The yeast Ura2 protein that catalyses the first two steps of pyrimidines biosynthesis accumulates not in the nucleus but in the cytoplasm, as shown by immunocytochemistry and Ura2-green fluorescent protein mapping.
The Ura2 multidomain protein catalyses the first two steps of pyrimidines biosynthesis in Saccharomyces cerevisiae. It consists of a 240 kDa polypeptide which contains carbamyl phosphate synthetase and aspartate transcarbamylase domains. The Ura2 protein was believed to be nucleoplasmic, since one of the aspartate transcarbamylase reaction products, monophosphate, was reported to be precipitated by lead ions inside nuclei. However, this ultracytochemical approach was recently shown to give artifactual lead polyphosphate precipitates, and the use of cerium instead of lead failed to reveal this nucleoplasmic localization. Ura2 localization has therefore been undertaken by means of three alternative approaches based on the detection of the protein itself: (a) indirect immunofluorescence of yeast protoplasts; (b) immunogold labelling of ultrathin sections of embedded yeast cells (both approaches using affinity purified primary antibodies directed against the 240 kDa Ura2 polypeptide chain, or against a 22 residue peptide specific of the carbamyl phosphate synthetase domain); and (c) direct fluorescence of cells expressing an Ura2-green fluorescent protein hybrid. All three approaches localize the bulk of Ura2 to the cytoplasm, whereas the signals associated with the nucleus, mitochondria or vacuoles are close to or at the background level.
Benoist
P
P
Instituto de Investigaciones Biomédicas, C.S.I.C, 4, Arturo Duperier, ES-28029 Madrid, Spain.
Feau
P
P
Pliss
A
A
Vorisek
J
J
Antonelli
R
R
Raska
I
I
Denis-Duphil
M
M
D000908:Antibodies, Fungal / Q000032:analysis; D000918:Antibody Specificity; D001221:Aspartate Carbamoyltransferase / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D002384:Catalysis; D002467:Cell Nucleus / Q000201:enzymology*; D003593:Cytoplasm / Q000201:enzymology*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D008164:Luminescent Proteins; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D009097:Multienzyme Complexes / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D011743:Pyrimidines / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins
D000908:Antibodies, Fungal; D008164:Luminescent Proteins; D009097:Multienzyme Complexes; D011743:Pyrimidines; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C110837:URA2 protein, S cerevisiae; D049452:Green Fluorescent Proteins; D001221:Aspartate Carbamoyltransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)