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. 2005 Oct 15;391(Pt 2):231-8.
doi: 10.1042/BJ20051001.

High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate

Dermott W O'Callaghan  1 , Lee P Haynes, Robert D Burgoyne
Affiliations

High-affinity interaction of the N-terminal myristoylation motif of the neuronal calcium sensor protein hippocalcin with phosphatidylinositol 4,5-bisphosphate

Dermott W O'Callaghan et al. Biochem J. .

Abstract

Many proteins are associated with intracellular membranes due to their N-terminal myristoylation. Not all myristoylated proteins have the same localization within cells, indicating that other factors must determine their membrane targeting. The NCS (neuronal calcium sensor) proteins are a family of Ca2+-binding proteins with diverse functions. Most members of the family are N-terminally myristoylated and are either constitutively membrane-bound or have a Ca2+/myristoyl switch that allows their reversible membrane association in response to Ca2+ signals. In the case of hippocalcin and NCS-1, or alternatively KChIP1 (K+ channel-interacting protein 1), their N-terminal myristoylation motifs are sufficient for targeting to distinct organelles. We have shown that an N-terminal myristoylated hippocalcin peptide is able to specifically reproduce the membrane targeting of hippocalcin/NCS-1 when introduced into permeabilized cells. The peptide binds to liposomes containing phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] with high affinity (K(d) 50 nM). Full-length hippocalcin also bound preferentially to liposomes supplemented with PtdIns(4,5)P2. Co-expression of hippocalcin-(1-14)-ECFP (enhanced cyan fluorescent protein) or NCS-1-ECFP partially displaced the expressed PH (pleckstrin homology) domain of phospholipase delta1 from the plasma membrane in live cells, indicating that they have a higher affinity for PtdIns(4,5)P2 than does this PH domain. The Golgi localization of the PH domain of FAPP1 (four-phosphate-adaptor protein 1), which binds to phosphatidylinositol 4-phosphate, was unaffected. The localization of NCS-1 and hippocalcin is likely to be determined, therefore, by their interaction with PtdIns(4,5)P2.

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Figures

Figure 1
Figure 1. Targeting via the myristoylation motifs of hippocalcin and KChIP1
(A) Comparison of the N-terminal sequences of human NCS-1, hippocalcin and KChIP1. Identical residues are boxed in red, and similar residues in yellow. (B) HeLa cells were co-transfected to express NCS-1 and hippocalcin-(1–14)–EGFP, fixed, and NCS-1 was detected by immunofluorescence. The overlapping localization is seen as yellow in the overlay image [NCS-1 in red, hippocalcin-(1–14)–EGFP in green]. (C) HeLa cells were co-transfected to express NCS-1–ECFP and KChIP1-(1–11)–EYFP. The overlay image shows NCS-1–ECFP in green and KChIP1-(1–11)–EYFP in red. The scale bar represents 10 μm.
Figure 2
Figure 2. Intracellular localization of biotinylated peptides
(A) Synthetic peptides used in the study. (B) Myr-hip-(2–14), corresponding to the N-terminus of the hippocalcin, was incubated with digitonin-permeabilized HeLa cells transfected with NCS-1–ECFP (i) or hipp(1–14)–EGFP (ii). Non-myristoylated, biotinylated hippocalcin peptide [hippocalcin-(2–14)] was also incubated with NCS-1–ECFP-transfected cells (iii). Myristoylated KChIP1 peptide was incubated with cells transfected with KChIP-1–EYFP. The biotinylated peptides were detected using Texas Red–streptavidin. Overlay images are displayed in colour, with expressed transfected constructs in green and peptides in red, and with regions of co-localization in yellow. The scale bar represents 10 μm.
Figure 3
Figure 3. Determination of the binding of peptides to immobilized phospholipids
The binding of peptides to phospholipids was determined using PIP-Strip™ binding. Positions of immobilized lipid species are indicated (left panel). Myr-hip-(2–14), hip-(2–14) or myristoylated KChIP1-(2–13) was incubated with PIP-Strip™ membranes and bound peptide was detected with streptavidin–horseradish peroxidase. The myr-hip-(2–14) peptide interacted with a number of immobilized lipid species. No binding of hippocalcin-(2–14) peptide or myr-KChIP1-(2–13) peptide to lipids was observed.
Figure 4
Figure 4. Binding of the myr-hip-(2–14) peptide to liposomes of various compositions
(A) Binding assay for analysis of peptide interaction with liposomes. The indicated peptides were incubated with or without liposomes. After centrifugation, sedimented material was spotted on to nitrocellulose filters, and bound peptide was detected by staining with Ponceau S. (B) Analysis of peptide binding to liposomes by quantitative densitometry. CES liposomes supplemented with 10% of the indicated PtdIns (PI) lipid were incubated with 90 nM myr-hip-(2–14). The highest level of binding was to liposomes supplemented with PtdIns(4,5)P2. PA, phosphatidic acid. (C) Determination of the affinity of myr-hip-(2–14) peptide for liposomes supplemented with PtdIns, PtdIns4P and PtdIns(4,5)P2. An increasing concentration of peptide was incubated in the presence of a fixed liposome concentration, and bound peptide was quantified by densitometry and comparison with that bound to control liposomes.
Figure 5
Figure 5. Binding of radiolabelled in vitro synthesized hippocalcin to liposomes
(A) Radiolabelled products from control and hippocalcin in vitro transcription/translation reactions. (B) Analysis of hippocalcin binding to liposomes. CES liposomes were supplemented with 10% of the indicated PtdIns (PI) lipid. After incubation with 10 μl of radiolabelled hippocalcin per reaction, the liposomes were washed and bound protein determined. (C) Binding of increasing amounts of radiolabelled hippocalcin from the in vitro transcription/translation mixture to CES liposomes supplemented with PtdIns(4,5)P2 in the presence of EGTA or with 1 μM free Ca2+.
Figure 6
Figure 6. Effects of expression of NCS-1 or the hippocalcin myristoylation motif on the plasma membrane association of PLCδ1-PH or the Golgi complex association of FAPP1-PH
HeLa cells were transfected to express GFP–PLCδ1-PH alone (A) or GFP–FAPP1-PH alone (B), or co-transfected with GFP–PLCδ1-PH plus NCS-1–ECFP (C, D) or hippocalcin-(1–14)–ECFP (E, F). Other HeLa cells were transfected to express GFP–FAPP1-PH along with NCS-1–ECFP (G, H) or hippocalcin-(1–14)–ECFP (I, J). The percentage of fluorescence due to GFP–PLCδ1-PH at the cell periphery in cells expressing GFP–PLCδ1-PH alone (control) or in the presence of NCS-1–ECFP or hippocalcin-(1–14)–ECFP was quantified (K). The scale bar represents 10 μm. Samples of cells transfected to express GFP–PLCδ1-PH, NCS-1–ECFP or hippocalcin-(1–14)–ECFP, singly or in combination, were probed by Western blotting using anti-GFP (L).
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References

    1. Resh M. D. Fatty acylation of proteins: new insights into membrane targeting of myristoylated and palmitoylated proteins. Biochim. Biophys. Acta. 1999;1451:1–16. - PubMed
    1. McCabe J. B., Berthiaume L. G. Functional roles for fatty acylated amino-terminal domains in subcellular localization. Mol. Biol. Cell. 1999;10:3771–3786. - PMC - PubMed
    1. McCabe J. B., Berthiaume L. G. N-terminal protein acylation confers localization to cholesterol, sphingolipid-enriched membranes but not lipid rafts/caveolae. Mol. Biol. Cell. 2001;12:3601–3617. - PMC - PubMed
    1. Burgoyne R. D., Weiss J. L. The neuronal calcium sensor family of Ca2+-binding proteins. Biochem. J. 2001;353:1–12. - PMC - PubMed
    1. Burgoyne R. D., O'Callaghan D. W., Hasdemir B., Haynes L. P., Tepikin A. V. Neuronal calcium sensor proteins: multitalented regulators of neuronal function. Trends Neurosci. 2004;27:203–209. - PubMed

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