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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

Identification of the NCX1 palmitoylation site. A) Membrane topology of mature NCX1.1 following cleavage of the signal peptide. The start and end of the large intracellular loop, and positions of the mutated cysteines are numbered. B) Individual cysteines and pairs of cysteines were mutated to alanine and transiently expressed in HEK cells. All mutants were palmitoylated with the exception of C739A. All acyl-RAC reactions were blotted for flotillin 2 (Flot2) to confirm efficient purification of palmitoylated proteins. C) Individual cysteines were reintroduced to NCX1 with a cys-less intracellular loop (cysteines 383, 387, 485, 557, 731, and 739 all mutated to alanine). Palmitoylation of NCX1 requires the presence of C739 only. M, mock transfected; Palm, proteins purified using acyl-RAC; RVM, rat ventricular myocytes; UF, unfractionated cell lysate.
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Figure 2: Identification of the NCX1 palmitoylation site. A) Membrane topology of mature NCX1.1 following cleavage of the signal peptide. The start and end of the large intracellular loop, and positions of the mutated cysteines are numbered. B) Individual cysteines and pairs of cysteines were mutated to alanine and transiently expressed in HEK cells. All mutants were palmitoylated with the exception of C739A. All acyl-RAC reactions were blotted for flotillin 2 (Flot2) to confirm efficient purification of palmitoylated proteins. C) Individual cysteines were reintroduced to NCX1 with a cys-less intracellular loop (cysteines 383, 387, 485, 557, 731, and 739 all mutated to alanine). Palmitoylation of NCX1 requires the presence of C739 only. M, mock transfected; Palm, proteins purified using acyl-RAC; RVM, rat ventricular myocytes; UF, unfractionated cell lysate.

Mentions: To identify the site of palmitoylation in NCX1, individual cysteines predicted to reside in or close to the cytoplasmic domains (see currently accepted topology, Fig. 2A) were mutated to alanine in the NCX1.1 canine cDNA, then expressed in HEK (Fig. 2B) and HeLa (not shown) cells. All cysteine-to-alanine mutants investigated displayed similar palmitoylation to WT NCX1 with the exception of mutant C739A, in which palmitoylation was abolished, strongly suggesting that this is the sole site of palmitoylation in NCX1.


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)

Identification of the NCX1 palmitoylation site. A) Membrane topology of mature NCX1.1 following cleavage of the signal peptide. The start and end of the large intracellular loop, and positions of the mutated cysteines are numbered. B) Individual cysteines and pairs of cysteines were mutated to alanine and transiently expressed in HEK cells. All mutants were palmitoylated with the exception of C739A. All acyl-RAC reactions were blotted for flotillin 2 (Flot2) to confirm efficient purification of palmitoylated proteins. C) Individual cysteines were reintroduced to NCX1 with a cys-less intracellular loop (cysteines 383, 387, 485, 557, 731, and 739 all mutated to alanine). Palmitoylation of NCX1 requires the presence of C739 only. M, mock transfected; Palm, proteins purified using acyl-RAC; RVM, rat ventricular myocytes; UF, unfractionated cell lysate.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Identification of the NCX1 palmitoylation site. A) Membrane topology of mature NCX1.1 following cleavage of the signal peptide. The start and end of the large intracellular loop, and positions of the mutated cysteines are numbered. B) Individual cysteines and pairs of cysteines were mutated to alanine and transiently expressed in HEK cells. All mutants were palmitoylated with the exception of C739A. All acyl-RAC reactions were blotted for flotillin 2 (Flot2) to confirm efficient purification of palmitoylated proteins. C) Individual cysteines were reintroduced to NCX1 with a cys-less intracellular loop (cysteines 383, 387, 485, 557, 731, and 739 all mutated to alanine). Palmitoylation of NCX1 requires the presence of C739 only. M, mock transfected; Palm, proteins purified using acyl-RAC; RVM, rat ventricular myocytes; UF, unfractionated cell lysate.
Mentions: To identify the site of palmitoylation in NCX1, individual cysteines predicted to reside in or close to the cytoplasmic domains (see currently accepted topology, Fig. 2A) were mutated to alanine in the NCX1.1 canine cDNA, then expressed in HEK (Fig. 2B) and HeLa (not shown) cells. All cysteine-to-alanine mutants investigated displayed similar palmitoylation to WT NCX1 with the exception of mutant C739A, in which palmitoylation was abolished, strongly suggesting that this is the sole site of palmitoylation in NCX1.

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