The activity of NTH1 enzyme is mediated by yeast 14-3-3 isoforms


D. Veisova1, L. Rezabkova1,2, V. Obsilova1


1Institute of Physiology Academy of Sciences of the Czech Republic, 14220 Prague, Czech Republic

2Department of  Physical and Macromolecular Chemistry, Faculty of Science, Charles University, 12843 Prague, Czech Republic


Trehalase (EC is an intrinsic glycoprotein of the small intestine and renal brush-border membranes that hydrolyzes α,α- trehalose (1-α-D-glucopyranosyl α-D-glucopyranoside) to two glucose molecules in animals [1]. Three trehalases have been identified in Saccharomyces cerevisiae so far: neutral trehalase 1 (NTH1), neutral trehalase 2 (NTH2) and acidic trehalase 1 (ATH1). NTH1 is responsible for trehalose degradation, which is accumulated after stress [3]. The activity of the NTH1 enzyme was just recently found to be mediated by BMH1 and BMH2 binding in yeast. Yeast BMH1 and BMH2 proteins (yeast 14-3-3 isoforms) form a complex with neutral trehalase after its phosphorylation by PKA. Either one of the two 14-3-3 yeast isoforms are required for complete activation of neutral trehalase (NTH1) [2]. However details concerning the mechanism of BMH-dependent activation of NTH1 remain still unknown.


We showed that PKA phosphorylates NTH1 in vitro on three Ser residues: 20, 21 and 83. To find out which site or sites are essential for the 14-3-3 binding  we produced NTH1 WT (both phosphorylated and non-phosphorylated), three NTH1 mutants containing single phosphorylation site, one double phosphorylated NTH1 mutant (at Ser20 and 21) and a mutant containing none of these studied phosphorylation sites as well. The interaction between BMH1 and BMH2 protein with enzyme NTH1 was monitored using native electrophoresis and sedimentation velocity measurements. The sedimentation equilibrium analysis was used to define the stoichiometry of NTH1/BMH complexes. Finally, we used enzyme kinetic measurements to monitor the BMH-dependent activation of NTH1.


1.    Y. Ouyang, Q. Xu, K. Mitsui, M. Motizuki, Z. Xu, BBRC, 379, (2009), 621-625.

2.     S. Panni, C. Landgraf, R. Volkmer-Engert, G. Cesareni, L. Castagnoli, FEMS Yeast Research 8, (2008) 53-63.

3.     E. Garre, E. Matallana, Microbiology., 155, (2009), 3092-3099.


This work was funded by Grant P207/11/0455 of the Grant Agency of the Czech Republic and Centre of Neurosciences LC554 of the Ministry of Education, Youth, and Sports of the Czech Republic and by Research Project AV0Z50110509 of the Academy of Sciences of the Czech Republic.