The lipid transfer activity of phosphatidylinositol transfer protein is sufficient to account for enhanced phospholipase C activity in turkey erythrocyte ghosts

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.

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Modifications of Cysteine Residues in the Solution and Membrane-Associated Conformations of Phosphatidylinositol Transfer Protein Have Differential Effects on Lipid Transfer Activity†

Biochemistry ◽

10.1021/bi0107896 ◽

2001 ◽

Vol 40 (31) ◽

pp. 9151-9158 ◽

Cited By ~ 4

Author(s):

Jacqueline M. Tremblay ◽

Hong Li ◽

Lynwood R. Yarbrough ◽

George M. Helmkamp

Keyword(s):

Cysteine Residues ◽

Lipid Transfer ◽

Differential Effects ◽

Transfer Protein ◽

Transfer Activity ◽

Phosphatidylinositol Transfer Protein ◽

Phosphatidylinositol Transfer

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

Cell ◽

10.1016/0092-8674(93)90471-2 ◽

1993 ◽

Vol 74 (5) ◽

pp. 919-928 ◽

Cited By ~ 158

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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Limited Proteolysis of Rat Phosphatidylinositol Transfer Protein by Trypsin Cleaves the C Terminus, Enhances Binding to Lipid Vesicles, and Reduces Phospholipid Transfer Activity

Journal of Biological Chemistry ◽

10.1074/jbc.271.35.21075 ◽

1996 ◽

Vol 271 (35) ◽

pp. 21075-21080 ◽

Cited By ~ 15

Author(s):

Jacqueline M. Tremblay ◽

Helmkamp M. George ◽

Lynwood R. Yarbrough

Keyword(s):

Limited Proteolysis ◽

Lipid Vesicles ◽

Transfer Protein ◽

Transfer Activity ◽

C Terminus ◽

Phosphatidylinositol Transfer Protein ◽

Phospholipid Transfer ◽

Phosphatidylinositol Transfer

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Phosphorylation of a Distinct Structural Form of Phosphatidylinositol Transfer Protein α at Ser166by Protein Kinase C Disrupts Receptor-mediated Phospholipase C Signaling by Inhibiting Delivery of Phosphatidylinositol to Membranes

Journal of Biological Chemistry ◽

10.1074/jbc.m405827200 ◽

2004 ◽

Vol 279 (45) ◽

pp. 47159-47171 ◽

Cited By ~ 20

Author(s):

Clive P. Morgan ◽

Alison Skippen ◽

Bruno Segui ◽

Andrew Ball ◽

Victoria Allen-Baume ◽

...

Keyword(s):

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Structural Form ◽

Transfer Protein ◽

Kinase C ◽

Phosphatidylinositol Transfer Protein ◽

Phosphatidylinositol Transfer

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[13] Purification of phosphatidylinositol transfer protein from brain cytosol for reconstituting G-protein-regulated phosphoinositide-specific phospholipase C-β

Heterotrimeric G-Protein Effectors - Methods in Enzymology ◽

10.1016/0076-6879(94)38015-5 ◽

1994 ◽

pp. 168-181 ◽

Cited By ~ 2

Author(s):

Geraint M.H. Thomas ◽

Emer Cunningham ◽

Shamshad Cockcroft

Keyword(s):

Phospholipase C ◽

G Protein ◽

Transfer Protein ◽

Phosphatidylinositol Transfer Protein ◽

Phosphatidylinositol Transfer ◽

Brain Cytosol

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

The Journal of Cell Biology ◽

10.1083/jcb.201907128 ◽

2020 ◽

Vol 219 (5) ◽

Cited By ~ 2

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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