Biochemical Characterization of Two Yeast Paralogous Proteins Mth1 and Std1
Glucose is the most abundant monosaccharide and preferred carbon and energy source for most cells. Many organisms have evolved sophisticated means to sense glucose and respond to it appropriately. The budding yeast, Saccharomyces cerevisiae senses glucose through two transmembrane proteins, Snf3 and Rgt2. In the presence of extracellular glucose Snf3 and Rgt2 generate an intracellular signal that leads to the degradation of Mth1 and Std1, thereby inducing the expression of hexose transporter genes ( HXT ) by inhibiting the function of Rgt1, a transcriptional repressor of HXT genes. Mth1 and Std1 are degraded via the Yck1/2 Kinase-SCF Grr1 -26S proteasome pathway triggered by the glucose sensors. RGT2-1 and SNF3-1 induce expression of HXT genes even in the absence of glucose. I show that RGT2-1 promotes ubiquitination and subsequent degradation of Mth1 and Std1 regardless of the presence of glucose. Site-specific mutagenesis reveals that conserved lysine residues of Mth1 and Std1 might serve as attachment sites for ubiquitin, and that the potential casein kinase (Yck1/2) consensus sites in Mth1 and Std1 are needed for their phosphorylation. The data provides biochemical evidence for glucose independent degradation of Mth1 and Std1. I further identified, the subcellular localization and the cellular compartment in which of Mth1 and Std1 are degraded in response to glucose. The data shows that, Mth1 and Std1 are present in nucleus when they are not degraded due to mutational blocks in the Snf3/Rgt2-Rgt1 pathway. Mth1 and Std1 could be degraded in both the nucleus and cytoplasm when its subcellular localization is artificially manipulated; however, glucose-induced degradation occurs only in the nucleus. I also demonstrate that membrane tethering of Yck1/2 plays no or little role in the degradation of Mth1. Transcriptomic analysis of mth1Δstd1Δ mutant identified new target genes for Mth1 and Std1 in new functional categories including mitochondrial/respiration genes, transporter genes and amino acid pathway genes in addition to HXT genes. This analysis provided insights into understanding the new functions of the two paralogous proteins Mth1 and Std1.