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Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue

Morris A. Kostiuk^*,1, Maria M. Corvi^*,1,2, Bernd O. Keller, Greg Plummer^*, Jennifer A. Prescher^,, Matthew J. Hangauer^,, Carolyn R. Bertozzi^,, Gurram Rajaiah^#, John R. Falck^# and Luc G. Berthiaume^*,3

^* Department of Cell Biology, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, Alberta, Canada;

Department of Pathology and Laboratory Medicine, Child & Family Research Institute, University of British Columbia, Vancouver, British Columbia, Canada;

Departments of Chemistry and Molecular Cell Biology and Howard Hughes Medical Institute, University of California, Berkeley, California, USA;

Materials Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, California, USA; and

^# Department of Biochemistry, University of Texas Southwestern, Dallas, Texas, USA

³Correspondence: Department of Cell Biology, University of Alberta, Faculty of Medicine and Dentistry, Edmonton, Alberta, T6G 2H7, Canada. E-mail: luc.berthiaume{at}ualberta.ca

Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.—Kostiuk, M. A., Corvi, M. M., Keller, B. O., Plummer, G., Prescher, J. A., Hangauer, M. J., Bertozzi, C. R., Rajaiah, G., Falck, J. R., Berthiaume, L. G. Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue.

Key Words: azido-fatty acid • carbamoyl phosphate synthetase 1 • Staudinger ligation • 3-hydroxy-3-methylglutaryl CoA synthase