Uracil-Induced Down-Regulation of the Yeast Uracil Permease

Effect of exogenous pyrimidines upon the FUR4transcript level. Total RNA was prepared from FL200 cells (wt^a) (lane 1), NC122sp6 cells (fur4Δ) untransformed (lane 2) or transformed with either pfF (FUR42μ) (lanes 6 to 8) or pgF (gal-FUR4 2μ) (lanes 9 and 10), NC217-5C cells (dhu1) (lanes 3 to 5), and and 23344C cells (wt^b) transformed with pFL38gF (gal-FUR4CEN) (lanes 11 and 12). Cells were grown to an A₆₀₀ of 0.5 on minimal medium without (−) or with 40 μg of either uracil (U) or cytosine (C)/ml or on rich medium (YP). The carbon source was glucose (lanes 1 to 8) or galactose (lanes 9 to 12). Ten micrograms of RNA was separated on a formaldehyde gel, blotted, and probed with a fragment from the FUR4 gene, theFCY2 gene, and the ACT1 gene. (A) Signals obtained with the PhosphorImager are presented. (B) Signals were quantified, and values for FUR4 mRNA, normalized toACT1 as an internal standard, are plotted as percentages of the value obtained for the same cells grown in the absence of pyrimidine.

Northern blot analysis of FUR4 mRNA level. (A) To measure mRNA decay, NC122sp6 cells expressing theFUR4 gene from plasmid pgF were subjected to a glucose arrest of galactose-induced transcription in the absence (●) or presence (○) of 40 μg of uracil per ml. RNA was extracted at the indicated times as described in Materials and Methods, and quantitative Northern blotting was performed as shown in Fig. 2. Values ofFUR4 mRNA, normalized to ACT1 as an internal standard, are plotted as percentages of the initial value at point zero. (B) NC122sp6 cells transformed with plasmid pfF were grown with galactose as the carbon source, glucose was added, and theFUR4 mRNA level was assayed at the indicated times as described for panel A. (C) NC122sp6 cells transformed with plasmid pfF were grown at 24°C and then subjected to a mild temperature shock to 36°C, and FUR4 mRNA was assayed at the indicated times as described for panel A.

Degradation of the permease upon exposure of cells to uracil. Strains 23344C (wild type [wt]) and 23344Cfur1Δ (fur1Δ) transformed with plasmid p195gF were grown at 30°C to an A₆₀₀ of 0.5, with galactose as the carbon source. Glucose was then added, and growth was followed in the absence (−) or presence (+) of 40 μg of uracil/ml. Protein extracts were prepared at the times indicated after the addition of glucose. Aliquots corresponding to 0.2 ml of culture were analyzed for uracil permease by Western immunoblotting. As already mentioned (48), potential dimers can be observed in the upper part of the gel. Bands just above the main signal correspond mostly to ubiquitin-permease conjugates over background (9), their steady-state abundance relative to the main signal is lower in galactose-grown cells (t₀) than during further growth in the presence of glucose.

Production of ubiquitin-permease conjugates is promoted in stimulated turnover conditions. NY279 (act1) cells transformed with plasmid p195gF were grown at 24°C to an A₆₀₀ of 0.5, with galactose as the carbon source. Glucose was then added, and 20 min later the cells were shifted to the nonpermissive temperature. Cells were brought rapidly to 36°C by dilution of the culture with an equal volume of medium prewarmed to 48°C and incubated in the absence (−) or presence (+) of 40 μg of uracil/ml (A) or 5 mM cysteine (B). Protein extracts were prepared from cells harvested at the times indicated after the shift to 36°C, and aliquots from 0.3 ml of culture were analyzed for uracil permease by Western immunoblotting. The putative dimers present at the top of the gel produced larger species in the presence of uracil.