Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.