A role for calcium in the regulation of neutral trehalase activity in the fission yeast Schizosaccharomyces pombe

Abstract

Neutral trehalases mobilize trehalose accumulated by fungal cells as a protective and storage carbohydrate. A structural feature of these enzymes is the presence of an EF-like motif similar to that shown by many Ca2+-binding proteins. In this study we provide direct evidence for physical binding of Ca2+ to neutral trehalase (Ntp1p) of the fission yeast Schizosaccharomyces pombe, and show that aspartic residues at positions 97 and 108 in the conserved putative Ca2+-binding motif of Ntp1p appear to be responsible for this interaction. Mutations in these residues do not interfere with the ability of Ntp1p to associate in vivo with trehalose-6-phosphate synthase, but prevent activation of neutral trehalase triggered by the addition of glucose or by subjecting cells to stressing conditions. Strains expressing Ntp1p variants that are unable to bind Ca2+ partially resemble those devoid of the ntp1+ gene in terms of trehalose hyperaccumulation. Gel filtration of cell extracts from wild-type cells after EDTA treatment or from cells containing Ntp1p with mutations in aspartic acid residues within the Ca2+-binding site revealed that Ntp1p eluted mainly in an inactive conformation instead of the dimeric or trimeric active form of the enzyme. These results suggest that activation of S. pombe Ntp1p under different conditions depends upon Ca2+ binding through the Ca2+-binding motif as a prerequisite for correct enzyme oligomerization to its active form. Given the high degree of conservation of the Ca2+ accommodation site, this might be a general mechanism regulating neutral trehalase activity in other yeasts and filamentous fungi.

Role of the EF-hand-like Motif in the 14-3-3 Protein-mediated Activation of Yeast Neutral Trehalase Nth1

“…The N-terminal part of S. cerevisiae contains sequence that closely resembles such an EF-hand motif (Fig. 1), suggesting the possibility that this sequence and the calcium binding play a role in the regulation of this enzyme activity (13). In this work, various techniques of structural biology, including HDX- a The C␣-C␣ interresidue distance constraints used were based on the length of the spacer arm, which is 7.7 Å for DSG and 11.…”

“…Franco et al (13) showed that the Ca 2ϩ -dependent activation of Nth1 from Schizosaccharomyces pombe is mediated by a conserved Ca 2ϩ -binding EF-hand-like motif that is also present in S. cerevisiae Nth1 (sequence 114 DTDKNYQITIED 125 ). To investigate the importance of this motif in S. cerevisiae pNth1 activation, we performed site-directed mutagenesis of several residues that correspond to both conserved and non-conserved positions from EF-hand motifs of numerous Ca 2ϩ -binding proteins (Fig.…”

“…It has been shown that the yeast S. cerevisiae possesses several different trehalases: the vacuolar acid trehalase Ath1 with a lower pH optimum of about 4.5, and the cytoplasmic neutral trehalases Nth1 and Nth2 with a pH optimum of about 7 (7)(8)(9)(10). The sequence comparison revealed that neutral trehalases from yeast S. cerevisiae and Kluyveromyces lactis possess, compared with other organisms, an N-terminal extension that contains several protein kinase A (PKA) phosphorylation sites as well as the EF-hand-like calcium binding motif, suggesting that this region is involved in the regulation of these enzymes' activity (11)(12)(13). Indeed, it has recently been shown that the activity of S. cerevisiae Nth1 is regulated by PKA phosphorylation, Ca 2ϩ , and the 14-3-3 protein binding (14 -16).…”

In Vivo Phosphorylation of Ser21 and Ser83 during Nutrient-induced Activation of the Yeast Protein Kinase A (PKA) Target Trehalase

“…Recently, mass spectrometry evidence was reported for phosphorylation of purified trehalase by PKA in vitro on Ser 20 , Ser 21 , Ser 60 , and Ser 83 (24). Schizosaccharomyces pombe neutral trehalase has a structure similar to that of S. cerevisiae trehalase and is rapidly activated under similar environmental conditions (25, 26). Mutagenesis of the putative PKA phosphorylation sites resulted in inactive trehalases unresponsive to environmental stimulation.…”

Molecular basis of the 14-3-3 protein-dependent activation of yeast neutral trehalase Nth1

“…Compared with trehalases from prokaryotic and higher eukaryotic organisms, yeast neutral trehalases possess an N-terminal extension that contains four cAMP-dependent protein kinase (PKA) phosphorylation sites (S20, S21, S60, and S83 in Saccharomyces cerevisiae Nth1) and the calcium-binding EF-hand-like motif (Fig. 1A), indicating significantly different regulation (17)(18)(19). It was subsequently shown that S. cerevisiae Nth1 is indeed activated in a PKA-and calcium-dependent manner in a process that requires interaction with the 14-3-3 protein through motifs containing phosphoserines pS60 and pS83 (20)(21)(22)(23).…”