Identification of an 11-residue portion of CTP–phosphocholine cytidylyltransferase that is required for enzyme–membrane interactions

CTP–phosphocholine cytidylyltransferase (CT) is a key regulatory enzyme in the biosynthesis of phosphatidylcholine (PC) in many cells. Enzyme–membrane interactions appear to play an important role in CT activation. A putative membrane-binding domain appears to be located between residues 236 and 293 from the N-terminus. To map the membrane-binding domain more precisely, glutathione S-transferase fusion proteins were prepared that contained deletions of various domains in this putative lipid-binding region. The fusion proteins were assessed for their binding of [3H]PC/oleic acid vesicles. Fusion proteins encompassing residues 267–277 bound to PC/oleic acid vesicles, whereas fragments lacking this region exhibited no specific binding to the lipid vesicles. The membrane-binding characteristics of the CT fusion proteins were also examined using intact lung microsomes. Only fragments encompassing residues 267–277 competed with full-length 125I-labelled CT, expressed in recombinant Sf9 insect cells, for microsomal membrane binding. To investigate the role of this region in PC biosynthesis, A549 and L2 cells were transfected with cDNA for CT mutants under the control of a glucocorticoid-inducible long terminal repeat (LTR) promoter. Induction of CT mutants containing residues 267–277 in transfectants resulted in reduced PC synthesis. The decrease in PC synthesis was accompanied by a shift in endogenous CT activity from the particulate to the soluble fraction. Expression of CT mutants lacking this region in A549 and L2 cells did not affect PC formation and subcellular distribution of CT activity. These results suggest that the CT region located between residues 267 and 277 from the N-terminus is required for the interaction of CT with membranes.

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Identification of a nuclear localization signal in the Plasmodium falciparum CTP: phosphocholine cytidylyltransferase enzyme

Scientific Reports ◽

10.1038/s41598-020-76829-1 ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Richard Izrael ◽

Lívia Marton ◽

Gergely N. Nagy ◽

Hajnalka L. Pálinkás ◽

Nóra Kucsma ◽

...

Keyword(s):

Plasmodium Falciparum ◽

Cell Line ◽

Nuclear Localization ◽

Nuclear Localization Signal ◽

Membrane Binding ◽

Binding Domain ◽

Regulatory Function ◽

Phosphocholine Cytidylyltransferase ◽

Phosphatidylcholine Biosynthesis ◽

Localization Signal

AbstractThe phospholipid biosynthesis of the malaria parasite, Plasmodium falciparum is a key process for its survival and its inhibition is a validated antimalarial therapeutic approach. The second and rate-limiting step of the de novo phosphatidylcholine biosynthesis is catalysed by CTP: phosphocholine cytidylyltransferase (PfCCT), which has a key regulatory function within the pathway. Here, we investigate the functional impact of the key structural differences and their respective role in the structurally unique pseudo-heterodimer PfCCT protein in a heterologous cellular context using the thermosensitive CCT-mutant CHO-MT58 cell line. We found that a Plasmodium-specific lysine-rich insertion within the catalytic domain of PfCCT acts as a nuclear localization signal and its deletion decreases the nuclear propensity of the protein in the model cell line. We further showed that the putative membrane-binding domain also affected the nuclear localization of the protein. Moreover, activation of phosphatidylcholine biosynthesis by phospholipase C treatment induces the partial nuclear-to-cytoplasmic translocation of PfCCT. We additionally investigated the cellular function of several PfCCT truncated constructs in a CHO-MT58 based rescue assay. In absence of the endogenous CCT activity we observed that truncated constructs lacking the lysine-rich insertion, or the membrane-binding domain provided similar cell survival ratio as the full length PfCCT protein.

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The Ca2+-dependent lipid binding domain of P120GAP mediates protein-protein interactions with Ca2+-dependent membrane-binding proteins. Evidence for a direct interaction between annexin VI and P120GAP.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)78723-7 ◽

1996 ◽

Vol 271 (52) ◽

pp. 33705

Author(s):

Alison J. Davis ◽

Jonathan T. Butt ◽

John H. Walker ◽

Stephen E. Moss ◽

Debra J. Gawler

Keyword(s):

Protein Interactions ◽

Binding Proteins ◽

Membrane Binding ◽

Direct Interaction ◽

Lipid Binding ◽

Binding Domain ◽

Protein Protein Interactions ◽

Annexin Vi

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The Ca2+-dependent Lipid Binding Domain of P120GAPMediates Protein-Protein Interactions with Ca2+-dependent Membrane-binding Proteinss

Journal of Biological Chemistry ◽

10.1074/jbc.271.40.24333 ◽

1996 ◽

Vol 271 (40) ◽

pp. 24333-24336 ◽

Cited By ~ 43

Author(s):

Alison J. Davis ◽

Jonathan T. Butt ◽

John H. Walker ◽

Stephen E. Moss ◽

Debra J. Gawler

Keyword(s):

Protein Interactions ◽

Membrane Binding ◽

Lipid Binding ◽

Binding Domain ◽

Protein Protein Interactions

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Analysis of the N-terminal binding domain of Goα

Biochemical Journal ◽

10.1042/bj3280023 ◽

1997 ◽

Vol 328 (1) ◽

pp. 23-31 ◽

Cited By ~ 11

Author(s):

Liliana BUSCONI ◽

M. Bradley DENKER

Keyword(s):

Amino Acids ◽

Cultured Cells ◽

Membrane Binding ◽

Receptor Activation ◽

Binding Domain ◽

Membrane Receptors ◽

Membrane Interactions ◽

Cos Cells ◽

Vitro Analysis

Signalling from membrane receptors through heterotrimeric G-proteins (Gα and Gβγ) to intracellular effectors is a highly regulated process. Receptor activation causes exchange of GTP for GDP on Gα and dissociation of Gα from Gβγ. Both subunits remain membrane-associated and interact with a series of other molecules throughout the cycle of activation. The N-terminal binding domain of Gα subunits interacts with the membrane by several partially defined mechanisms: the anchoring of Gα to the more hydrophobic Gβγ subunits, the interaction of N-terminal lipids (palmitate and/or myristate) with the membrane, and attachment of amino acid regions to the membrane {amino acids 11-14 of Goα (D[11-14]); Busconi, Boutin and Denker (1997) Biochem. J. 323, 239-244}. We characterized N-terminal mutants of Goα with known Gβγ-binding properties for the ability to interact with phospholipid vesicles and membranes prepared from cultured cells (acceptor membranes). In vitro analysis allows membrane interactions that are important to the activated and depalmitoylated state of Gα to be characterized. Subcellular localization was also determined in transiently transfected COS cells. All of the mutant proteins are myristoylated, and differences in myristoylation do not account for changes in membrane binding. Disrupting the N-terminal α-helix of Goα with a proline point mutation at Arg-9 (R9P) does not affect interactions with Gβγ on sucrose-density gradients but significantly reduces acceptor membrane binding. Deletion of amino acids 6-15 (D[6-15]; reduced Gβγ binding) or deletion of amino acids 3-21 (D[3-21]); no detectable Gβγ binding) further reduces acceptor membrane binding. When expressed in COS cells, R9P and D[6-15] are localized in the membrane similar to wild-type Goα as a result of the contribution from palmitoylation. In contrast, D[3-21] is completely soluble in COS cells, and no palmitoylation is detected. The binding of Goα and mutants translated in vitro to liposomes indicates that Goα preferentially binds to neutral phospholipids (phosphatidylcholine). R9P and D[11-14] bind to phosphatidylcholine liposomes like Goα, but D[6-15] exhibits no detectable binding. Taken together, these studies suggest that interactions of the N-terminus of Gα subunits with the membrane may be affected by both membrane proteins and lipids. A detailed understanding of Gα-membrane interactions may reveal unique mechanisms for regulating signal transduction.

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Conformation and Lipid Binding Properties of Four Peptides Derived from the Membrane-Binding Domain of CTP:Phosphocholine Cytidylyltransferase†

Biochemistry ◽

10.1021/bi980340l ◽

1998 ◽

Vol 37 (26) ◽

pp. 9509-9519 ◽

Cited By ~ 39

Author(s):

Joanne E. Johnson ◽

N. Madhusudhana Rao ◽

Sek-Wen Hui ◽

Rosemary B. Cornell

Keyword(s):

Membrane Binding ◽

Lipid Binding ◽

Binding Domain ◽

Binding Properties ◽

Ctp:Phosphocholine Cytidylyltransferase

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Receptor-dependent compartmentalization of PPIP5K1, a kinase with a cryptic polyphosphoinositide binding domain

Biochemical Journal ◽

10.1042/bj20101437 ◽

2011 ◽

Vol 434 (3) ◽

pp. 415-426 ◽

Cited By ~ 30

Author(s):

Nikhil A. Gokhale ◽

Angelika Zaremba ◽

Stephen B. Shears

Keyword(s):

Mammalian Cells ◽

Consensus Sequence ◽

Lipid Vesicles ◽

Lipid Binding ◽

Binding Domain ◽

Site Directed Mutagenesis ◽

Inositol Pyrophosphate ◽

Signalling Molecules ◽

Catalytically Active ◽

Inositol Pyrophosphates

The inositol pyrophosphates are multifunctional signalling molecules. One of the families of enzymes that synthesize the inositol pyrophosphates are the Vip1/PPIP5Ks (PP-InsP5 kinases). The kinase domains in Vip1/PPIP5Ks have been mapped to their N-terminus. Each of these proteins also possess a phosphatase-like domain of unknown significance. In the present study, we show that this phosphatase-like domain is not catalytically active. Instead, by using SPR (surface plasmon resonance) to study protein binding to immobilized lipid vesicles, we show that this domain is specialized for binding PtdIns(3,4,5)P3 (PPIP5K1 Kd=96 nM; PPIP5K2 Kd=705 nM). Both PtdIns(3,4)P2 and PtdIns(4,5)P2 are significantly weaker ligands, and no significant binding of PtdIns(3,5)P2 was detected. We confirm the functional importance of this domain in inositol lipid binding by site-directed mutagenesis. We present evidence that the PtdIns(3,4,5)P3-binding domain is an unusual hybrid, in which a partial PH (pleckstrin homology) consensus sequence is spliced into the phosphatase-like domain. Agonist-dependent activation of the PtdIns 3-kinase pathway in NIH 3T3 cells drives translocation of PPIP5K1 from the cytosol to the plasma membrane. We have therefore demonstrated receptor-regulated compartmentalization of inositol pyrophosphate synthesis in mammalian cells.

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