Abstract
Glucose, which is the most preferred carbon source for the yeast Saccharomyces cerevisiae, is transported across the plasma membrane into cells by hexose transporter (Hxt) proteins. The Hxt proteins are encoded by a multigene family consisting of 20 members. It was shown previously that HXT1-4 and HXT6-7 encode the major
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Hxt proteins and contribute mainly to glucose transport, whereas the remaining Hxt proteins have other or unknown functions. Expression of the major HXT genes is regulated by the extracellular glucose concentration. Once glucose is inside the cell it is either metabolized or stored inside the cell as reserve carbohydrates. Trehalose, which is one of the reserve carbohydrates, accumulates when the growth rate of cells decreases as a consequence of glucose exhaustion or conditions that are disadvantageous for growth. Earlier studies suggested that Tps1p, an important protein involved in trehalose production, might control glucose transport. Thus, trehalose accumulation and glucose transport seem to be connected.
In this thesis the regulation of HXT5 expression, and the role of Hxt5 in trehalose accumulation, were studied in more detail. It appeared that expression of HXT5 is not regulated by the availability of extracellular glucose, but by the growth rate of yeast cells, which is defined as the time it takes for cells to divide. In order to determine how the growth rate exactly regulates HXT5 expression on the transcriptional level, putative regulatory elements in the promoter of the HXT5 gene were mutated. In this manner transcription factors were no longer able to bind to these elements to regulate HXT5 expression. The results indicated that one of the two so-called Stress Responsive Elements (STREs) in the promoter of HXT5 was extremely important for regulation of HXT5 expression, whereas HXT5 expression was additionally regulated by two Hap2/3/4/5 complex binding elements. Earlier studies in our laboratory revealed that accumulation of trehalose is also regulated by the growth rate of cells. Therefore, the role of Hxt5 in trehalose accumulation was studied in more detail. Strikingly, the expression pattern of HXT5 and the trehalose accumulation pattern were identical during a variety of conditions of slow growth. Furthermore, a common signal transduction pathway, being the cAMP/PKA pathway, regulates expression of HXT5 and TPS1. However, in cells deleted for hxt5 trehalose accumulation was decreased to only 80% of wildtype levels, indicating that Hxt5 may indeed contribute to, but is not exclusively involved in trehalose accumulation. In order to determine potential other functions of Hxt5 besides glucose transport, DNA microarray experiments were performed to identify genes with expression patterns similar to HXT5 during a specific condition of slow growth. This method was chosen, because genes with similar expression patterns may encode proteins that are involved in similar processes. Besides genes involved in trehalose accumulation, also genes involved in respiration, protein folding, stress responses and carbon metabolism were expressed similar to HXT5. A high percentage of these genes were enriched in STREs in their promoter region. Finally, the factors and processes that may contribute to regulation of the growth rate and growth rate-regulated expression of genes in Saccharomyces cerevisiae are discussed
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