The FASEB Journal, Vol 2, 2982-2989, Copyright © 1988 by The Federation of American Societies for Experimental Biology

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Intracellular location of the multidomain protein CAD in mammalian cells

MG Chaparian and DR Evans
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Michigan 48201.

The first three steps of mammalian de novo pyrimidine biosynthesis are catalyzed by the multifunctional protein CAD, consisting of glutamine- dependent carbamylphosphate synthetase, aspartate transcarbamylase, and dihydroorotase. The intracellular distribution of CAD in two hamster cell lines, BHK 21 and BHK 165-23 (a strain in which the CAD gene was selectively amplified), was determined by differential centrifugation and by two different cytochemical immunolocalization methods. Ammonia- dependent carbamylphosphate synthetase I was found in both cell types at a concentration of 0.01% of the total cell protein, so its distribution was also determined as a control for possible cross- reactivity of the CAD antibody probes and as a mitochondrial marker. CAD was localized in the cytoplasmic compartment and almost completely excluded from the nucleus. A punctate staining pattern suggested that it was not uniformly dispersed throughout the cytosol (unlike typical soluble proteins) but was associated with subcellular organelles. Although there was a slight tendency for CAD to be localized in the vicinity of the nuclear envelope, the amount of staining was much less than expected from differential centrifugation, which showed that 30% of the protein was found in the nuclear fraction. No interactions with other subcellular components could be detected by centrifugation. It is possible, however, that CAD is associated with subcellular structures that cosediment with the nuclei. Despite a 150-fold increase in CAD concentration in the over-producing cells, the distribution of the protein was unaltered. CAD was not concentrated near the mitochondria where the next enzyme of the de novo pathway, dihydroorotate dehydrogenase, is localized, which indicates that the intermediate dihydroorotate is not channeled, but rather dissociates from CAD and diffuses through the bulk cellular fluid.

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