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 Mthl and Stdl, thereby inducing the expression of hexose transporter genes (EXT) by inhibiting the function of Rgtl, a transcriptional repressor of HXT genes. Mthl and Stdl are degraded via the Yckl/2 Kinase-SCFGrrl-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 Mthl and Stdl regardless of the presence of glucose. Sitespecific mutagenesis reveals that conserved lysine residues of Mthl and Stdl might serve as attachment sites for ubiquitin, and that the potential casein kinase (Yckl/2) consensus sites in Mthl and Stdl are needed for their phosphorylation. The data provides biochemical evidence for glucose independent degradation of Mthl and Stdl. I further identified, the subcellular localization and the cellular compartment in which of Mthl and Stdl are degraded in response to glucose. The data shows that, Mthl and Stdl are present in nucleus when they are not degraded due to mutational blocks in the SnO/Rgt2-Rgtl pathway. Mthl and Stdl 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 Yckl/2 plays no or little role in the degradation of Mthl. Transcriptomic analysis of mthlAstdlA mutant identified new target genes for Mthl and Stdl 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 Mthl and Stdl

Role of Casein Kinase 1 in the Glucose Sensor-Mediated Signaling Pathway in Yeast

BackgroundIn yeast, glucose-dependent degradation of the Mth1 protein, a corepressor of the glucose transporter gene (HXT) repressor Rgt1, is a crucial event enabling expression of several HXT. This event occurs through a signaling pathway that involves the Rgt2 and Snf3 glucose sensors and yeast casein kinase 1 and 2 (Yck1/2). In this study, we examined whether the glucose sensors directly couple with Yck1/2 to convert glucose binding into an intracellular signal that leads to the degradation of Mth1. ResultsHigh levels of glucose induce degradation of Mth1 through the Rgt2/Snf3 glucose signaling pathway. Fluorescence microscopy analysis indicates that, under glucose-limited conditions, GFP-Mth1 is localized in the nucleus and does not shuttle between the nucleus and cytoplasm. If glucose-induced degradation is prevented due to disruption of the Rgt2/Snf3 pathway, GFP-Mth1 accumulates in the nucleus. When engineered to be localized to the cytoplasm, GFP-Mth1 is degraded regardless of the presence of glucose or the glucose sensors. In addition, removal of Grr1 from the nucleus prevents degradation of GFP-Mth1. These results suggest that glucose-induced, glucose sensor-dependent Mth1 degradation occurs in the nucleus. We also show that, like Yck2, Yck1 is localized to the plasma membrane via C-terminal palmitoylation mediated by the palmitoyl transferase Akr1. However, glucose-dependent degradation of Mth1 is not impaired in the absence of Akr1, suggesting that a direct interaction between the glucose sensors and Yck1/2 is not required for Mth1 degradation. ConclusionGlucose-induced, glucose sensor-regulated degradation of Mth1 occurs in the nucleus and does not require direct interaction of the glucose sensors with Yck1/2

Biochemical Evidence for Glucose-Independent Induction of \u3ci\u3eHXT\u3c/i\u3e Expression in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

The yeast glucose sensors Rgt2 and Snf3 generate a signal in response to glucose that leads to degradation of Mth1 and Std1, thereby relieving repression of Rgt1-repressed genes such as the glucose transporter genes (HXT). Mth1 and Std1 are degraded via the Yck1/2 kinase-SCFGrr1-26S proteasome pathway triggered by the glucose sensors. Here, we show that RGT2-1 promotes ubiquitination and subsequent degradation of Mth1 and Std1 regardless of the presence of glucose. Site-specific mutagenesis reveals that the conserved lysine residues of Mth1 and Std1 might serve as attachment sites for ubiquitin, and that the potential casein kinase (Yck1/2) sites of serine phosphorylation might control their ubiquitination. Finally, we show that active Snf1 protein kinase in high glucose prevents degradation of Mth1 and Std1

Reverse Recruitment: The Nup84 Nuclear Pore Subcomplex Mediates Rap1/Gcr1/Gcr2 Transcriptional Activation

The recruitment model for gene activation presumes that DNA is a platform on which the requisite components of the transcriptional machinery are assembled. In contrast to this idea, we show here that Rap1/Gcr1/Gcr2 transcriptional activation in yeast cells occurs through a large anchored protein platform, the Nup84 nuclear pore subcomplex. Surprisingly, Nup84 and associated subcomplex components activate transcription themselves in vivo when fused to a heterologous DNA-binding domain. The Rap1 coactivators Gcr1 and Gcr2 form an important bridge between the yeast nuclear pore complex and the transcriptional machinery. Nucleoporin activation may be a widespread eukaryotic phenomenon, because it was first detected as a consequence of oncogenic rearrangements in acute myeloid leukemia and related syndromes in humans. These chromosomal translocations fuse a homeobox DNA-binding domain to the human homolog (hNup98) of a transcriptionally active component of the yeast Nup84 subcomplex. We conclude that Rap1 target genes are activated by moving to contact compartmentalized nuclear assemblages, rather than through recruitment of the requisite factors to chromatin by means of diffusion. We term this previously undescribed mechanism reverse recruitment and discuss the possibility that it is a central feature of eukaryotic gene regulation. Reverse recruitment stipulates that activators work by bringing the DNA to an nuclear pore complex-tethered platform of assembled transcriptional machine components

Massively parallel reporter assay confirms regulatory potential of hQTLs and reveals important variants in lupus and other autoimmune diseases

Summary: We designed a massively parallel reporter assay (MPRA) in an Epstein-Barr virus transformed B cell line to directly characterize the potential for histone post-translational modifications, i.e., histone quantitative trait loci (hQTLs), expression QTLs (eQTLs), and variants on systemic lupus erythematosus (SLE) and autoimmune (AI) disease risk haplotypes to modulate regulatory activity in an allele-dependent manner. Our study demonstrates that hQTLs, as a group, are more likely to modulate regulatory activity in an MPRA compared with other variant classes tested, including a set of eQTLs previously shown to interact with hQTLs and tested AI risk variants. In addition, we nominate 17 variants (including 11 previously unreported) as putative causal variants for SLE and another 14 for various other AI diseases, prioritizing these variants for future functional studies in primary and immortalized B cells. Thus, we uncover important insights into the mechanistic relationships among genotype, epigenetics, and gene expression in SLE and AI disease phenotypes

Motif mimetic of epsin perturbs tumor growth and metastasis

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