The role of Mep2 in yeast pseudohyphal growth

PhD ThesisIn response to limiting levels of nitrogen in the environment, the diploid yeast Saccharomyces cerevisiae undergoes a dimorphic switch from yeast like growth to filamentous pseudohyphal growth. During this morphological change yeast cells grow as elongated chains of cells attached to each other away from the colony to forage for nutrients. Earlier studies have established the two major signalling pathways that regulate pseudohyphal growth include the MAP Kinase and cAMP-PKA pathways. The Mep2 ammonium transporter is an indispensable but poorly understood element of the pseudohyphal pathway. Although the role of Mep2 in this dimorphic switch has been recognized, the precise molecular mechanisms that link ammonium transport to this dimorphic switch is still unclear. Two distinct models of Mep2 function have been proposed. In the first, pH model, import of substrate during ammonium transport (either ammonium ion, ammonia gas or ammonia gas plus proton) would result in localised cytosolic pH changes which is sensed by an appropriate signal transduction pathway. In the second, transceptor model, Mep2 behaves like a transceptor by undergoing a conformational change during ammonium transport allowing it to physically engage a downstream signalling partner to initiate pseudohyphal growth. The pH model was tested which demonstrates that Mep2 signalling is independent of intracellular pH changes. The genetic screen to identify potential interaction partners of Mep2 identified an interaction between Mep2 and the 14-3-3 protein Bmh1. This interaction has been confirmed using western analysis of membrane fractions and demonstrated that this interaction is lost in signalling deficient Mep2 mutants. The 14-3-3 protein binding site in Mep2 has been identified which is required for the Mep2 dependent activation of the MAP Kinase pathway during pseudohyphal growth. A model for Mep2 sensing is proposed where Mep2 recruits signalling components to the membrane enabling cells to establish polarity where Mep2 is most active.BBSR

MAX randomisation: designed, non-degenerate saturation mutagenesis of armadillo repeat proteins.

Non-degenerate saturation mutagenesis is critical to library composition both in terms of library size and amino acid representation. Unlike conventional methodologies,non-degeneracy permits equal representation of all encoded amino acids and effectively eliminates termination codons from saturated positions. We have previously described both ‘MAX’ randomisation, which saturates physically separated codons and ‘ProxiMAX’ randomisation, which saturates contiguous codons. Both allow the additional advantage of encoding only required amino acids without reference to codon sequence, but their methodologies are quite different. Whilst MAX randomisation employs a manual process of selectional hybridisation between individual oligonucleotides and a conventionally-randomised template, ProxiMAX relies on saturation cycling; repeated cycles of blunt-ended ligation, Type IIS restriction and PCR amplification. MAX randomisation is thus typically employed in the research laboratory to engineer active sites of enzymes (or α-helices within other proteins), whilst ProxiMAX is now a chiefly commercial, automated process employed in antibody engineering. Here we present the application of MAX randomisation to engineer libraries of Armadillo Repeat Proteins (ArmRPs), α-helical proteins that selectively bind extended peptides. We have utilised the MAX randomisation technique to engineer ArmRPs for the generation of gene libraries encoding multiple repeat modules, saturating seven positions and encoding between 4 and 18 amino acids within each location, achieving excellent correlation between the design and observed specifications. We also present early developments to extend MAX randomisation into the realm of multiple contiguous codons

The Energy-Signaling Hub SnRK1 Is Important for Sucrose-Induced Hypocotyl Elongation

Emerging seedlings respond to environmental conditions such as light and temperature to optimize their establishment. Seedlings grow initially through elongation of the hypocotyl, which is regulated by signaling pathways that integrate environmental information to regulate seedling development. The hypocotyls of Arabidopsis (Arabidopsis thaliana) also elongate in response to sucrose. Here, we investigated the role of cellular sugar-sensing mechanisms in the elongation of hypocotyls in response to Suc. We focused upon the role of SnRK1, which is a sugar-signaling hub that regulates metabolism and transcription in response to cellular energy status. We also investigated the role of TPS1, which synthesizes the signaling sugar trehalose-6-P that is proposed to regulate SnRK1 activity. Under light/dark cycles, we found that Suc-induced hypocotyl elongation did not occur in tps1 mutants and overexpressors of KIN10 (AKIN10/SnRK1.1), a catalytic subunit of SnRK1. We demonstrate that the magnitude of Suc-induced hypocotyl elongation depends on the day length and light intensity. We identified roles for auxin and gibberellin signaling in Suc-induced hypocotyl elongation under short photoperiods. We found that Suc-induced hypocotyl elongation under light/dark cycles does not involve another proposed sugar sensor, HEXOKINASE1, or the circadian oscillator. Our study identifies novel roles for KIN10 and TPS1 in mediating a signal that underlies Suc-induced hypocotyl elongation in light/dark cycles

Circadian Entrainment in Arabidopsis by the Sugar-Responsive Transcription Factor bZIP63.

Synchronization of circadian clocks to the day-night cycle ensures the correct timing of biological events. This entrainment process is essential to ensure that the phase of the circadian oscillator is synchronized with daily events within the environment [1], to permit accurate anticipation of environmental changes [2, 3]. Entrainment in plants requires phase changes in the circadian oscillator, through unidentified pathways, which alter circadian oscillator gene expression in response to light, temperature, and sugars [4-6]. To determine how circadian clocks respond to metabolic rhythms, we investigated the mechanisms by which sugars adjust the circadian phase in Arabidopsis [5]. We focused upon metabolic regulation because interactions occur between circadian oscillators and metabolism in several experimental systems [5, 7-9], but the molecular mechanisms are unidentified. Here, we demonstrate that the transcription factor BASIC LEUCINE ZIPPER63 (bZIP63) regulates the circadian oscillator gene PSEUDO RESPONSE REGULATOR7 (PRR7) to change the circadian phase in response to sugars. We find that SnRK1, a sugar-sensing kinase that regulates bZIP63 activity and circadian period [10-14] is required for sucrose-induced changes in circadian phase. Furthermore, TREHALOSE-6-PHOSPHATE SYNTHASE1 (TPS1), which synthesizes the signaling sugar trehalose-6-phosphate, is required for circadian phase adjustment in response to sucrose. We demonstrate that daily rhythms of energy availability can entrain the circadian oscillator through the function of bZIP63, TPS1, and the KIN10 subunit of the SnRK1 energy sensor. This identifies a molecular mechanism that adjusts the circadian phase in response to sugars.FAPESP, The Royal Society, the Bristol Centre for Agricultural Innovation, the Peter und Traudl Engelhorn-Stiftung, the National Council of Technological and Scientific Development (CNPq) (Brazil), and Consejo Nacional de Ciencia y Tecnología (Mexico