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Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling.

Reilly L, Howie J, Wypijewski K, Ashford ML, Hilgemann DW, Fuller W - FASEB J. (2015)

Bottom Line: Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1.However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild-type exchangers are inactive) and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients.The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs.

View Article: PubMed Central - PubMed

Affiliation: *Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, United Kingdom; and Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

No MeSH data available.


Related in: MedlinePlus

Impact of palmitoylation on NCX1 trafficking and subcellular localization. A) Neither pharmacological nor genetic inhibition of NCX1 palmitoylation alters cell surface expression of NCX1 in stably transfected FT-293 cells (2-BP, 100 µM, applied overnight). B) Fractionation based on detergent solubility indicates YFP-NCX1-IC is membrane-anchored when palmitoylated in transiently transfected HEK cells. C) Subcellular distribution of YFP-NCX1-IC in transiently transfected HEK cells. Cys-less YFP-NCX1-IC is distributed similar to YFP alone. WT and C739only YFP-NCX1-IC localize to an intracellular compartment but do not colocalize with the endoplasmic reticulum marker DsRed-ER. D) WT and C739only YFP-NCX1-IC colocalize with the Golgi marker Grasp65-mCherry in transiently transfected HEK cells. E) NCX1 localizes to buoyant caveolin enriched microdomains in rat ventricular myocytes. F) Both palmitoylated and nonpalmitoylated NCX1 are present in buoyant caveolin enriched microdomains prepared from rat ventricular myocytes. 2-BP, 2-bromopalmitate; CS, cell surface protein; SM, starting cell lysate.
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Figure 3: Impact of palmitoylation on NCX1 trafficking and subcellular localization. A) Neither pharmacological nor genetic inhibition of NCX1 palmitoylation alters cell surface expression of NCX1 in stably transfected FT-293 cells (2-BP, 100 µM, applied overnight). B) Fractionation based on detergent solubility indicates YFP-NCX1-IC is membrane-anchored when palmitoylated in transiently transfected HEK cells. C) Subcellular distribution of YFP-NCX1-IC in transiently transfected HEK cells. Cys-less YFP-NCX1-IC is distributed similar to YFP alone. WT and C739only YFP-NCX1-IC localize to an intracellular compartment but do not colocalize with the endoplasmic reticulum marker DsRed-ER. D) WT and C739only YFP-NCX1-IC colocalize with the Golgi marker Grasp65-mCherry in transiently transfected HEK cells. E) NCX1 localizes to buoyant caveolin enriched microdomains in rat ventricular myocytes. F) Both palmitoylated and nonpalmitoylated NCX1 are present in buoyant caveolin enriched microdomains prepared from rat ventricular myocytes. 2-BP, 2-bromopalmitate; CS, cell surface protein; SM, starting cell lysate.

Mentions: The pharmacological specificity of 2-bromopalmitate, widely used as an inhibitor of Asp-His-His-Cys motifs containing palmitoyl acyl transferases, has recently been questioned (40). We therefore used both genetic and pharmacological approaches to assess the impact of palmitoylation on trafficking of NCX1 through the secretory pathway in FT-293 cells stably expressing WT and C739A NCX1. Neither 2-bromopalmitate (100 µM, applied overnight) nor mutation C739A influenced cell surface localization of NCX1 (assessed using membrane impermeable amine-reactive biotinylation reagents, Fig. 3A).


Palmitoylation of the Na/Ca exchanger cytoplasmic loop controls its inactivation and internalization during stress signaling.

Reilly L, Howie J, Wypijewski K, Ashford ML, Hilgemann DW, Fuller W - FASEB J. (2015)

Impact of palmitoylation on NCX1 trafficking and subcellular localization. A) Neither pharmacological nor genetic inhibition of NCX1 palmitoylation alters cell surface expression of NCX1 in stably transfected FT-293 cells (2-BP, 100 µM, applied overnight). B) Fractionation based on detergent solubility indicates YFP-NCX1-IC is membrane-anchored when palmitoylated in transiently transfected HEK cells. C) Subcellular distribution of YFP-NCX1-IC in transiently transfected HEK cells. Cys-less YFP-NCX1-IC is distributed similar to YFP alone. WT and C739only YFP-NCX1-IC localize to an intracellular compartment but do not colocalize with the endoplasmic reticulum marker DsRed-ER. D) WT and C739only YFP-NCX1-IC colocalize with the Golgi marker Grasp65-mCherry in transiently transfected HEK cells. E) NCX1 localizes to buoyant caveolin enriched microdomains in rat ventricular myocytes. F) Both palmitoylated and nonpalmitoylated NCX1 are present in buoyant caveolin enriched microdomains prepared from rat ventricular myocytes. 2-BP, 2-bromopalmitate; CS, cell surface protein; SM, starting cell lysate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608915&req=5

Figure 3: Impact of palmitoylation on NCX1 trafficking and subcellular localization. A) Neither pharmacological nor genetic inhibition of NCX1 palmitoylation alters cell surface expression of NCX1 in stably transfected FT-293 cells (2-BP, 100 µM, applied overnight). B) Fractionation based on detergent solubility indicates YFP-NCX1-IC is membrane-anchored when palmitoylated in transiently transfected HEK cells. C) Subcellular distribution of YFP-NCX1-IC in transiently transfected HEK cells. Cys-less YFP-NCX1-IC is distributed similar to YFP alone. WT and C739only YFP-NCX1-IC localize to an intracellular compartment but do not colocalize with the endoplasmic reticulum marker DsRed-ER. D) WT and C739only YFP-NCX1-IC colocalize with the Golgi marker Grasp65-mCherry in transiently transfected HEK cells. E) NCX1 localizes to buoyant caveolin enriched microdomains in rat ventricular myocytes. F) Both palmitoylated and nonpalmitoylated NCX1 are present in buoyant caveolin enriched microdomains prepared from rat ventricular myocytes. 2-BP, 2-bromopalmitate; CS, cell surface protein; SM, starting cell lysate.
Mentions: The pharmacological specificity of 2-bromopalmitate, widely used as an inhibitor of Asp-His-His-Cys motifs containing palmitoyl acyl transferases, has recently been questioned (40). We therefore used both genetic and pharmacological approaches to assess the impact of palmitoylation on trafficking of NCX1 through the secretory pathway in FT-293 cells stably expressing WT and C739A NCX1. Neither 2-bromopalmitate (100 µM, applied overnight) nor mutation C739A influenced cell surface localization of NCX1 (assessed using membrane impermeable amine-reactive biotinylation reagents, Fig. 3A).

Bottom Line: Surprisingly, palmitoylation does not influence trafficking or localization of NCX1 to surface membranes, nor does it strongly affect the normal forward or reverse transport modes of NCX1.However, exchangers that cannot be palmitoylated do not inactivate normally (leading to substantial activity in conditions when wild-type exchangers are inactive) and do not promote cargo-dependent endocytosis that internalizes 50% of the cell surface following strong G-protein activation or large Ca transients.The palmitoylated cysteine in NCX1 is found in all vertebrate and some invertebrate NCX homologs.

View Article: PubMed Central - PubMed

Affiliation: *Division of Cardiovascular and Diabetes Medicine, Medical Research Institute, College of Medicine, Dentistry, and Nursing, University of Dundee, Dundee, United Kingdom; and Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas, USA.

No MeSH data available.


Related in: MedlinePlus