Abstract
Mitochondrial enzymes involved in energy transformation are organized into multiprotein complexes that channel the reaction intermediates for efficient ATP production. Three of the mammalian urea cycle enzymes: N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) reside in the mitochondria. Urea cycle is required to convert ammonia into urea and protect the brain from ammonia toxicity. Urea cycle intermediates are tightly channeled in and out of mitochondria, indicating that efficient activity of these enzymes relies upon their coordinated interaction with each other, perhaps in a cluster. This view is supported by mutations in surface residues of the urea cycle proteins that impair ureagenesis in the patients, but do not affect protein stability or catalytic activity. We find the NAGS, CPS1, and OTC proteins in liver mitochondria can associate with the inner mitochondrial membrane (IMM) and can be co-immunoprecipitated. Our in-silico analysis of vertebrate NAGS proteins, the least abundant of the urea cycle enzymes, identified a protein-protein interaction region present only in the mammalian NAGS protein—“variable segment,” which mediates the interaction of NAGS with CPS1. Use of super resolution microscopy showed that NAGS, CPS1 and OTC are organized into clusters in the hepatocyte mitochondria. These results indicate that mitochondrial urea cycle proteins cluster, instead of functioning either independently or in a rigid multienzyme complex.
Highlights
Mitochondria are ATP producing organelles where multi-enzyme complexes catalyze reactions of the TCA cycle, fatty acid beta-oxidation and oxidative phosphorylation
Using a combination of protein structural analysis, mapping of patient mutations, liver mitochondrial fractionation, co-immunoprecipitation of urea cycle enzymes, and superresolution microscopy we provide structural evidence that N-acetylglutamate synthase (NAGS), carbamylphosphate synthetase 1 (CPS1), and ornithine transcarbamylase (OTC) enzymes interact and form clusters in the mitochondria
Between 0.5 and 1 μg of immunoprecipitated proteins were resolved using SDSPAGE, and probed with primary antibodies raised against NAGS, CPS1, or OTC followed by the HPRT-conjugated secondary antibody
Summary
Mitochondria are ATP producing organelles where multi-enzyme complexes catalyze reactions of the TCA cycle, fatty acid beta-oxidation and oxidative phosphorylation To perform these diverse functions, mitochondria maintain the inner and outer mitochondrial membrane-bound compartments. Using a combination of protein structural analysis, mapping of patient mutations, liver mitochondrial fractionation, co-immunoprecipitation of urea cycle enzymes, and superresolution microscopy we provide structural evidence that NAGS, CPS1, and OTC enzymes interact and form clusters in the mitochondria. This evidence offers direct support that the urea cycle is yet another mitochondrial function that relies upon compartmentalization by the formation of a protein cluster
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