Natalie S. Cohen, Ph.D.

Molecular Pharmacology and Toxicology

Cellular Organization of Enzyme Systems

Many enzymes which are soluble under test tube conditions form multicomponent complexes under the unique conditions existing in the living cell. The cytoskeleton and intracellular membranes form a scaffold for the attachment of organized, functionally related enzymes. The organization of soluble enzyme systems is a functionally significant feature of all eucaryotic cells. Our research is aimed at identifying some of the underlying mechanisms, using the urea cycle of rat liver as an experimental model.

The pathway of urea synthesis consists of five enzymes; the first two are in the mitochondrial matrix, and the next three in the cytoplasm. Although all the enzymes are soluble, we have shown that they are highly organized in situ; intermediates are channeled between enzymes and compartments, and the cytoplasmic enzymes are sequentially organized at the mitochondrial membrane. In addition, the mRNAs of two of the cytoplasmic enzymes are localized next to mitochondria, indicating that translation of these proteins occurs at their final intracellular site.

We are studying mRNA localization by determining if the mRNAs are enriched in a particular population of polysomes, perhaps associated with mitochondria and/or a specific cytoskeletal element, and by identifying both the mRNA targeting sequences and the cellular component(s) that serve as the target. We will eventually examine the functional effects of perturbing mRNA localization.

We are studying how the proper localization of the enzyme proteins is maintained by examining their interactions in situ with each other, and/or with some (as yet unidentified) structural component. The approaches involve using a) reversible cross-linking reagents and immunological techniques; b) the two-hybrid system of gene expression.

Selected Publications

1. Cheung, C-W., Cohen, N.S., and Raijman, L. Channeling of urea cycle intermediates in situ in permeabilized hepatocytes. J. Biol. Chem. 264, 4038-4044 (1989).
2. Cohen, N.S., Cheung, C-W., Sijuwade, E., and Raijman, L. Kinetics of carbamoyl phosphate synthase (ammonia) and ornithine carbamoyltransferase in permeabilized mitochondria. Biochem. J., 282, 173-180 (1992).
3. Cohen, N.S. Intracellular localization of the mRNAs of argininosuccinate synthetase and argininosuccinate lyase around liver mitochondria, visualized by in situ reverse transcription-polymerase chain reaction. J. Cell. Biochem. 61, 81-96 (1996).