The insertion of two amino acids into a transcriptional inducer converts it into a galactokinase

Abstract

The transcriptional induction of the GAL genes of Saccharomyces cerevisiae occurs when galactose and ATP interact with Gal3p. This protein-small molecule complex associates with Gal80p to relieve its inhibitory effect on the transcriptional activator Gal4p. Gal3p shares a high degree of sequence homology to galactokinase, Gal1p, but does not itself possess galactokinase activity. By constructing chimeric proteins in which regions of the GAL1 gene are inserted into the GAL3 coding sequence, we have been able to impart galactokinase activity upon Gal3p as judged in vivo and in vitro. Remarkably, the insertion of just two amino acids from Gal1p into the corresponding region of Gal3p confers galactokinase activity onto the resultant protein. The chimeric protein, termed Gal3p+SA, retains its ability to efficiently induce the GAL genes. Kinetic analysis of Gal3p+SA reveals that the K(m) for galactose is similar to that of Gal1p, but the K(m) for ATP is increased. The chimeric enzyme was found to have a decreased turnover number in comparison to Gal1p. These results are discussed in terms of both the mechanism of galactokinase function and that of transcriptional induction.

Figure 1

Sequence comparison of galactokinase molecules. The numbers at the top refer to the Gal3p amino acid sequence. Sc, Saccharomyces cerevisiae; Ec, Escherichia coli; Bs, Bacillus subtilis; Ca, Candida albicans; Hi, Haemophilus influenzae; St, Salmonella typhimurium; Kl, Kluyveromyces lactis; At, Arabidopsis thaliana; Hs, Homo sapiens; St, Streptomyces lividans. Amino acids have been colored according to their properties. Blue indicates positively charge amino acids (H, K, R), red indicates negatively charged residues (D, E), green indicates polar neutral residues (S, T, N, Q), grey indicates nonpolar aliphatics (A, V, L, I, M), and purple indicates nonpolar aromatic residues (F, Y, W). Brown is used to indicate proline and glycine, whereas yellow indicates cysteine.

Figure 2

The activity of Gal3p deletion mutations. (A) Plasmids expressing the wild-type Gal3p protein or the deletion derivative indicated were transformed into a yeast strain in which the GAL3 gene had been disrupted. Each of the strains were then grown on either raffinose (Raf) or galactose (Gal) as the sole carbon source, and cell growth was monitored after 3 days of growth at 30°C. (B) The effect of various Gal3p deletion derivatives on the expression of GAL10. Northern blot analysis of GAL10 and 18S RNA expression was monitored daily after the cells were transferred to a galactose-containing medium. (C) Western blot analysis of Gal3p and the deletion derivatives. A yeast strain deleted for GAL1, GAL3, and GAL80 was transformed with the appropriate Gal3p expression vector and grown on galactose for 2 days. Western blot analysis was performed on whole cell extracts using either Gal3p polyclonal antibodies or 30C12, a mouse monoclonal antibody raised against Gal3p.

Figure 3

The insertion of two amino acids into Gal3p converts it into a galactokinase. Plasmids expressing Gal1p, Gal3p, or the indicated chimera were transformed into yeast strains lacking GAL1 (Δ1), GAL3 (Δ3), or both GAL1 and GAL3 (Δ1,Δ3). Cells were then plated onto media containing either raffinose (R) or galactose (G) as the sole carbon source. Growth was monitored after 3 days incubation at 30°C. The chimeras are numbered to indicate the amino acids from Gal3p that have been replaced by the corresponding sequences from Gal1p.

Figure 4

The activity of Gal3p+SA in vitro. In vitro transcription and primer extension reactions were performed as described previously (9). All reactions contained 1.5 mM galactose and 1 mM ATP and, where indicated, 3 nM Gal4p (amino acids 1–93 fused to 768–881), 3 nM Gal80p and either Gal3p (75, 25, and 8 nM in lanes 4, 5, and 6, respectively), Gap3p+SA (75, 25, and 8 nM in lanes 7, 8, and 9, respectively), or Gal1p (3,000, 1,000, and 333 nM in lanes 10, 11, and 12 respectively).