The kinetics of changes occurring in colonies during their acid/alkali transition. (A) Giant colonies of BY4742 growing on GM-BKP. Phases 1 (7 d), 2 (9 d), 3 (10 d), 4 (10.5 d), 5 (11 d) to 6 (12 d) are also indicated by colored rectangles above the photos. (B) Cells exhibiting characteristic neutral red staining in individual phases 1–6. Magnification, ×3000. (C) Changes in total intracellular concentrations of individual amino acids during phases 1–6. Color of the bars corresponds to phases 1–6 as indicated in A. (D) Ratio of the concentrations of individual amino acids in vacuoles and in the cytoplasm during phases 1–6. (E) Northern hybridization of RNAs isolated from colonies occurring in stages 1–6. Probes for genes that were induced in microarray experiment are indicated by a red rectangle, those that were repressed by a green rectangle, and the nonregulated control, RPL28, by a black rectangle.

The expression variations in colonies during their acid/alkali transition. The time-course transcription profiles of 200 genes, the expression of which significantly changed in at least one phase during the acid/alkali transition, have been arranged according to their functions. Information obtained from YPD, SGD, YTPdb, MIPS, and the literature cited in the text were used for functional classification. The genes ATO1 (YCR010c), ATO2 (YNR002c), and ATO3 (YDR384c) were defined in this work (see text and Figure 4). The numbers and color bars at the top of the clusters define the phases. The change in expression is indicated by a colored square (color bar on the bottom right). The corresponding values are on Table 2S at http://www.biologie.ens.fr/fr/genetiqu/puces/publications/ATO/index.html. Three ribosomal protein-coding genes (RPL) and the ACT1 gene for actin represent the nonregulated genes.

Colonies of mutants in genes induced during the acid/alkali transition exhibit defect in ammonia production. (A) Fifteen-day-old giant colonies of the parental strain BY4742 and of the ydr384c mutant growing on GM-BKP. (B) Ammonia production by giant colonies of BY4742 and pho89, ynr002c, ydr384c, ycr010c, and sok2 mutants. The ammonia produced by colonies growing on GM agar was absorbed (during indicated intervals) and measured as described. Color scale indicates visible acid/alkali transition of the parental strain. (C) Morphology of 20-d-old monocolonies of BY4742 and the sok2 growing on GM agar. (D) Northern analysis of MSN4 gene expression in giant colonies of BY4742 and the sok2. RNA was isolated from colonies either in the acid phase (9-d-old, yellow rectangle) or in the alkali phase (13-d-old, violet rectangle).

S. cerevisiae members of YaaH family are involved in ammonia production in colonies. (A) Region of homology of Ycr010p with the C. elegans putative ammonium transporter, P54145. Blue letters indicate the similarity. (B) The regions of similarity of YaaH family proteins with the putative ATS of Ycr010p. Most conservative amino acids (red). Nonconserved amino acids (black). Yeast sp. and amino acids conserved among yeast genes (green). Bacterial sp. and sequences conserved among bacterial genes (blue). The accession numbers of individual proteins are on Figure 4 at www.biologie.ens.fr/yeast-publi.html. The consensus sequence is indicated below, h, hydrophobic amino acids (A, C, F, G, H, I, K, L, M, R, T, V, W, Y), s, small amino acids (A, C, D, G, N, P, S, T, V), u, tiny amino acids (A, G, S), and l, aliphatic amino acids (I, L, V). (C) TMHMM prediction of the transmembrane localization of Ycr010p. Numbers indicate amino acids. Blue, region of homology with C. elegans P54145; violet, region of homology with ammonium transporter signature (ATS). (D) Average distance tree of YaaH family proteins. Ura3p of S. cerevisiae was used as a nonrelated protein control. (E) Ammonium production by cells from acid or alkali colonies of BY4742 and mutants ycr010c, ydr384c, and ynr002c. Samples of cells suspended in 10 mM MES (pH 6.0) were taken at 0, 5, 10, 15, 20, and 25 min of incubation. (F) Ammonium production by BY4742 cells from alkali colonies at different pH values. Samples of cells suspended in 10 mM MES of different pH (4, 5, 6, or 7) were taken at 0, 5, 10, 15, 20, and 25 min of incubation.

Model of metabolic changes within S. cerevisiae colonies during the acid/alkali transition. Summary of the important changes occurring in colonies in phases 1, 3, 4, and 5. A detailed description of the individual processes is given in the results and in Figure 2. The proposed relationships between the processes are described in the DISCUSSION. Red color indicates the activated expression of a particular functional gene group, green color indicates the repression. MT, mitochondria; PX, peroxisome; V, vacuole; OXPH, oxidative phosphorylation system; TCA, enzymes of tricarboxylate (citrate) cycle; ESR, environmental stress response factors; SUL, transporters of sulfate; PHO, transporters of phosphate; DCA, transporters of carboxylic acids; ATO, yeast members of YaaH family; AA, amino acids; OAA, oxaloacetate.

Model of the yeast glyoxylate bypass pathway and its possible connection with other predicted changes. Red part indicates the activated glyoxylate bypass. Green part indicates repressed part of the TCA cycle and oxidative phosphorylation. Notice that the limiting enzymes of glyoxylate bypass and oxaloacetate turn over are Cit3 and Icl2 (activated in phase 5) and Idh1/Idh2 (repressed in phase 5). The expression of ACO1 gene is twofold repressed in phase 5, but this enzyme seems to be very highly expressed in the cells (our own measurements), and this repression may not be detrimental to the glyoxylate bypass efficiency. The gene expression pattern suggests a switch from TCA degradative cycle to glyoxylate bypass at phase 5 of the acid/alkali transition.