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S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines

The insulin-responsive aminopeptidase (IRAP) was recently identified as an S-acylated protein in adipocytes and other tissues. However, there is currently no information on the extent of S-acylation of this protein, the residues that are modified, or the effects of S-acylation on IRAP localisation....

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Autores principales: Werno, Martin W., Chamberlain, Luke H.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510526/
https://www.ncbi.nlm.nih.gov/pubmed/26198666
http://dx.doi.org/10.1038/srep12413
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author Werno, Martin W.
Chamberlain, Luke H.
author_facet Werno, Martin W.
Chamberlain, Luke H.
author_sort Werno, Martin W.
collection PubMed
description The insulin-responsive aminopeptidase (IRAP) was recently identified as an S-acylated protein in adipocytes and other tissues. However, there is currently no information on the extent of S-acylation of this protein, the residues that are modified, or the effects of S-acylation on IRAP localisation. In this study, we employ a semi-quantitative acyl-RAC technique to show that approximately 60% of IRAP is S-acylated in 3T3-L1 adipocytes. In contrast, S-acylation of GLUT4, a glucose transporter that extensively co-localises with IRAP, was approximately five-fold lower. Site-directed mutagenesis was employed to map the sites of S-acylation on IRAP to two cysteine residues, one of which is predicted to lie in the cytoplasmic side of the single transmembrane domain and the other which is just upstream of this transmembrane domain; our results suggest that these cysteines may be modified in a mutually-exclusive manner. Although S-acylation regulates the intracellular trafficking of several transmembrane proteins, we did not detect any effects of mutating the modified cysteines on the plasma membrane localisation of IRAP in HEK293T cells, suggesting that S-acylation is not essential for the movement of IRAP through the secretory pathway.
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spelling pubmed-45105262015-07-28 S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines Werno, Martin W. Chamberlain, Luke H. Sci Rep Article The insulin-responsive aminopeptidase (IRAP) was recently identified as an S-acylated protein in adipocytes and other tissues. However, there is currently no information on the extent of S-acylation of this protein, the residues that are modified, or the effects of S-acylation on IRAP localisation. In this study, we employ a semi-quantitative acyl-RAC technique to show that approximately 60% of IRAP is S-acylated in 3T3-L1 adipocytes. In contrast, S-acylation of GLUT4, a glucose transporter that extensively co-localises with IRAP, was approximately five-fold lower. Site-directed mutagenesis was employed to map the sites of S-acylation on IRAP to two cysteine residues, one of which is predicted to lie in the cytoplasmic side of the single transmembrane domain and the other which is just upstream of this transmembrane domain; our results suggest that these cysteines may be modified in a mutually-exclusive manner. Although S-acylation regulates the intracellular trafficking of several transmembrane proteins, we did not detect any effects of mutating the modified cysteines on the plasma membrane localisation of IRAP in HEK293T cells, suggesting that S-acylation is not essential for the movement of IRAP through the secretory pathway. Nature Publishing Group 2015-07-22 /pmc/articles/PMC4510526/ /pubmed/26198666 http://dx.doi.org/10.1038/srep12413 Text en Copyright © 2015, Macmillan Publishers Limited http://creativecommons.org/licenses/by/4.0/ This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
spellingShingle Article
Werno, Martin W.
Chamberlain, Luke H.
S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines
title S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines
title_full S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines
title_fullStr S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines
title_full_unstemmed S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines
title_short S-acylation of the Insulin-Responsive Aminopeptidase (IRAP): Quantitative analysis and Identification of Modified Cysteines
title_sort s-acylation of the insulin-responsive aminopeptidase (irap): quantitative analysis and identification of modified cysteines
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4510526/
https://www.ncbi.nlm.nih.gov/pubmed/26198666
http://dx.doi.org/10.1038/srep12413
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