Life-cycle-dependent changes of aspartate carbamoyltransferase localization in membranes of Saccharomyces cerevisiae--centrifugal elutriation and ultracytochemical study.
Exponential culture of a Saccharomyces 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 detected in 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 glycoproteins via the secretory pathway remains to be explained. To confirm the in situ detection of ACTase activity in membranes, we assayed the enzyme activity in both the 10,000 g sediment and supernatant prepared from yeast homogenate precentrifuged at 3000 g. From 23 to 43% of ACTase activity was detected in the sediments including membranes of wild-type and ACTase-overexpressing strains.
Vorísek
J
J
Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Noaillac-Depeyre
J
J
Denis-Duphil
M
M
D001221:Aspartate Carbamoyltransferase / Q000302:isolation & purification*; D002451:Cell Compartmentation; D002455:Cell Division; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology* / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000201:enzymology* / Q000648:ultrastructure; D015388:Organelles / Q000201:enzymology* / Q000648:ultrastructure; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development / Q000648:ultrastructure
D001221:Aspartate Carbamoyltransferase