Characterization of Mutants Devoid of Neutral Trehalase Activity in the Fission Yeast Schizosaccharomyces pombe : Partial Protection from Heat Shock and High-Salt Stress

Abstract

Exposure of cells of Schizosaccharomyces pombe to heat shock or osmotic upshift results in an increased level of neutral trehalase activity, which is responsible for hydrolysis of intracellular trehalose. We constructed S. pombe mutants lacking neutral trehalase activity by gene replacement at the newly defined ntp1 + locus. Analysis of these mutants revealed that a twofold increase in trehalose accumulation, enhanced acquired thermoresistance, and marked salt tolerance characterized their ability to grow in liquid and solid media. Analysis of the expression of the trehalase gene under heat shock and osmotic upshift revealed the transcriptional activation ofntp1 + in response to both stresses.

Role for trehalase during germination of spores in the fission yeast Schizosaccharomyces pombe

“…In a previous work we described that ntp1-disrupted strains of S. pombe, which are devoid of neutral trehalase activity, were able to sporulate [12]. In the present study we analysed the germination of these spores in liquid medium and found marked di¡erences when compared to wild-type spores.…”

“…Obviously, this can be explained by the occurrence in spores of other trehalase activity. The existence of additional neutral trehalase activity is di¤cult to rationalise since it escapes biochemical detection in ntp1-disrupted cells and no sig-ni¢cant homology was detected with other S. pombe genes during cloning of ntp1 as to suggest the existence of related enzymes [12]. Instead, a candidate enzyme to account for these results would be the sporulation-speci¢c acid trehalase described in spores and asci of the ¢ssion yeast [8,9].…”

“…Neutral trehalase was assayed as described previously [12]. Acid trehalase was assayed at pH 4.2 as indicated in [9].…”

“…Neutral trehalase is post-translationally activated upon increases in the level of cAMP, and its corresponding gene ntp1 has been cloned and sequenced [11]. Disruption mutants in this gene show better survival than wild-type cells after multiple stresses, including dehydration, osmostress and heat shock [12]. Another trehalase, which appears to be sporulation-specific, shows optimal activity at acidic pH and it is found in walls of asci and spores of the ¢ssion yeast [8,9].…”

Molecular interaction of neutral trehalase with other enzymes of trehalose metabolism in the fission yeast Schizosaccharomyces pombe

“…These data indicate that, in addition to the monomeric forms, at least two distinct complexes exist containing significant amounts of all three proteins. One corresponds to the high molecular mass complex (700-800 kDa) and the other to a more diffusely spread population corresponding to lower molecular mass range (80-250 kDa, fractions [12][13][14][15][16][17]. To clarify which of these protein complexes exhibit neutral trehalase activity, we prepared log-phase cultures from strain C335 and subjected them to thermal (40°C, 1 h) or osmotic (0.75 M NaCl, 2 h) stress.…”