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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

NCX1 is palmitoylated in heart, brain and kidney. A) Palmitoylated proteins were purified from homogenates of heart, brain, and kidney and immunoblotted as shown. Note the extended incubation at 40°C during acyl-RAC causes partial aggregation of NCX1 resulting in 2 species of ∼120 and 150 kDa resolved by SDS-PAGE. B) Fraction of NCX1.1 palmitoylated in ventricular muscle. A small fraction of NCX1.1 is not palmitoylated in ventricular muscle. NCX1.1 enrichment in acyl-RAC reactions was normalized to the constitutively palmitoylated protein caveolin 3 (Cav3). n = 5, mean ± sem shown. C) NCX1.1 is palmitoylated in human-induced pluripotent stem cells differentiated into cardiac myocytes. P, proteins purified by acyl-RAC; Palm, proteins purified using acyl-RAC in either the presence (+) or absence (−) of hydroxylamine (ha); UF, unfractionated cell lysate; UB, material not captured by acyl-RAC.
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Figure 1: NCX1 is palmitoylated in heart, brain and kidney. A) Palmitoylated proteins were purified from homogenates of heart, brain, and kidney and immunoblotted as shown. Note the extended incubation at 40°C during acyl-RAC causes partial aggregation of NCX1 resulting in 2 species of ∼120 and 150 kDa resolved by SDS-PAGE. B) Fraction of NCX1.1 palmitoylated in ventricular muscle. A small fraction of NCX1.1 is not palmitoylated in ventricular muscle. NCX1.1 enrichment in acyl-RAC reactions was normalized to the constitutively palmitoylated protein caveolin 3 (Cav3). n = 5, mean ± sem shown. C) NCX1.1 is palmitoylated in human-induced pluripotent stem cells differentiated into cardiac myocytes. P, proteins purified by acyl-RAC; Palm, proteins purified using acyl-RAC in either the presence (+) or absence (−) of hydroxylamine (ha); UF, unfractionated cell lysate; UB, material not captured by acyl-RAC.

Mentions: Analysis of the rat neural palmitoyl proteome has previously identified NCX1 and NCX2 as being palmitoylated (38), but did not address whether a biologically meaningful fraction of NCX was modified. We purified palmitoylated proteins using resin-assisted capture of acylated proteins (acyl-RAC) from homogenates of rat brain, kidney, and heart (Fig. 1A). NCX1 was robustly palmitoylated in all tissues examined, implying that multiple splice variants are palmitoylated. We determined what fraction of the cardiac splice variant NCX1.1 is palmitoylated in isolated rat ventricular myocytes by comparing the enrichment of NCX1 following acyl-RAC with the enrichment of the stoichiometrically palmitoylated protein caveolin 3 (39). A small fraction of NCX1 was not purified by acyl-RAC, but all caveolin 3 was captured. The results indicate that ∼60% of NCX1 is palmitoylated in ventricular muscle (Fig. 1B). We also confirmed palmitoylation of NCX1.1 in human-induced pluripotent stem cells differentiated into cardiac myocytes (Fig. 1C).


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)

NCX1 is palmitoylated in heart, brain and kidney. A) Palmitoylated proteins were purified from homogenates of heart, brain, and kidney and immunoblotted as shown. Note the extended incubation at 40°C during acyl-RAC causes partial aggregation of NCX1 resulting in 2 species of ∼120 and 150 kDa resolved by SDS-PAGE. B) Fraction of NCX1.1 palmitoylated in ventricular muscle. A small fraction of NCX1.1 is not palmitoylated in ventricular muscle. NCX1.1 enrichment in acyl-RAC reactions was normalized to the constitutively palmitoylated protein caveolin 3 (Cav3). n = 5, mean ± sem shown. C) NCX1.1 is palmitoylated in human-induced pluripotent stem cells differentiated into cardiac myocytes. P, proteins purified by acyl-RAC; Palm, proteins purified using acyl-RAC in either the presence (+) or absence (−) of hydroxylamine (ha); UF, unfractionated cell lysate; UB, material not captured by acyl-RAC.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: NCX1 is palmitoylated in heart, brain and kidney. A) Palmitoylated proteins were purified from homogenates of heart, brain, and kidney and immunoblotted as shown. Note the extended incubation at 40°C during acyl-RAC causes partial aggregation of NCX1 resulting in 2 species of ∼120 and 150 kDa resolved by SDS-PAGE. B) Fraction of NCX1.1 palmitoylated in ventricular muscle. A small fraction of NCX1.1 is not palmitoylated in ventricular muscle. NCX1.1 enrichment in acyl-RAC reactions was normalized to the constitutively palmitoylated protein caveolin 3 (Cav3). n = 5, mean ± sem shown. C) NCX1.1 is palmitoylated in human-induced pluripotent stem cells differentiated into cardiac myocytes. P, proteins purified by acyl-RAC; Palm, proteins purified using acyl-RAC in either the presence (+) or absence (−) of hydroxylamine (ha); UF, unfractionated cell lysate; UB, material not captured by acyl-RAC.
Mentions: Analysis of the rat neural palmitoyl proteome has previously identified NCX1 and NCX2 as being palmitoylated (38), but did not address whether a biologically meaningful fraction of NCX was modified. We purified palmitoylated proteins using resin-assisted capture of acylated proteins (acyl-RAC) from homogenates of rat brain, kidney, and heart (Fig. 1A). NCX1 was robustly palmitoylated in all tissues examined, implying that multiple splice variants are palmitoylated. We determined what fraction of the cardiac splice variant NCX1.1 is palmitoylated in isolated rat ventricular myocytes by comparing the enrichment of NCX1 following acyl-RAC with the enrichment of the stoichiometrically palmitoylated protein caveolin 3 (39). A small fraction of NCX1 was not purified by acyl-RAC, but all caveolin 3 was captured. The results indicate that ∼60% of NCX1 is palmitoylated in ventricular muscle (Fig. 1B). We also confirmed palmitoylation of NCX1.1 in human-induced pluripotent stem cells differentiated into cardiac myocytes (Fig. 1C).

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