The hexokinase gene is required for transcriptional regulation of the glucose transporter gene RAG1 in Kluyveromyces lactis.

Abstract

The R4GI gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAGS, and RAG8 genes are trans-acting genes controlling the expression of the RAG) gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAGS gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. ragS mutants showed no detectable hexokinase or glucokinase activity, suggesting that the sugar kinase activity encoded by this gene is the only hexokinase in K. lactis. Both high-and low-affinity transport systems of glucose were affected in ragS mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG) gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the K2 gene of S. cerevisiae and complementation of hxikl hxk2 mutations of S. cerevisiae by the RAGS gene showed that RAGS and HXK2 were equivalent for sugar-phosphorylating activity but that RA4GS could not restore glucose repression in the S. cerevisiae hexokinase mutants.

Hexokinase as a sugar sensor in higher plants.

“…AtHXKI and AIHXK2 share 82% nucleotide identity, and AtHXKI and AtHXK2 share 85% amino acid identity. Results of data base searches and sequence comparisons showed that AtHXK proteins share significant sequence identity with human (Nishi et al, 1992) and rat (Magnuson et al, 1989) GLK (34 to 35%) and yeast (Stachelek et al, 1986;Prior et al, 1993) HXKs (36 to 38%) ( Figure 2). Conserved ATP and sugar binding domains (Bork et al, 1993) were found in the deduced protein sequences of both AtHXK genes (Figure 2).…”

“…A t h x k Z H w a n R a t Y e a m t l Y e a m t l Y e a r t 3 A t h x k l A t h x k Z H w a n R a t Y e a r t l Y e a m t Z Y e a m t 3 A comparison of predicted amino acid sequences of Arabidopsis AtHXK1 and AtHXK2, human GLK (Nishi et al, 1992), rat GLK (Magnuson et al, 1989), S. cerevisiae HXKl (Yeastl) and HXK2 (Yeast2) (Stachelek et al, 1986), and Kluyveromyces lactis RAG5 (Yeast3) (Prior et al, 1993) is shown. The ATP binding site (Bork et al, 1993) includes underlined regions 1, 2, and A, indicating the conserved phosphate 1 and 2 and adenosine interaction regions, respectively.…”

“…It is possible that specific HXK proteins are required for sugar signaling in each organism, and carbon flux might not be critical for triggering glucose repression. Furthermore, it has been shown that the genes encoding HXK from milk yeast (Kluyveromyces lactis) (Prior et al, 1993) and from fission yeast (Schizosaccharomyces pombe) (J.-C. Jang and J. Sheen, unpublished data) can complement the catalytic but not the regulatory function of YHXK2 in the S. cerevisiae hxkl hxk2 double mutant. These results further support the notion that glucose signaling may be uncoupled from sugar metabolism in yeasts and plants.…”

Secretion of Mouse α-Amylase fromKluyveromyces lactis

“…cerevisiae shuttle vector, pSK1, has replication origins from K. lactis (S11 fragment of pKD1) and S. cerevisiae (ori of 2 m) plasmids. This shuttle plasmid was originally constructed by Prior et al (1993) to characterize K. lactis homologous gene complementing an S. cerevisiae mutation and vice versa. Here, we constructed the shuttle secretion vector, pMI6, by inserting a mouse -amylase secretion cassette encoded by the 3·2 kb SalI-BamHI fragment of pMI5 (PGK promoter-signal sequence of the 128 kDa killer precursor--amylase-PGK terminator) into SalI/ BamHI-digested pSK1.…”

“…This fragment was subcloned into BamHI-SalI-digested pSK1 (the K. lactis/S. cerevisiae shuttle vector; Prior et al, 1993) to construct pMI6. The yeast was transformed using the Li + -salt described by Ito et al (1983).…”