31P NMR studies of intracellular pH and phosphate metabolism during cell division cycle of Saccharomyces cerevisiae.

Abstract

We have analyzed changes in intracellular pH and phosphate metabolism during the cell cycle of Saccharomyces cerevisiae (NCYC 239) by using high-resolution 31P NMR spectroscopy. High-density yeast cultures (2 x 108 cells per ml) were arrested prior to "start" by sequential glucose deprivation, after which they synchronously replicated DNA and divided after a final glucose feeding. Oxygenation of arrested cultures in the absence ofglucose led to increased levels ofsugar phosphates and ATP and an increase in intracellular pH. However, these conditions did not initiate cell cycle progression, indicating that energization is not used as an intracellular signal for initiation ofthe cell division cycle and that the cells need exogenous carbon sources for growth. Glucose refeeding initiated an alkaline intracellular pH transient only in the synchronous cultures, showing that increased intracellular pH accompanies the traversal of start. Changes in phosphate flow and utilization also were observed in the synchronous cultures. In particular, there was increased consumption of external phosphate during DNA synthesis. When external phosphate levels were low, the cells consumed their internal polyphosphate stores. This shows that, under these conditions, polyphosphate acts as a phosphate supply. 31P NMR spectroscopy is rapidly gaining in importance as a technique for the noninvasive analysis ofphosphorus-containing metabolites and intracellular pH in vivo. Since the first 31p NMR spectra ofyeast were published in 1975 (1), sensitivity and resolution have been improved to allow for the investigation of glycolytic mutants (2), the control of glycolysis (3), and compartmentation of pH in ascospores (4). In this communication, we present the results of a 31P NMR analysis of synchronous suspensions of yeast cells in which we have investigated intracellular pH and phosphorus metabolism during the cell division cycle.Yeast are an ideal system for investigating the cell division cycle. We have found that they can be maintained for long periods at the high densities (2 x 108 cells per ml) necessary to obtain good signal-to-noise in the NMR experiments. In addition, many temperature-sensitive cell division cycle mutants of Saccharomyces cerevisiae are available, allowing for more detailed analyses in the future.Concepts of the cell division cycle in yeast have changed in recent years. Using the cell division cycle mutant system, Hartwell and his colleagues (5-10) have identified the presence of two loosely coupled subcycles, each comprising a series of discrete, interdependent steps. One subcycle involves the nuclear events of DNA replication and nuclear division; the other consists of the cytoplasmic events governing bud emergence and growth. The subcycles converge prior to mitosis and diverge again after a point called "start"; this point bears some similarity to the R point ofmammalian cells (11). Although much is known with regard to the morphologic and genetic aspects of "start," its biochemistry is relatively unknown. ...

Cytoplasmic pH and the regulation of the dictyostelium cell cycle

“…Thus, in the slime mold Physarum polycephalum pHi rises rapidly just before mitosis (Gerson and Burton, 1977), whereas in the protozoan Tetrahymena there are two alkaline pHr transients per cell cycle (Gillies and Deamer, 1979). A transient alkalinization has also been observed in synchronous cultures of yeast (Gillies et al, 1981). Taken together, these findings have raised the possibility that pHr has a regulatory role in the mitotic cycleof eukaryotic cells.…”

Physiological control of repressible acid phosphatase gene transcripts in Saccharomyces cerevisiae.

“…Changes in phosphate flow and utilization have also been observed in synchronous cultures of S. cerevisiae (15,16 (15,16). During this same period of the cell cycle, APase has been shown to have a step-wise increase in activity (33) associated with the morphological changes occurring during bud emergence (13,28).…”

“…The derepression of exocellular APase results in the generation of Pi from available external phosphoester substrates (41,46) followed by its intracellular incorporation. Derepression of the other enzymes results in polyP degradation (16,49) followed by utilization of the liberated Pi in the biosynthesis of nucleic acids and phospholipids (18). Thus, two potential precursor pools of Pi are available upon derepression.…”

The calcium-dependent ATP-Mg/Pi mitochondrial carrier is a target of glucose-induced calcium signalling in Saccharomyces cerevisiae

“…One possible inducing signal could be ATP, because it is a product of glycolysis. However, glucose-starved cells contain high levels of ATP [37,38], but do not divide, making ATP an unlikely inducing signal. Our hypothesis is that one of the possible signals is not ATP itself, but the entry of ATP into mitochondria.…”