CERIUM-BASED ULTRACYTOCHEMICAL LOCALIZATION OF ASPARTATE-TRANSCARBAMYLASE ACTIVITY IN THE CELL-MEMBRANE COMPLEX OF SACCHAROMYCES-CEREVISIAE

Aspartate transcarbamylase (ATCase) activity was localized ultracytoch emically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate. We prefixed yeas t cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity. Cells were washed and incubated with ATCase subst rates (aspartate, carbamyl phosphate) plus cerium chloride, and postfi xed by osmium tetroxide. In cells from exponential batch cultures, dep osits of cerium phosphate delineated simultaneously or alternatively m embranes of the secretory pathway: nuclear envelope, endoplasmic retic ulum, Golgi complex and the plasmalemma; mitochondrial membranes and i ntramitochondrial fibrous component were labelled as well. Deposits of cerium phosphate were never observed in the nucleoplasm. Cells incuba ted in the absence of cerium or ATCase substrates and mutant S. cerevi siae cells lacking ATCase activity served as controls. Small round ele ctron-dense condensates were found to be randomly distributed within s ome cells, both in control and experimental runs, in the nucleoplasm, cytoplasm and mitochondrial matrix and represented undefined osmicated endogenous compounds. Our results suggest that the synthesis of pyrim idine precursors occurs in membranes, where compounds such as UDP-gluc ose and CDP-diglycerides are needed for membrane and/or yeast cell wal l synthesis. The possible contribution of ATCase activity found in the nuclear envelope to nucleic acid synthesis remains to be clarified. ( C) 1997 Elsevier Science Ltd.