The yeast, Saccharomyces cerevisiae, has an ENOX1 activity with a period length of 24 min similar to that of other eukaryotes. In contrast to other eukaryotes, however, Saccharomyces cerevisiae has a second ENOX1-like activity with a period length of 25 min. The latter is distinguishable from the traditional ENOX1 on the basis of the longer period length along with resistance to an ENOX1 inhibitor, simalikalactone D, and failure to be phased by melatonin. In addition, two periods are apparent in measurements of oxygen consumption indicating that the consumption of oxygen to water occurs independently by homodimers of both of the two forms of ENOX. Based on the measurements of glyceraldehyde-3- phosphate dehydrogenase, S. cerevisiae exhibits circadian activity maxima at 24 and 25 h together with a 40 h period possibly representing the 40 min metabolic rhythm of yeast not observed in our measurement of oxygen consumption and normally observed only with continuous cultures. The findings are indicative of at least three independent time-keeping systems being operative in a single cell.
ECTO-NOX designates a family of cell surface proteins that exhibit a time-keeping function, oxidize reduced coenzyme Q or NAD(P)H and carry out protein disulfide-thiol interchange [
At least three members of the ECTO-NOX (ENOX) family have been described: 1) the human constitutive NADH oxidase (ENOX1 or cNOX) (GenBank Accession No. EF432052), expressed in normal cells and located on chromosome 13q14.11; 2) the human tumor-associated NADH oxidase (ENOX2 or tNOX) (GenBank Accession No. AF207881), present on the surface of invasive cancer cells and located on chromosome Xq25-q26.2 and 3) the age-related NADH oxidase (arNOX or ENOX3), which is present only in individuals after the age of 30, in late passage cultured cells and in senescent plant parts [
ECTO-NOX proteins differ from all other NAD(P)H oxidases in that they are able to reduce pyridine nucleotides in the absence of bound flavin [
Although enzymatic activity has been most often measured by monitoring NAD(P)H oxidation at 340 nm, the physiological substrates appear to be quinols (i.e., ubiquinols or phytoquinols) [10,11]. The NADH binding site is on the outside of the cell where there is usually little or no NAD(P)H. Quinones, such as Coenzyme Q, act as trans-plasma membrane shuttles to transfer reducing equivalents from cytosolic NAD(P)H to external acceptors, including molecular oxygen and protein disulfide. ENOX1 and ENOX2 lack iron or iron sulfur clusters. However, the copper binding motifs revealed from sequence analysis and site-directed mutagenesis are necessary for electron transfer to molecular oxygen [6,7]. Based on size exclusion chromatography, the soluble forms of ENOX2 are predominantly dimers. Thereforethe ENOX proteins may be viewed as homodimeric proteins containing four coppers per dimer, thus carrying out four electron transfers to molecular oxygen as required to form water [
ECTO-NOX proteins exhibit a second catalytic activity that of protein disulfide-thiol interchange despite the absence of the CXXC motif common to most members of the protein disulfide isomerase family [5,6,12]. The two activities, hydroquinone oxidation and protein disulfide-thiol interchange, alternate with a period length that is characteristic for each family member. ENOX1 exhibits oscillations with a period length of 24 min, that of ENOX2 with a period length of about 22 min and that of arNOX with a period length of about 26 min [
Several lines of evidence demonstrate that ENOX1 and ENOX2 proteins are involved in at least two important cell functions. They drive the enlargement phase of cell growth and they are components of the biological clock. The regulation of the circadian oscillatory system is mainly ascribed to ENOX1. The two enzymatic activities catalyzed by ENOX1 alternate within a 24 min period: the first activity rests after 12 min and the second one begins in a cycle that confers a time-keeping function to the protein. This oscillation pattern is entrainable and temperature independent. Cells transfected with mutated ENOX cDNAs, which have oscillation period lengths of 22, 36 or 42 min, exhibited circadian period lengths of 22, 36 or 42 h for the cellular circadian biochemical marker, glyceraldehyde-3-phosphate dehydrogenase [
ENOX proteins are ubiquitous at the surface of all cells thus far studied [
In this report, we have identified two different constitutive oscillatory ENOX proteins of S. cerevisiae one with a period length of 24 min and a second with a period length of about 25 min. Both proteins catalyze the oxidation of both externally supplied NAD(P)H to NAD(P)+ and hydroquinone and carry out protein disulfide interchange. These two forms of ENOX activity together with an already known ultradian rhythm with a period length of 40 min in S. cerevisiae generate three independent sets of potential ultradian time keeping oscillations.
The yeast strains were maintained on YEPD agar plates and stored at 4˚C. Cells were grown at room temperature with shaking in rich media (YEPD) for 1 - 2 days to attain ca 2 × 108 cells/ml. ENOX proteins of yeast are exposed at the external surface of the yeast cells such that activities may be measured with unbroken cells. Additionally, the yeast cells were heated for 1 h at 70˚C to inactivate heat-sensitive enzymes prior to analysis. This was to prevent yeast contamination in the open laboratory. ENOX proteins resist heating at 70˚C for one h. Results were similar with unheated yeast cells.
Heat-inactivated yeast cells (ca. 107 cells in 50 µl) were combined with 2.5 ml 50 mM Tris-Mes buffer (pH 7.0), 2 mM KCN and 150 µM NADH. To refold and renature the ENOX proteins of the heat-inactivated yeast, reduced glutathione (GSH) was added to a final concentration of 100 µM, and incubated for 10 min to reduce disulfides to allow for refolding. Next H2O2 was added to a final concentration of 0.03% followed by incubation for 10 min to reform disulfides to stabilize the protein in a correctly folded conformation.
NADH oxidation was measured in a Hitachi Model U3210 spectrophotometer at 340 nm, continuously for one min at 1.5 min intervals for two h using an extinction coefficient of 6.21 mM−1∙cm−1. Inhibitors were added at 60 min and data were collected for a further 60 min. A second machine of the same model with the vehicle alone added at 60 min served as the control.
Reduced coenzyme Q10 was prepared fresh by addition of an equal volume of 0.25% NaBH4 under nitrogen to a 7.5 mM stock solution of coenzyme Q10 in ethanol. After several min of incubation, 0.l volume of 0.1 N HC1 was added to degrade the excess NaBH4. The reaction mixture for measuring the oxidation of the reduced coenzyme Q10 (Q10H2) contained heat-inactivated yeast, ca. 107 cells in 2.5 ml of 50 mM Tris-Mes buffer, pH 7.0 and 0.08% Triton X-100 to maintain the Q10H2 in suspension. The reaction was started with the addition of 40 µl of 5 mM Q10H2. The disappearance of reduced CoQ was measured at both 290 and 410 nm using an extinction coefficient of 0.805 mM−1∙cm−1.
Heat-inactivated yeast (ca. 107 cells in 50 µl) were added to 2.5 ml of 50 mM Tris-Mes, pH 7.0. The reaction was pre-incubated with 0.5 µmol of 2.2’-dithiodipyridine (DTDP) in 5 µl DMSO to oxidize residual thiols. After 10 min of incubation, a further 3.5 µmol of DTDP were added in 35 µl of DMSO to start the reaction. The increase in absorbance due to the cleavage of DTDP was monitored at 340 nm using a millimolar extinction coefficient of 6.21.
Yeast were grown in 200 ml minimal SD minus uracil or SR minus uracil media (Open Biosystems, Huntsville, AL) on a shaker at 200 rpm at room temperature for 2 - 3 days at 25˚C. Samples of 50 µl were collected every 2 h and snap frozen at −80˚C. Time windows were used to collect the samples, with the flow between collections returned to the flask. Although this was a batch culture, the small sample sizes did not reduce the total volume of the culture significantly, and each sample had approximately the same total protein content.
To prepare the samples for the assay, the yeast were thawed and 50 µl of Y-PER (Yeast Protein Extraction Reagent, Pierce) were added and the solution was vortexed for 20 min to lyse the yeast and release the cell contents. Glyceraldehyde-3-phosphate dehydrogenase activity was assayed in a reaction solution containing 100 µl lysed yeast, 0.1 M Tris-HC1, 0.5 mM EGTA, pH 8.0, 1 mM Na2HAsO4, 2 mM NAD+ and 3 mM glyceroldehyde-3-phosphate. NAD+ reduction was determined from the increases in absorbance at 340 nm at 37˚C measured over 5 min using a millimolar extinction coefficient of 6.21.
Results were analyzed using fast Fourier transform and decomposition fits [
Standard NAD(P)H oxidase activity assays in which the disappearance of NADH was measured over one min at 1.5 min intervals in heat-inactivated yeast revealed two sets of oscillations based on the recurrence of two maxima separated by 6 min of similar but not identical period lengths. One set denoted by ① and ② in
increases the complexity of the data over that of mammalian cells and plants which normally exhibit only a single set of oscillations with a period length of 24 min.
To resolve the two oscillatory patterns, fast Fourier (
To link the two ENOX activities and the extra maxima corresponding to a 40 min ultradian period to the circadian clock, glyderaldehyde-3-phosphate dehydrogenase (GAPDH) activity was used. GAPDH activity peaks once within each 24 h period making it an ideal marker of circadian day length [
As measured by cleavage of the substrate 2,2’-dithiodipyridine (DTDP), three maxia separated by 4.5 min were generated by mammalian ENOX1 [
Although externally-supplied NADH was used to assay ENOX activity, hydroquinones resident in the plasma membrane are most likely the natural substrate [
*Decomposition analyses decompose a time series into trend, cyclic, and error components. The measured values of
Melatonin shifts the period of ENOX1 in mammalian cells [
sumed exactly 24 min following addition of melatonin 1.5 min out of phase with the original 24 min oscillations. In contrast, melatonin addition did not affect the oscillations with the 25 min period length (A and B) (
An extract from the leaves and bark of Quassia amara L. containing the ENOX 1 inhibitor, simalikalactone D, that inhibits the activity of ENOX1 in mammalian and plant preparations [
The mammalian form of ENOX1 binds 2 moles of copper/mole of protein and copper is essential for its activity
[
TFA and bathocuproine treated preparations by addition of copper. In the presence of TFA, both ENOX1 activities were present (
The period lengths of the two ENOX forms in yeast were
temperature independent (
A hallmark of the ENOX family of proteins is that of protease resistance. When the yeast ENOX forms were assayed after digestion with proteinase K or trypsin, the activity was unaffected even when unfolded in the presence of trifluoroacetic acid prior to addition of protease (
Organisms grown in heavy water (deuterium oxide or D2O) exhibit a circadian day lengthened in period length by about 25% [23,24]. When mammalian ENOX was assayed in heavy water, it exhibited a period length of about 30 min [
1Period length monitored over time intervals as long as 6 h varied with a cumulative error of less than one min; 2Nanomoles/min/106 cells.
*Trifluoroacetic acid.
min for the maxima labeled ① and ② and of approximately 32 min (31.25 min) for the maxima labeled A and B (
Mammalian ENOX1 exhibits several defining characteristics to indicate that yeast contain not one but two ENOX forms similar to the mammalian ENOX1. The ENOX activities of yeast are at the external surface of the plasma membrane so that activity may be measured with intact cells using external NADH, a membrane impermeant substrate. The activities are protease and heat (80˚C) resistant in keeping with data from mammalian sources [26-28]. In mammalian and plant preparations of ENOX1, 5 maxima are seen, in which the first two dominant maxima are separated by 6 min from each other, and the remaining 3 subordinate maxima are separated from each other and from the first two maxima by 4.5 min [
Analysis of the statistical validity of asymmetric, nonsinusoidal oscillatory patterns such as those exhibited by ENOX proteins is limited primarily to Fast Fourier and decomposition (seasonal forecasting) types of analyses [
were resolved into two recurrent patterns, one with a period length of 24 min and one with a period length of 25 min. Three statistical measures of reproducibility MAPE (mean absolute percentage error), MAD (mean average deviation) and MSD (mean standard deviation) comparing successive periods from the same data sets revealed a mean standard deviation of 5% or less for each of the several data sets amenable to this type of analysis.
Both of the two patterns of ENOX activity, one with a period length of approximately 24 min and one with a period length of approximately 25 min share features in common with mammalian ENOX1. In addition to NADH oxidation, both activities cleave the dithiodipyridine (DTDP) substrate indicative of protein disulfidethiol exchange and oxidize hydroquinone indicating that both proteins share the dual functionality of other ECTONOX family members. These alternating activities are unprecedented in the biochemical literature and, thus far, are unique to the ENOX family of proteins [1,15].
More compelling evidence came from measurements of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity as an indicator of circadian day length [
Despite important similarities, the two yeast ENOX activities are not identical. Upon addition of melatonin, mammalian ENOX1 exhibits a new maximum exactly 24 min after melatonin addition as in characteristic of ENOX1 of other organisms [
The 24 h rhythm in GAPDH activity corresponded to the 24 min ENOX1 activity as observed in other organisms, and accounted for one of the two ENOX activities demonstrated here for heat-inactivated yeast. It was blocked by simalikalactone D and phased by melatonin whereas the activity with the 25 min period length was not. Thus we suggest that the 25 h rhythm corresponds to a second ENOX activity, which in addition to a slightly longer (25 min) period length, responded differently from ENOX1 to ENOX modulators.
In contrast to melatonin and simalikalactone D which affected only the ENOX1 with the 24 min period, the periods of both ENOX forms of S. cerevisiae were lengthened by assay in D2O. The latter underscores the vital role of water associated with the ENOX1 proteinbound copper hexahydrate in the oscillatory process [
Among the first defined clock-related oscillation in yeast, known as the respiratory oscillation, was a 40 min cycle of dissolved oxygen levels in continuous culture [20-22,32-35]. After establishing that the oscillations were driven by some aspect of ethanol metabolism [
Maxima separated by 40 min and not assignable to either the ENOX form with the 24 min period or the ENOX form with the 25 min period were normally not observed. Only in
It is interesting that in yeast, ENOX1 inhibitors do not inhibit growth, as they do in mammalian and plant cells [