (PITPs) are a small family of proteins that bind and transfer PI monomers from one cellular compartment to another in vitro. Studies in yeast cells suggested that PITP proteins are essential for the biosynthesis of phosphoinositides. Mammalian cells class I PITP has two members, PITPα and PITPβ. These two isoforms are 77% identical in primary sequence and are 94% homologous. It is notable that PITPα is approximately 7-fold more abundant than PITPβ in murine platelets. To characterize the role of each PITP isoform in platelet activation, we generated mice containing conditional null mutations within the gene of each isoform specifically in their megakaryocytes, and consequently to knock out these proteins in their platelets. Mice lacking individual platelet PITP isoforms have approximately 25% lower platelet counts than their littermate controls. Mice lacking both PITP isoforms have platelet counts that are 45% lower than wild type littermates, but otherwise have normal blood counts and appear phenotypically normal. Although the loss of either PITP isoform caused only mild ex vivo platelet function defects, loss of both isoforms led to significant impairment of cell spreading, aggregation, and secretion. To distinguish the role of both PITP isoforms in platelet phosphoinositide production, we 32P-labeled platelets ex vivo, and then analyzed by thin layer chromatography the concentration of individual phosphoinositides. Despite the fact that PITPα is far more abundant than PITPβ, we found that the loss of either isoform impaired the synthesis of PI(4)P and PI(4,5)P2 by 40–50% in either resting or thrombin stimulated platelets. To determine whether PITPs mediate their effect on phosphoinositide synthesis via PI transfer activity, we analyzed in vitro phospholipid kinase activity in lysates of knockout platelets using either PI or PI(4)P as the exogenous substrate. We reasoned that providing abundant quantities of exogenous substrate should eliminate the need for any transfer activity, and any effect of PITP on phosphoinositide synthesis in this circumstance would instead be due to an effect of PITPs on phospholipid kinase activity. We observed that the loss of either PITPα or PITPβ resulted in decreased synthesis of PIP and PI(4,5)P2 in vitro. Interestingly, even though PITPβ is far less abundant than PITPα, PITPβ is required for the majority (approximately 70%) of thrombin induced PI(4,5)P2 synthesis in vitro. In contrast, PITPα is required for 60% of thrombin induced PI(4)P synthesis. As expected, we could reverse the phosphoinositide synthesis defect by adding back recombinant PITPα to the PITPα-null platelet lysates or by adding back recombinant PITPβ to the PITPβ-null platelet lysates. Finally, we analyzed whether the loss of either PITP isoform affected the ability of the production of the second messenger IP3 (a product of PLC-mediated hydrolysis of PI(4,5)P2). We observed that the PITPα-null mutation caused an 80% decrease of thrombin-induced IP3 formation, and the PITPβ-null mutation caused a 56% loss in IP3 production. Together, the data demonstrate that although both PITPα and PITPβ are required for phosphoinositides synthesis and IP3 formation, they appear to have non-redundant functions. PITPα plays a larger role in maintaining PI(4)P levels and PLC signaling, and PITPβ contributes more to PI(4,5)P2 synthesis. Both isoforms cooperate together to promote platelet activation. Most importantly, our work demonstrates that despite their name, PhosphatidylInositol Transfer Proteins (PITPs) do more than just transfer phospholipids. They also possess critical cofactor activity for the synthesis of phosphoinositides. Disclosures: No relevant conflicts of interest to declare.

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