, Volume 120, Issue 2, pp 141-149

Differential extraction of soluble pools from the cytosol and the vacuoles of yeast (Candida utilis) using DEAE-dextran

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Abstract

The plasma membrane of Candida utilis cells was rapidly disrupted by a small dose of DEAE-dextran. The vacuolar membranes, in contrast, remained intact under isotonic conditions. Therefore, the cytosolic pool could be extracted in a first step, and in a second step, after disruption of the vacuoles, the vacuolar pool. The two extracts were studied in cells grown on different nitrogen sources, namely ammonium, arginine, ornithine, citrulline, glycine, and proline.

The amount of soluble amino acids in Candida cells varies considerably depending on the nitrogen source. This is largely caused by the variation in size of the vacuolar pool (0.8–2.4 mmol per g protein) containing nearly all nitrogen-rich amino acids such as arginine and ornithine, whereas the size of the cytoplasmic pool, holding most of the glutamic acid, is fairly constant (1.3 mmol per g protein). Upon nitrogen starvation the vacuolar pool was reduced much more than the cytosolic pool. A storage and buffer function of the vacuolar pool was also indicated by the much slower turnover of the vacuolar than of the cytosolic glutamine in an isotope labelling experiment. Potassium, sodium, orthophosphate, ATP, and other substances absorbing at 260 nm were found predominantly in the cytosolic extracts. Extraction of uniformly 14C-labelled cells showed that the total soluble pool of the cells contained about 10% of the total carbon. Of this about 45% was in the vacuolar the rest in the cytosolic extract. The labelled extracts were further characterized by ion exchange chromatography.