Cerium-based ultracytochemical localization of aspartate transcarbamylase activity in the cell membrane complex of Saccharomyces cerevisiae.
Aspartate transcarbamylase (ATCase) activity was localized ultracytochemically in the yeast Saccharomyces cerevisiae by precipitation of its reaction product orthophosphate as cerium phosphate. We prefixed yeast cells with ice-cold 1% glutaraldehyde for 30 min which preserved 80% of ATCase activity. Cells were washed and incubated with ATCase substrates (aspartate, carbamyl phosphate) plus cerium chloride, and postfixed by osmium tetroxide. In cells from exponential batch cultures, deposits of cerium phosphate delineated simultaneously or alternatively membranes of the secretory pathway: nuclear envelope, endoplasmic reticulum, Golgi complex and the plasmalemma; mitochondrial membranes and intramitochondrial fibrous component were labelled as well. Deposits of cerium phosphate were never observed in the nucleoplasm. Cells incubated in the absence of cerium or ATCase substrates and mutant S. cerevisiae cells lacking ATCase activity served as controls. Small round electron-dense condensates were found to be randomly distributed within some 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 pyrimidine precursors occurs in membranes, where compounds such as UDP-glucose and CDP-diglycerides are needed for membrane and/or yeast cell wall synthesis. The possible contribution of ATCase activity found in the nuclear envelope to nucleic acid synthesis remains to be clarified.
Vorísek
J
J
Institute of Microbiology, Academy of Sciences of Czech Republic, Praha, Czech Republic.
Gas
N
N
Denis-Duphil
M
M
D001221:Aspartate Carbamoyltransferase / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D002563:Cerium; D006651:Histocytochemistry / Q000379:methods*; D007854:Lead; D008854:Microscopy, Electron; D009154:Mutation; D010710:Phosphates; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure*; D013194:Staining and Labeling / Q000379:methods
D010710:Phosphates; D007854:Lead; D002563:Cerium; D001221:Aspartate Carbamoyltransferase; C048083:cerium phosphate