Glucose Uptake Kinetics and Transcription of HXTGenes in Chemostat Cultures of Saccharomyces cerevisiae

Abstract

The kinetics of glucose transport and the transcription of all 20 members of the HXT hexose transporter gene family were studied in relation to the steady state in situ carbon metabolism of Saccharomyces cerevisiae CEN.PK113-7D grown in chemostat cultures. Cells were cultivated at a dilution rate of 0.10 h ؊1 under various nutrient-limited conditions (anaerobically glucose-or nitrogen-limited or aerobically glucose-, galactose-, fructose-, ethanol-, or nitrogen-limited), or at dilution rates ranging between 0.05 and 0.38 h ؊1 in aerobic glucose-limited cultures. Transcription of HXT1-HXT7 was correlated with the extracellular glucose concentration in the cultures. Transcription of GAL2, encoding the galactose transporter, was only detected in galactoselimited cultures. SNF3 and RGT2, two members of the HXT family that encode glucose sensors, were transcribed at low levels. HXT8 -HXT17 transcripts were detected at very low levels. A consistent relationship was observed between the expression of individual HXT genes and the glucose transport kinetics determined from zero-trans influx of 14 C-glucose during 5 s. This relationship was in broad agreement with the transport kinetics of Hxt1-Hxt7 and Gal2 deduced in previous studies on single-HXT strains. At lower dilution rates the glucose transport capacity estimated from zerotrans influx experiments and the residual glucose concentration exceeded the measured in situ glucose consumption rate. At high dilution rates, however, the estimated glucose transport capacity was too low to account for the in situ glucose consumption rate.

Expression and activity of the Hxt7 high‐affinity hexose transporter of Saccharomyces cerevisiae

“…When the expression of HXT6 and HXT7 was assessed independently of one another in wild-type strains, they were shown to be differentially regulated; in these cases HXT7 expression was much higher than HXT6 expression (Diderich et al, 1999;Reifenberger et al, 1997;P. van Hoek, J. Pronk and A. Kruckeberg, unpublished results).…”

“…Total RNA was isolated from the yeast cells by acid-phenol extraction as described (Diderich et al, 1999). RNA samples were separated by electrophoresis in 1% agarose formaldehyde gels (Sambrook et al, 1989).…”

“…RNA samples were separated by electrophoresis in 1% agarose formaldehyde gels (Sambrook et al, 1989). Transfer to nylon membranes, prehybridization, hybridization with HXT7-and PDA1-specific oligonucleotide probes, and washing were performed as described (Diderich et al, 1999).…”

“…HXT7 is also transcribed under the widest variety of conditions; its mRNA is abundant in cells approaching the diauxic shift during batch culture on glucose medium (DeRisi et al, 1997;Diderich et al, 1999) and during batch culture on galactose and ethanol/glycerol media (Liang and Gaber, 1996). It is also transcribed under a wide variety of chemostat-cultivation conditions in both a wildtype laboratory strain and an industrial baker's yeast strain (Diderich et al, 1999;P.…”

The amino‐terminal tail of Hxt11 confers membrane stability to the Hxt2 sugar transporter and improves xylose fermentation in the presence of acetic acid

“…These HXT genes are differentially regulated at the levels of expression and post‐translational inactivation in the response to sugar concentration (Boles and Hollenberg, 1997; Kruckeberg et al, 1999). For instance, yeast expresses the high‐affinity glucose transporter encoding HXT2 and HXT7 genes at low‐glucose conditions (Diderich et al, 1999). Importantly, transcription of these genes is repressed at high glucose concentration, while under those conditions, Hxt2 and Hxt7 proteins at the membrane are targeted to the vacuole for degradation (Kruckeberg et al, 1999; Ye et al, 2001).…”