Evidence for a modulation of neutral trehalase activity by Ca2+ and cAMP signaling pathways in Saccharomyces cerevisiae

Saccharomyces cerevisiae neutral trehalase (encoded by NTH1) is regulated by cAMP-dependent protein kinase (PKA) and by an endogenous modulator protein. A yeast strain with knockouts of CMK1 and CMK2 genes (cmk1cmk2) and its isogenic control (CMK1CMK2) were used to investigate the role of CaM kinase II in the in vitro activation of neutral trehalase during growth on glucose. In the exponential growth phase, cmk1cmk2 cells exhibited basal trehalase activity and an activation ratio by PKA very similar to that found in CMK1CMK2 cells. At diauxie, even though both cells presented comparable basal trehalase activities, cmk1cmk2 cells showed reduced activation by PKA and lower total trehalase activity when compared to CMK1CMK2 cells. To determine if CaM kinase II regulates NTH1 expression or is involved in post-translational modulation of neutral trehalase activity, NTH1 promoter activity was evaluated using an NTH1-lacZ reporter gene. Similar ß-galactosidase activities were found for CMK1CMK2 and cmk1cmk2 cells, ruling out the role of CaM kinase II in NTH1 expression. Thus, CaM kinase II should act in concert with PKA on the activation of the cryptic form of neutral trehalase. A model for trehalase regulation by CaM kinase II is proposed whereby the target protein for Ca2+/CaM-dependent kinase II phosphorylation is not the neutral trehalase itself. The possible identity of this target protein with the recently identified trehalase-associated protein YLR270Wp is discussed

Shared control of maltose and trehalose utilization in Candida utilis

Trehalose biosynthesis and its hydrolysis have been extensively studied in yeast, but few reports have addressed the catabolism of exogenously supplied trehalose. Here we report the catabolism of exogenous trehalose by Candida utilis. In contrast to the biphasic growth in glucose, the growth of C. utilis in a mineral medium with trehalose as the sole carbon and energy source is aerobic and exhibits the Kluyver effect. Trehalose is transported into the cell by an inducible trehalose transporter (K M of 8 mM and V MAX of 1.8 µmol trehalose min-1 mg cell (dry weight)-1. The activity of the trehalose transporter is high in cells growing in media containing trehalose or maltose and very low or absent during the growth in glucose or glycerol. Similarly, total trehalase activity was increased from about 1.0 mU/mg protein in cells growing in glucose to 39.0 and 56.2 mU/mg protein in cells growing in maltose and trehalose, respectively. Acidic and neutral trehalase activities increased during the growth in trehalose, with neutral trehalase contributing to about 70% of the total activity. In addition to the increased activities of the trehalose transporter and trehalases, growth in trehalose promoted the increase in the activity of alpha-glucosidase and the maltose transporter. These results clearly indicate that maltose and trehalose promote the increase of the enzymatic activities necessary to their catabolism but are also able to stimulate each other's catabolism, as reported to occur in Escherichia coli. We show here for the first time that trehalose induces the catabolism of maltose in yeast

Data analysis procedures for pulse ELDOR measurements of broad distance distributions

Jeschke G, Panek G, Godt A, Bender A, Paulsen H. Data analysis procedures for pulse ELDOR measurements of broad distance distributions. APPLIED MAGNETIC RESONANCE. 2004;26(1-2):223-244.The reliability of procedures for extracting the distance distribution between spins from the dipolar evolution function is studied with particular emphasis on broad distributions. A new numerically stable procedure for fitting distance distributions with polynomial interpolation between sampling points is introduced and compared to Tikhonov regularization in the dipolar frequency and distance domains and to approximate Pake transformation. Distance distributions with only narrow peaks are most reliably extracted by distance-domain Tikhonov regularization, while frequency-domain Tikhonov regularization is favorable for distributions with only broad peaks. For the quantification of distributions by their mean distance and variance, Hermite polynomial interpolation provides the best results. Distributions that contain both broad and narrow peaks are most difficult to analyze. In this case a high signal-to-noise ratio is strictly required and approximate Pake transformation should be applied. A procedure is given for renormalizing primary experimental data from protein preparations with slightly different degrees of spin labelling, so that they can be compared directly. Performance of all the data analysis procedures is demonstrated on experimental data for a shape-persistent biradical with a label-to-label distance of 5 nm, for a [2]catenane with a broad distance distribution, and for a doubly spin-labelled double mutant of plant light harvesting complex II