Biology subjects

Bhat, P. J.

Publications and source records attributed to Bhat, P. J..

5 recordsLinked to original sources

bioRxiv2024

Co-lethality of LEU2 and BAP2 is carbon source dependent and is overcome by BUL1 deletion in Saccharomyces cerevisiae

In a leucine requiring Saccharomyces cerevisiae strain, deletion of BAP2, a high affinity leucine permease confers lethality in presence of fermentable sugars, but not other carbon sources. Deletion of BUL1, an adaptor overcomes this lethality. BUL1 deletion augments leucine import through accumulation of Bap3 and Bap2 in the plasma membrane and reduces their turnover. We suspect BUL1 as a cognate adaptor for ubiquitylation of these permeases.

genetics↗

bioRxiv2022

Epistasis between synonymous and nonsynonymous mutations in Dictyostelium discoideum ammonium transporter amtA drives functional complementation in Saccharomyces cerevisiae.

Role of Horizontal Gene Transfer (HGT) in evolution transcends across the three domains of life. Ammonium transporters are present in all species and therefore offer an excellent paradigm to study protein evolution following HGT. While investigating HGT through complementation assay, we observed that synonymous and nonsynonymous mutations follow an epistastic relationship. As a proxy for HGT, we attempted to complement a mep1mep2mep3{Delta} strain of S. cerevisiae (triple deletion strain) which cannot grow on ammonium as a sole nitrogen source below a concentration of 3 mM, with amtA of D. discoideum. As the wild type amtA did not complement, we isolated two mutant derivatives of amtA that complemented the triple deletion strain of S. cerevisiae. amtA M1 bears three nonsynonymous and two synonymous substitutions and these substitutions are necessary for its functionality. amtA M2 bears two nonsynonymous and one synonymous substitution, all of which are necessary for functionality. These mutants were then studied at phenotypic, cell biological, and biochemical level. Interestingly, AmtA M1 transports ammonium but does not confer toxicity to methylamine while AmtA M2 transports ammonium as well as confers methylamine toxicity, demonstrating functional diversification. Based on the results presented, we suggest that protein evolution cannot be fathomed by studying nonsynonymous and synonymous substitutions separately. This is because, protein evolution entails an interaction between synonymous and nonsynonymous substitution, which seems to have gone unnoticed thus far. Above observations have significant implications in various facets of biological processes and are discussed in detail. HighlightsO_LIAmmonium transporters (Amts) from bacteria to humans complement in yeast C_LIO_LIAmtA of D. discoideum does not complement yeast defective for ammonium uptake C_LIO_LISynonymous & nonsynonymous mutations are essential for AmtA functionality in yeast C_LIO_LIConformational differences underlie functionality & functional diversification C_LIO_LIProtein evolution entails interaction of synonymous & nonsynonymous mutations C_LI

genetics↗

bioRxiv2022

Yeast galactokinase in closed conformation can switch between catalytic and signal transducer states.

S.cerevisiae galactokinase (ScGal1p), in closed conformation catalyzes the phosphorylation of galactose to galactose 1-phopshate using ATP as the phosphate donor as well as allosterically activates the GAL genetic switch in response to galactose and ATP as ligands. How both kinase and signaling activities of ScGal1p are associated with closed conformation of the protein is not understood. Conformational sampling of ScGal1p indicated that this protein samples closed kinase and closed non-kinase conformers. Closed non-kinase conformers are catalytically incompetent to phosphorylate galactose and act as a bonafide signal transducer. It was observed that toggling of side chain of highly conserved K266 of ScGal1p between S171and catalytic base D217 is responsible for transitioning of ScGal1p between signal transducer and kinase states. Interestingly in ScGal3p, the paralog of ScGal1p, which has only signal transduction activity and lacks kinase activity, a H bond between a non-conserved Y433, and a highly conserved Y57, gets broken during MD simulation. The corresponding H-bond present in ScGal1p between residues Y441 and Y63 respectively, remains intact throughout the simulations of ScGal1p.Therefore, we predicted that K266 and Y441 have a role in bifunctionality of ScGal1p. To test the above predictions, we monitored the signaling and kinase activity of ScGal1K266Rp and ScGal1Y441Ap variants. Signaling activity increased in both ScGal1Y441Ap and ScGal1K266Rp variants as compared to ScGal1wtp, whereas the kinase activity increased in ScGal1Y441Ap, but decreased in ScGal1K266Rp Based on the above, we propose that K266 and Y441 are crucial for conferring bifunctionality to ScGal1p. Author summaryGalactokinase of S.cerevisiae(ScGal1p), the first enzyme of Leloir pathway of galactose metabolism, phosphorylates galactose using ATP as the phosphate donor. ScGal1p also functions as a signal transducer of GAL regulon wherein galactose and ATP allosterically activate galactokinase. The active form of galactokinase, then sequesters the repressor ScGal80p, to activate the GAL switch. ScGal1p has a single site each for binding to galactose and ATP. How ScGal1p, a monomeric protein, performs the above two mutually exclusive activities using the same set of substrates/ligands, with the same site acting as the active site for enzymatic activity as well as allosteric site for signal transduction activity is unclear. Our findings are that this protein has a distinct conformational state for functioning as a signal transducer and a distinct conformational state for functioning as a kinase. A highly conserved lysine residue (K266) present only in fungal galactokinases, triggers the interconversion between catalysis and signal transduction states. This interconversion is subdued by H bond between Y441 and Y63. These studies suggest that the two activities of ScGal1p are fine tuned by evolution to regulate metabolism through transcriptional control.

genetics↗

bioRxiv2021

Cellular heterogeneity and MTH1 play key roles in galactose mediated signaling of the GAL switch to utilize the disaccharide melibiose

Yeast metabolizes the disaccharide melibiose by hydrolyzing it into equimolar concentrations of glucose and galactose by MEL1-encoded -galactosidase. Galactose metabolizing genes (including MEL1) are induced by galactose and repressed by glucose, which are the products of melibiose hydrolysis. Therefore, how melibiose catabolization and utilization take place by circumventing the glucose repression is an enigma. Other than the galactose metabolizing genes MTH1, a negative regulator of glucose signal pathway has Gal4p binding sites and is induced by galactose and repressed by high glucose concentration. But, at low or no glucose MTH1 along with its paralogue STD1 represses hexose transporters, that are involved in glucose transport. This sort of tuning of glucose and galactose regulation motivated us to delineate the role of MTH1 as a regulator of MEL1 expression and melibiose utilization. The deletion mutant of MTH1 shows growth defect on melibiose and this growth defect is enhanced upon the deletion of both MTH1 and its paralogue STD1. Microscopy and flowcytometry analysis, suggest, that even though MEL1 and GAL1 promoter are under Gal4p and Gal80p regulation, upon deletion of MTH1 it hampers only MEL1 expression, but not the GAL1 gene expression. By using 2-Deoxy galactose toxicity assay, we observed phenotypic heterogeneity in cells grown on melibiose i.e. after cleaving of melibiose a fraction of cell population utilizes glucose and another fraction utilizes galactose and coexist together. Understanding GAL/MEL gene expression patterns in melibiose will have great implication to understand various other complex sugar utilizations, tunable gene expressions and complex feedback gene regulations. SignificanceSugar metabolism is an important phenomenon to understand the regulation of gene expression. Glucose is the most preferred carbon source. Yeast follows glycolytic pathways like cancer cells for metabolism of sugars and understanding this will throw more light to the metabolism of cancer cells. In this communication we observed cell-to-cell heterogeneity in yeast cells playing a key role in metabolism of a complex disaccharide melibiose, which gets cleaved into glucose and galactose by -galactosidase. Glucose represses -galactosidase and galactose induces it. Because of the heterogeneous population of cells one fraction consumes glucose liberated by melibiose hydrolysis, therefore it is not sufficient to repress -galactosidase and other GAL genes. Therefore, GAL genes are expressed and help in metabolizing melibiose and galactose.

microbiology↗

bioRxiv2020

Membrane localization of paralogous leucine permeases Bap2 and Bap3 is regulated by Bul1

Timeliness in expression and degradation of the nutrient permeases is crucial for any organism. In Saccharomyces cerevisiae, post translational regulation of nutrient permeases such as trafficking and turnover are poorly understood. We found that loss of a leucine permease BAP2, but not other permeases lead to severe growth retardation when the carbon source is glucose or galactose but not glycerol and lactate. Leucine prototrophy suppressed the retardation, showing BAP2 and LEU2 are synthetically lethal. We discovered that loss of BUL1, an arrestin involved in trafficking of diverse permeases suppressed this lethality. The suppression required another leucine permease, BAP3. Our results suggest that BUL1 downregulate permeases BAP2 and BAP3 present in plasma membrane through Rsp5 dependent endocytosis. We speculate that by regulating leucine import BUL1 regulates the activity of TORC1.

genetics↗