Title: Identification and characterization of Sul1 and Sul2 in Saccharomyces cerevisiae as first sulfate sensors in cell biology
Other Titles: Identificatie en karakterisatie van Sul1 en Sul2 in Saccharomyces cerevisiae als eerste sulfaat sensoren in de celbiologie
Authors: Kankipati, Harish Nag; S0209684
Issue Date: 4-Apr-2014
Abstract: Saccharomyces cerevisiae is being used for many years by humans in the production of bread, beer and wine. It is now also used for the production of renewable energy sources such as bioethanol. Apart from being vital for the economy of the beverage sector, it is also the simplest eukaryotic model organism which has been most extensively studied to understand the basics of life. Many cellular processes in S. cerevisiae are well conserved in higher organisms, providing an advantage for researchers to easily study these cellular processes. In nature, cells are exposed to fluctuating environmental conditions to which they have the ability to adjust because of a myriad of adaptive mechanisms obtained over millions of years of evolution. In particular, microorganisms such as S. cerevisiae, are continuously exposed to rapid environmental changes from mere physiochemical properties such as temperature to nutrient availability. It is therefore very crucial for these organisms to coordinate their life cycle with these changes for their survival and perpetuation of their species.Nutrients play a pivotal role in the growth and proliferation of cells. They are used as energy sources and building blocks to scaffold all cellular components. Many studies have shown that nutrients also act as signaling molecules which trigger various signaling pathways inside the cells which lead to adaptations needed for the survival. Nutrient limitation conditions trigger cells to enter a low proliferative state characterized by phenotypes indicative of low protein kinase A (PKA). During this state, cells substitute growth and proliferation mechanisms by cell survival mechanisms such as increased carbohydrate storage, reduced ribosomal synthesis and increased expression of stress tolerant proteins which provide resistance in the unfavorable conditions. These phenotypic responses are reversed when the missing nutrient is restored in the medium, suggesting a strong signaling role for the nutrients in activating/deactivating growth and proliferation versus survival pathways.Recently, it has been shown that nutrient-specific receptor-like proteins called transceptors (because of their dual transporter-receptor function) scan for specific nutrients and trigger pathways leading to growth and proliferation upon their sensing of nutrient availability. As explained above, the absence of a single nutrient can trigger cells to enter a quiescent state in which among other processes, they induce the expression of specific nutrient transceptors at the plasma membrane. Reappearance of the missing nutrient in the medium is sensed by the nutrient-specific transceptor/s which then rapidly activates the PKA signaling cascade. In S. cerevisiae, transceptor sensing of nutrients and subsequent PKA activation is always dependent on the presence of a fermentable sugar. In contrast to classical PKA activation, this activation occurs independently of intracellular cAMP increase. Transceptor-dependent PKA activating pathways have been collectively designated as Fermentative Growth Medium (FGM) pathway to differentiate them from the classical cAMP-dependent activation of PKA pathway. Nutrient transceptors such as Gap1 and Pho84 have, for example, been shown to activate FGM signaling pathway upon sensing amino acids and phosphate, respectively.In this work, we have studied the effect of re-addition of sulfur to sulfur-starved cells. We have investigated the mechanisms of sulfate sensing and identified two sulfate transceptors that trigger PKA activation leading to cell growth and proliferation upon sulfate availability. We first optimized the sulfur starvation conditions by studying accumulation of trehalose and entry of cells in quiescent state. We then confirmed activation of the PKA signaling pathway upon re-addition of sulfate to sulfur starved cells. We verified this activation by analyzing the upregulation and downregulation of determined PKA downstream targets. Furthermore, we confirmed that sulfate signaling is mediated by Sul1 and Sul2, two previously known high affinity sulfate transporters. This uncovered novel signaling functions for Sul1,2 in addition to their previously known transporting function.Using two different approaches, we further demonstrated that the signaling function of Sul1 and Sul2 can occur independently from their transporting function. We first identified a non-transporting agonist, D-glucosamine 2-sulfate, which triggers PKA signaling in spite of not being transported into the cell. Then we identified residues in Sul1 and Sul2, whose mutation blocks the transporting ability of these two proteins without eliminating their sulfate-sensing and PKA signaling capacities. Moreover, these approaches confirmed that transport of sulfate into the cell is not necessary to trigger transceptor-mediated signaling and that metabolism of sulfate is not involved in this mode of PKA activation. After characterizing sulfate transporters Sul1 and Sul2 as sulfate transceptors, we have shown that these plasma membrane proteins undergo substrate-mediated endocytosis and are downregulated in the same way as classical receptors. Re-addition of sulfate to sulfate-starved cells downregulated sulfate transceptors both at the transcriptional and post transcriptional levels. We also identified that the conserved C-terminus STAS domain of the sulfate transceptors is important for both transport and sensor functions of Sul1 and Sul2.Finally, in this study we have additionally discovered and annotated ORF YIL166c as a low affinity sulfate transporter which we referred as Siu1. In this work we show that this transporter can compensate for the lack of Sul1 and Sul2 and allow growth of a sul1Δ sul2Δ mutant in the presence of high sulfate concentrations.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Molecular Microbiology and Biotechnology Section - miscellaneous

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