Karel van Dam

A functional genomics strategy that uses metabolome data to reveal the phenotype of silent mutations

A large proportion of the 6,000 genes present in the genome of Saccharomyces cerevisiae, and of those sequenced in other organisms, encode proteins of unknown function. Many of these genes are “silent,” that is, they show no overt phenotype, in terms of growth rate or other fluxes, when they are deleted from the genome. We demonstrate how the intracellular concentrations of metabolites can reveal phenotypes for proteins active in metabolic regulation. Quantification of the change of several metabolite concentrations relative to the concentration change of one selected metabolite can reveal the site of action, in the metabolic network, of a silent gene. In the same way, comprehensive analyses of metabolite concentrations in mutants, providing “metabolic snapshots,” can reveal functions when snapshots from strains deleted for unstudied genes are compared to those deleted for known genes. This approach to functional analysis, using comparative metabolomics, we call FANCY—an abbreviation for functional analysis by co-responses in yeast.

On the mechanism of activation of the ATPase in chloroplasts

Abstract 1. The Mg 2+ -ATPase induced in chloroplasts by light and trypsin is subjected to the same energetic feedback control as is the light-dependent dithioerythritol-induced Mg 2+ -ATPase. 2. The modification by dithioerythritol or by trypsin of the membrane-bound chloroplast coupling factor is not readily reversible. After treatment in the light with dithioerythritol and subsequent washing, a stable chloroplast preparation is obtained which potentially can be energized by ATP. 3. The decay of the ATPase-active conformation in the dark in the absence of ATP is caused by a non-energy linked, Mg 2+ -stimulated degradation; it can be reversed by a short light trigger. 4. Addition of ATP to dithioerythritol-treated chloroplasts in the dark initiates, under appropriate conditions, a rapid autocatalytic membrane conformation towards the ATPase-active form.

The fluorescent properties of acridines in the presence of chloroplasts or liposomes. On the quantitative relationship between the fluorescence quenching and the transmembrane proton gradient

Abstract The fluorescent properties of 9-aminoacridine were studied in chloroplasts and phospholipid liposomes. In energized chloroplasts it was found that the percentage of fluorescence quenching was dependent on both the 9-aminoacridine concentration and the chlorophyll concentration. On the other hand, it was independent of the osmolarity of the medium. In phospholipid liposomes the dependence of the fluorescence quenching on the concentration of 9-aminoacridine was similar to that in chloroplasts. Moreover, the fluorescence quenching depended on the presence of charged compounds in the membrane being larger in negatively charged than in positively charged liposomes. The fluorescence of both the monoamine 9-amino-6-chloro-2-methoxyacridine and the diamine atebrin is quenched more extensively than that of 9-aminoacridine. Although the percentage of fluorescence quenching of both atebrin and 9-aminoacridine is dependent on the outside pH, the relationship between the fluorescence quenching of the two probes under similar conditions is not pH-dependent. It is concluded that calculation of ΔpH from the percentage of fluorescence quenching of fluorescent amines is not meaningful, that the osmotic volume of chloroplasts is not involved in the quenching process and, consequently, that the interaction between the acridines and energized membranes is more likely to occur at the level of the membrane proper.

Sustained oscillations in free-energy state and hexose phosphates in yeast

In a population of intact cells of the yeast Saccharomyces cerevisiae the dynamics of glycolytic metabolism were investigated under the condition of sustained oscillations. At 5‐s intervals cells were quenched in −40°C methanol, extracted and the intracellular concentrations of glycolytic metabolites, adenine nucleotides and phosphate were analysed. Oscillations were found for the glycolytic intermediates glucose 6‐phosphate, fructose 6‐phosphate and fructose 1,6‐bisphosphate. At variance with earlier reports on transient glycolytic oscillations, some intermediates further down the glycolytic pathway did not oscillate significantly, even though NADH did. In addition, the adenylate energy charge and the free energy of ATP hydrolysis oscillated significantly. Dynamic coupling through the latter may be responsible for this effective compartmentation of glycolytic dynamics.

Around the growth phase transition S. cerevisiae's make-up favours sustained oscillations of intracellular metabolites

Under a limited set of hitherto incompletely defined conditions, inhibition of respiration has been shown to cause transient oscillations in NAD(P)H fluorescence of yeast cells. In this paper, we apply a new method [1992, Anal. Biochem. 204, 118‐132] for extraction of intracellular metabolites. This method involves spraying the cells into −40°C methanol; the neutral pH allows extraction of nearly all intracellular metabolites, including NADH. Close to the shift from glucose to ethanol as a growth substrate, the cells acquire a make‐up amenable to sustained oscillations in intracellular concentrations of NADH and glycolytic intermediates such as glucose‐6‐phosphate. NADH was found to oscillate between 200 μM and 400 μM intracellular concentration. The cellular make‐up determining the tendency to oscillate is ‘remembered’ by the cells after three hours of starvation.

Functional analysis of the hexose transporter homologue HXT5 in Saccharomyces cerevisiae

The HXT5 gene encodes a functional hexose transporter that has moderate affinity for glucose (Km=10 mM), moderate to low affinity for fructose (Km=40 mM) and low affinity for mannose (Km>100 mM). The sole presence of Hxt5p in an otherwise hexose transport null mutant is sufficient to sustain a flux through glycolysis from glucose to fermentative products. However, the presence of HXT5 as the sole hexose transporter gene results in extremely poor growth on glucose, which suggests the involvement of glucose repression in the transcriptional regulation of HXT5. From Northern blot analysis on the members of the HXT family and studies with HXT5 tagged with the green fluorescent protein (GFP), it is evident that HXT5 is transcribed and translated during conditions of relatively slow growth, during growth on non‐fermentable carbon sources and in particular during sporulation. In wild‐type batch cultivations on fermentable carbon sources, Hxt5p is abundant in stationary phase or after depletion of the fermentable carbon source, which seems independent of the carbon source. The deletion of HXT5 does not result in a clear phenotype. A shift of stationary phase cells to fresh glucose medium resulted in somewhat slower resumption of growth in the hxt5 deletion strain compared to the wild‐type strain. The abundance of Hxt5p during stationary phase, sporulation and low glucose conditions suggests that HXT5 is a ‘reserve’ transporter, which might be involved in the initial uptake of glucose after the appearance of glucose. Other possible functions of the protein encoded by HXT5 will be discussed in the context of the results. Copyright

Physiological Properties of Saccharomyces cerevisiae from Which Hexokinase II Has Been Deleted

ABSTRACT Hexokinase II is an enzyme central to glucose metabolism and glucose repression in the yeast Saccharomyces cerevisiae. Deletion of HXK2, the gene which encodes hexokinase II, dramatically changed the physiology of S. cerevisiae. The hxk2-null mutant strain displayed fully oxidative growth at high glucose concentrations in early exponential batch cultures, resulting in an initial absence of fermentative products such as ethanol, a postponed and shortened diauxic shift, and higher biomass yields. Several intracellular changes were associated with the deletion of hexokinase II. Thehxk2 mutant had a higher mitochondrial H+-ATPase activity and a lower pyruvate decarboxylase activity, which coincided with an intracellular accumulation of pyruvate in the hxk2 mutant. The concentrations of adenine nucleotides, glucose-6-phosphate, and fructose-6-phosphate are comparable in the wild type and the hxk2 mutant. In contrast, the concentration of fructose-1,6-bisphosphate, an allosteric activator of pyruvate kinase, is clearly lower in the hxk2mutant than in the wild type. The results suggest a redirection of carbon flux in the hxk2 mutant to the production of biomass as a consequence of reduced glucose repression.

Yeast cells with a specific cellular make-up and an environment that removes acetaldehyde are prone to sustained glycolytic oscillations.

Glycolytic oscillations can be induced by adding glucose to starved Saccharomyces cerevisiae cells and, after a steady state has been established, cyanide. Transient oscillations or limit‐cycle oscillations can be induced depending on the growth phase in which the cells are harvested. To find what causes these differences in the dynamic behaviour, we analyzed glycolytic enzyme activities at different growth phases. The hexokinase activity increased by a factor of three after growth substrate transition from glucose to ethanol; the other measured activities remained constant. Cyanide was found not only to block respiration, but also to trap acetaldehyde. Both cyanide actions appear necessary for the occurrence of sustained glycolytic oscillations.

The energy level associated with the light-triggered Mg2+-dependent ATPase in spinach chloroplasts

Abstract Light-induced Mg2+-ATPase activity of chloroplasts and the pH difference (ΔpH) across the thylakoid membrane maintained by this activity are measured simultaneously under varying conditions of preillumination time and dark decay time. It is shown that with increasing ATPase activity, ΔpH reaches a maximal level which is determined by the degree of uncoupling of the thylakoid membrane.

On the origin of the limited control of mitochondrial respiration by the adenine nucleotide translocator

A thermodynamic control theory previously developed has been applied to mitochondrial oxidative phosphorylation with emphasis on the role of delta microH and coupling and within the paradigm of delocalized chemiosmotic coupling. The basis for the observed distribution of flux control over the participating enzymes is shown to lie in the relative magnitudes of so-called delta microH elasticity coefficients, i.e., the delta microH dependencies of the different mitochondrial processes. In particular the relatively strong delta microH dependence of mitochondrial respiration is responsible for the significant role of the adenine nucleotide translocator in the control of oxidative phosphorylation. Uncoupling decreases the control exerted by this translocator on respiration but increases that exerted on phosphorylation.