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

Abstract

High-affinity hexose transport is required for efficient utilization of low hexose concentrations by the baker's yeast Saccharomyces cerevisiae. These low concentrations occur during the late exponential phase of batch growth on hexoses, during hexose-limited chemostat or fed-batch culture, or during growth on sugars such as sucrose and raffinose that are hydrolysed to hexoses outside the cell. The expression of the Hxt7 high-affinity glucose transporter of S. cerevisiae was examined during batch growth on glucose medium in a wild-type strain and a strain expressing only HXT7 (i.e. with null mutations in HXT1-HXT6). In the wild-type strain, HXT7 transcription was repressed at high glucose and was detected when the glucose in the culture approached depletion. In the HXT7-only strain, transcription of HXT7 was constitutive throughout the glucose growth phase and was increased further at low glucose concentrations. After glucose depletion, the levels of HXT7 mRNA declined rapidly in both strains. In contrast, the Hxt7 protein was relatively stable after glucose depletion. By monitoring the subcellular localization of an Hxt7::GFP fusion protein it was observed that Hxt7 was localized in the plasma membrane, even when expressed at high glucose concentrations in the HXT7-only strain. After glucose depletion Hxt7 was gradually endocytosed and targeted to the vacuole for degradation. The Hxt7::GFP fusion protein was a fully functional hexose transporter with a catalytic centre activity of approximately 200/sec. It is concluded that repression of HXT7 and degradation of Hxt7 at high glucose concentrations is dependent on a high glucose transport capacity.