Arthur L. Kruckeberg

Functional analysis of the hexose transporter homologue HXT5 in Saccharomyces cerevisiae

The HXT5 gene encodes a functional hexose transporter that has moderate affinity for glucose (Km=10 mM), moderate to low affinity for fructose (Km=40 mM) and low affinity for mannose (Km>100 mM). The sole presence of Hxt5p in an otherwise hexose transport null mutant is sufficient to sustain a flux through glycolysis from glucose to fermentative products. However, the presence of HXT5 as the sole hexose transporter gene results in extremely poor growth on glucose, which suggests the involvement of glucose repression in the transcriptional regulation of HXT5. From Northern blot analysis on the members of the HXT family and studies with HXT5 tagged with the green fluorescent protein (GFP), it is evident that HXT5 is transcribed and translated during conditions of relatively slow growth, during growth on non‐fermentable carbon sources and in particular during sporulation. In wild‐type batch cultivations on fermentable carbon sources, Hxt5p is abundant in stationary phase or after depletion of the fermentable carbon source, which seems independent of the carbon source. The deletion of HXT5 does not result in a clear phenotype. A shift of stationary phase cells to fresh glucose medium resulted in somewhat slower resumption of growth in the hxt5 deletion strain compared to the wild‐type strain. The abundance of Hxt5p during stationary phase, sporulation and low glucose conditions suggests that HXT5 is a ‘reserve’ transporter, which might be involved in the initial uptake of glucose after the appearance of glucose. Other possible functions of the protein encoded by HXT5 will be discussed in the context of the results. Copyright

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

ABSTRACT Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. Thehxk2 mutant had a higher mitochondrial H+-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.

Intracellular localization of an active green fluorescent protein-tagged Pho84 phosphate permease in Saccharomyces cerevisiae.

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the carboxy‐terminus of the Pho84 phosphate permease of Saccharomyces cerevisiae. Both components of the fusion protein displayed their native functions and revealed a cellular localization and degradation of the Pho84‐GFP chimera consistent with the behavior of the wild‐type Pho84 protein. The GFP‐tagged chimera allowed for a detection of conditions under which the Pho84 transporter is localized to its functional environment, i.e. the plasma membrane, and conditions linked to relocation of the protein to the vacuole for degradation. By use of the methodology described, GFP should be useful in studies of localization and degradation also of other membrane proteins in vivo.

Hexokinase regulates kinetics of glucose transport and expression of genes encoding hexose transporters in Saccharomyces cerevisiae

Glucose transport kinetics and mRNA levels of different glucose transporters were determined in Saccharomyces cerevisiae strains expressing different sugar kinases. During exponential growth on glucose, a hxk2 null strain exhibited high-affinity hexose transport associated with an elevated transcription of the genes HXT2 and HXT7, encoding high-affinity transporters, and a diminished expression of the HXT1 and HXT3 genes, encoding low-affinity transporters. Deletion of HXT7 revealed that the high-affinity component is mostly due to HXT7; however, a previously unidentified very-high-affinity component (K(m) = 0.19 mM) appeared to be due to other factors. Expression of genes encoding hexokinases from Schizosaccharomyces pombe or Yarrowia lipolytica in a hxk1 hxk2 glk1 strain prevented derepression of the high-affinity transport system at high concentrations of glucose.

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

High‐affinity hexose transport is required for efficient utilization of low hexose concentrations by the bakers 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. Copyright

Utilization of green fluorescent protein as a marker for studying the expression and turnover of the monocarboxylate permease Jen1p of Saccharomyces cerevisiae

Green fluorescent protein (GFP) from Aequorea victoria was used as an in vivo reporter protein when fused to the C-terminus of the Jen1 lactate permease of Saccharomyces cerevisiae. The Jen1 protein tagged with GFP is a functional lactate transporter with a cellular abundance of 1670 molecules/cell, and a catalytic-centre activity of 123 s(-1). It is expressed and tagged to the plasma membrane under induction conditions. The factors involved in proper localization and turnover of Jen1p were revealed by expression of the Jen1p-GFP fusion protein in a set of strains bearing mutations in specific steps of the secretory and endocytic pathways. The chimaeric protein Jen1p-GFP is targeted to the plasma membrane via a Sec6-dependent process; upon treatment with glucose, it is endocytosed via END3 and targeted for degradation in the vacuole. Experiments performed in a Deltadoa4 mutant strain showed that ubiquitination is associated with the turnover of the permease.

How do yeast cells sense glucose

A glucose-sensing mechanism has been described in Saccharomyces cerevisiae that regulates expression of glucose transporter genes. The sensor proteins Snf3 and Rgt2 are homologous to the transporters they regulate. Snf3 and Rgt2 are integral plasma membrane proteins with unique carboxy-terminal domains that are predicted to be localized in the cytoplasm. In a recent paper Ozcan and colleagues [Ozcan S, et al. EMBO J 1998; 17:2556-2773 (Ref. 1)] present evidence that the cytoplasmic domains of Snf3 and Rgt2 are required to transmit a glucose signal. They provide additional evidence to support their earlier assertion [Ozcan S, et al. Proc Natl Acad Sci USA 1996;93:12428-12432 (Ref. 2)] that glucose transport via Snf3 and Rgt2 is not involved in glucose sensing but, rather, that these proteins behave like glucose receptors. Other examples of transporter homologs with regulatory functions have recently been described in fungi as well [Madi L, et al. Genetics 1997; 146:499-508 (Ref. 3). and Didion T, et al. Mol Microbiol 1998;27:643-650 (Ref. 4)]. The identification of this class of nutrient sensors is an important step in elucidating the complex of regulatory mechanisms that leads to adaptation of fungi to different environments.

Mutagenic and functional analysis of the C-terminus of Saccharomyces cerevisiae Pho84 phosphate transporter

A widely accepted mechanism for selective degradation of plasma membrane proteins is via ubiquitination and/or phosphorylation events. Such a regulated degradation has previously been suggested to rely on the presence of a specific SINNDAKSS sequence within the protein. Modification of a partly conserved SINNDAKSS‐like sequence in the C‐terminal tail of the Pho84 phosphate transporter, in combination with C‐terminal fusion of green fluorescent protein or a MYC epitope, were used to evaluate the presence of this sequence and its role in the regulated degradation. The functional Pho84 mutants in which this SINNDAKSS‐like sequence was altered or truncated were subjected to degradation like that of the wild type, suggesting that degradation of the Pho84 protein is regulated by factors other than properties of this sequence.

The Yeast Phosphate Transporting System

The life of Saccharomyces cerevisiae with about 6000 putative genes and gene products distributed over several intracellular compartments is maintained and regulated by a complicated interplay between its different cellular components. For the cell to respond to different internal and external signals, proteins are synthesised and routed to their functional localisation. Phosphate starvation is such a signal leading to derepression of several genes in the PHO-regulon of the yeast cell (Oshima, 1997). A de novo production of proteins active in cellular phosphate acquisition under these limiting conditions results in a continued viability of the cells.