The Hexokinase Gene Is Required for Transcriptional Regulation of the Glucose Transporter Gene RAG1 in Kluyveromyces lactis

Abstract

The RAG1 gene of Kluyveromyces lactis encodes a low-affinity glucose/fructose transporter. Its transcription is induced by glucose, fructose, and several other sugars. The RAG4, RAG5, and RAG8 genes are trans-acting genes controlling the expression of the RAG1 gene. We report here the characterization of one of these genes, RAG5. The nucleotide sequence of the cloned RAG5 gene indicated that it encodes a protein that is homologous to hexokinases of Saccharomyces cerevisiae. rag5 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 rag5 mutants. The defect of the low-affinity component was found to be due to a block of transcription of the RAG1 gene by the hexokinase mutation. In vivo complementation of the rag5 mutation by the HXK2 gene of S. cerevisiae and complementation of hxk1 hxk2 mutations of S. cerevisiae by the RAG5 gene showed that RAG5 and HXK2 were equivalent for sugar-phosphorylating activity but that RAG5 could not restore glucose repression in the S. cerevisiae hexokinase mutants.

Regulation of primary carbon metabolism in Kluyveromyces lactis

“…RAG5 encodes the single hexokinase of K. lactis [18]. It may influence RAG1 expression because induction by glucose requires glucose phosphorylation.…”

Changing course: Glucose starvation drives nuclear accumulation of Hexokinase 2 in S. cerevisiae

“…cerevisiae , which did not occur in K . lactis [ 42 , 43 ]. Kl Hxk1 is like both Sc Hxk1 (70% identity) and Sc Hxk2 (73% identity) ( S4A Fig ), but it is slightly more similar to Sc Hxk2, as is evident when structures of these three enzymes are superimposed ( S4B–S4D Fig ) [ 41 ].…”

“…In K. lactis, the sole Hxk1 ortholog corresponds to both ScHxk1 and ScHxk2. ScHxk1 and ScHxk2 are paralogs that arose from a whole genome duplication in S. cerevisiae, which did not occur in K. lactis [42,43]. KlHxk1 is like both ScHxk1 (70% identity) and ScHxk2 (73% identity) (S4A Fig), but it is slightly more similar to ScHxk2, as is evident when structures of these three enzymes are superimposed (S4B-S4D Fig) [41].…”

Mutants with Enhanced Cellobiose-Fermenting Ability from Thermotolerant Kluyveromyces marxianus DMKU 3-1042, Which Are Beneficial for Fermentation with Cellulosic Biomass

“…Since at least three genes, HGT1, KHT2 and RAG1, for transporters that are able to uptake glucose [38][39][40] were found to have either nonsense or missense mutations in SY14 and SY15-2, their glucose-fermenting abilities were examined in YAYD medium (Figure 1). As expected, SY14 and SY15-2 showed large reductions of glucose utilization and ethanol production.…”