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.