Catabolite inactivation of the high‐affinity hexose transporters Hxt6 and Hxt7 of Saccharomyces cerevisiae occurs in the vacuole after internalization by endocytosis¹

Abstract

After addition of high concentrations of glucose, rates of high-affinity glucose uptake in Saccharomyces cerevisiae decrease rapidly. We found that the high-affinity hexose transporters Hxt6 and Hxt7 are subject to glucose-induced proteolytic degradation (catabolite inactivation). Degradation occurs in the vacuole, as Hxt6/7 were stabilized in proteinase Adeficient mutant cells. Degradation was independent of the proteasome. The half-life of Hxt6 and Hxt7 strongly increased in end4, ren1 and act1 mutant strains, indicating that the proteins are delivered to the vacuole by endocytosis. Moreover, both proteins were also stabilized in mutants defective in ubiquitination. However, the initial signal that triggers catabolite inactivation is not relayed via the glucose sensors Snf3 and Rgt2.z 1998 Federation of European Biochemical Societies.

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

“…It has been demonstrated previously that Hxt7 is rapidly endocytosed and degraded in the vacuole when induced (raffinose-grown) cells are starved for nitrogen and treated with high glucose concentrations (Krampe et al, 1998). However, our results demonstrate that high glucose was not sufficient to stimulate endocytosis of Hxt7 when the protein was inappropriately expressed at high glucose concentrations during mid-exponential growth in the HXT7-only strain.…”

“…The proteins were transferred to nitrocellulose membranes at 25 V for 30 min, 50 V for 30 min and 100 V for 30 min at 4uC. The blots were sequentially incubated with anti-GFP rabbit antiserum diluted 1 : 10 000 (Molecular Probes) or antiHxt7 antiserum diluted 1 : 250 (Krampe et al, 1998), and with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody diluted 1 : 3000 (Bio-Rad). Detection was carried out by chemiluminescence using the SuperSignal chemiluminescent substrate kit (Pierce).…”

“…Particularly in the case of the HXT7-only strain in early exponential growth (at high residual glucose concentrations), the location of the protein is an important question, since under these conditions the protein is not expressed in wildtype strains. Moreover, in non-growing cells Hxt7 undergoes endocytosis and vacuolar degradation in response to high glucose concentrations (Krampe et al, 1998). In order to monitor the expression and targeting of the Hxt7 protein, a chimeric Hxt7::GFP protein was engineered and examined by fluorimetry and epifluorescence microscopy.…”

“…Moreover, the affinity of Hxt7 for glucose is modulated in response to both genetic and nutritional factors (Reifenberger et al, 1997;Walsh et al, 1996;this report). And finally, the Hxt7 protein is subject to endocytosis and proteolysis in the vacuole upon treatment of nitrogen-starved cells with high concentrations of glucose (Krampe et al, 1998).…”

Characterization of the xylose-transporting properties of yeast hexose transporters and their influence on xylose utilization

“…Construction of plasmids. The 0n4 kb SacI\SpeI MET25 promoter fragment of multicopy plasmid p426MET25 (Mumberg et al, 1994) was replaced by a 0n4 kb DNA fragment containing an HXT7 promoter fragment from k392 bp to k1 bp that was amplified by PCR with primers P426H7-1 (5h-CTAGAGCTCG TAGGAACAAT TTCGG-3h) and P426H7-2 (5h-CGACTAGTGT GATGGTGATG GTGATG-CATG TTAACTTTTT GATTAAAATT AAAAAAACTT3h), and plasmid YEpkHXT7 (Krampe et al, 1998) as the template, resulting in plasmid p4H7. All monosaccharidetransporter genes were cloned by recombination-cloning into p4H7 using the strategies described by Wieczorke et al (1999).…”