Abstract
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.
Highlights
Protein S-acylation is a post-translational modification (PTM) involving the attachment of palmitate and other fatty acids to cysteine residues of proteins via thioester linkage
Analysis of insulin-responsive aminopeptidase (IRAP) immunoreactivity in the recovered fractions was consistent with ~60% of this protein being S-acylated, whereas the level of S-acylation of GLUT4 was estimated to be less than 12% (Fig. 1)
As S-acylation has been suggested to regulate the intracellular trafficking of GLUT419, it will be interesting in follow-up work to investigate if this modification is differentially regulated on GLUT4 and IRAP
Summary
Protein S-acylation ( referred to as palmitoylation) is a post-translational modification (PTM) involving the attachment of palmitate and other fatty acids to cysteine residues of proteins via thioester linkage. S-acylation is mediated by a family of membrane-bound enzymes comprising 24 isoforms in mammals[3,4]. These palmitoyl-transferases (PATs) are referred to as zDHHC enzymes due to the presence of a conserved DHHC (Asp-His-His-Cys) amino acid motif present within a zinc-finger cysteine-rich domain[5,6,7]. More recent studies identified additional targets of the S-acylation machinery in adipocytes, including GLUT4 and the insulin-responsive aminopeptidase (IRAP)[17], a protein that co-localises extensively with GLUT4 and regulates the formation and/or trafficking of insulin-responsive GLUT4 storage vesicles[18]. The sites of S-acylation on IRAP or how this modification affects the localisation of the protein are not known. There is no information on the likely extent of S-acylation of IRAP and how this compares with proteins such as GLUT4
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