Biochemical Evidence of Slo1 Protein Internal Myristoylation: Involvement of a Hydroxyester Chemical Bond

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Myristic and/or palmitic acid incorporation to proteins is a mean by which cells tether proteins to the intracellular leaflet of plasma membranes. Two types of protein myristoylation have been reported; one occurs co-translationally at the N-terminus (e.g. c-Src) and the other post-translationally at an internal amino-acid residue. Here, we tested whether Slo1 might undergo post-translational myristoylation as it lacks an N-terminal consensus site for myristoylation. HEK-293T cells expressing Slo1 or c-Src (positive control) were metabolically radiolabeled with [3H]-myristic acid and subjected to immunoprecipitation; radiolabeled proteins were detected by autoradiography. Our data show that Slo1 incorporates [3H]-myristic acid (n=5) via a post-translational mechanism as assessed by the lack of effect upon inhibition of protein synthesis with cyclohexamide. As control, cyclohexamide treatment reduced c-Src myristoylation confirming its co-translational incorporation (n=3). Next, we sought to determine what type of chemical bond is involved in Slo1 protein myristoylation. Hydroxylamine (NH2OH) at pH10 but not Tris-HCl at pH10 (negative control) or NH2OH at pH 7, cleaves hydroxyester bonds. Treatment of [3H]-myristoyl-Slo1 with NH2OH, pH10 but neither treatment with Tris-HCl at pH10 nor NH2OH at pH7, completely removed incorporated myristic acid from Slo1 (n=3). Possible palmitoylation of Slo1 via a thioester bond was excluded because treatment of labeled Slo1 with NH2OH at pH7 which cleaves thioester bonds or 1.4 M β-mercaptoethanol, a reducing agent, did not alter the signal. Further, we did not observe Slo1 labeling using [3H]-palmitate (n=2). These data strongly support an involvement of a hydroxyester chemical bond between myristic acid and Slo1 S/T/Y residue(s). In conclusion, we show for the first time that Slo1 protein is post-translationally myristoylated at an internal site. This myristoylation might play a role in controlling Slo1 channel structure, function or trafficking. Supported by NIH.