Abstract
Exponential culture of aSaccharomyces cerevisiae strain with overexpressed aspartate carbamoyltransferase activity (ACTase) was chilled in ice and fractionated by centrifugal elutriation to several cell populations of increasing cell mass. The enzyme activity which belongs to the pyrimidine biosynthesis pathway, was detectedin situ by a specific ultracytochemical reaction: the ACTase byproduct, monophosphate, was precipitated by cerium ions to cerium phosphate. During the outgrowth of nonbudding daughter cells (zero cells) the label appeared first in membranes of nuclear envelope and of mitochondria. In larger zero cells, this label appeared also in the endoplasmic reticulum, microvesicles and plasmalemma. In budding mother cells, the label was conspicuous in the whole cell-membrane complex. In most aged cells the ACTase activity was not detectable. The presence of ACTase activity in membranes of compartments conveying glycoproteinsvia the secretory pathway remains to be explained. To confirm thein situ detection of ACTase activity in membranes, we assayed the enzyme activity in both the 10 000g sediment and supernatant prepared from yeast homogenate precentrifuged at 3000g. From 23 to 43% of ACTase activity was detected in the sediments including membranes of wild-type and ACTase-overexpressing strains.
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Abbreviations
- CP:
-
carbamoyl phosphate
- ACTase:
-
aspartate carbamoyltransferase (EC 2.1.3.2)
- CPSaseArg :
-
CPSase from arginine pathway
- DHOase:
-
dihydroorotase (EC 3.5.2.3)
- YNBG:
-
YNB plus glucose
- CAD:
-
CPSase-ACTase-DHOase multidomain protein
- CPSase:
-
carbamoyl phosphate synthetase (EC 6.3.5.5)
- CPSaseUra :
-
CPSase from uracil pathway
- YNB:
-
yeast nitrogen base without amino acids
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Voříšek, J., Noaillac-Depeyre, J. & Denis-Duphil, M. Life-cycle-dependent changes of aspartate carbamoyltransferase localization in membranes ofSaccharomyces cerevisiae—Centrifugal elutriation and ultracytochemical study. Folia Microbiol 44, 289–294 (1999). https://doi.org/10.1007/BF02818549
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DOI: https://doi.org/10.1007/BF02818549