Functional Expression of Phosphagen Kinase Systems Confers Resistance to Transient Stresses in Saccharomyces cerevisiae by Buffering the ATP Pool

Abstract

Phosphagen kinase systems provide different advantages to tissues with high and fluctuating energy demands, in particular an efficient energy buffering system. In this study we show for the first time functional expression of two phosphagen kinase systems in Saccharomyces cerevisiae, which does not normally contain such systems. First, to establish the creatine kinase system, in addition to overexpressing creatine kinase isoenzymes, we had to install the biosynthesis pathway of creatine by co-overexpression of L-arginine:glycine amidinotransferase and guanidinoacetate methyltransferase. Although we could achieve considerable creatine kinase activity, together with more than 3 mM intracellular creatine, this was not sufficient to confer an obvious advantage to the yeast under the specific stress conditions examined here. Second, using arginine kinase, we successfully installed an intracellular phosphagen pool of about 5 mM phosphoarginine. Such arginine kinase-expressing yeast showed improved resistance under two stress challenges that drain cellular energy, which were transient pH reduction and starvation. Although transient starvation led to 50% reduced intracellular ATP concentrations in wild-type yeast, arginine kinase overexpression stabilized the ATP pool at the pre-stress level. Thus, our results demonstrate that temporal energy buffering is an intrinsic property of phosphagen kinases that can be transferred to phylogenetically very distant organisms.The availability of biochemical energy, with ATP as the primary energy currency, is fundamental to most cellular processes. Although ATP and its congeners are involved in literally hundreds of biochemical reactions, the intracellular concentration of ATP is generally kept very constant at about 2-5 mM, depending on organisms and tissues, with a turnover rate of the ATP pool that is in the range of a few seconds (1). Hence, metabolic ATP generation in a cell must be balanced tightly with ATP-consuming processes. Small deviations from the standard cellular concentrations of free ATP, ADP, and AMP serve important regulatory roles in fine tuning this delicate balance.This balance between energy-consuming and -producing processes is particularly challenged in tissues that experience periods of high and fluctuating energy demand, such as brain, heart, or skeletal muscle. To maintain constant ATP levels, these tissues express creatine kinase (CK 1 ; EC 2.7.3.2) that uses creatine (Cr) to create a metabolically inert pool of phosphocreatine (PCr). Among other functions, this PCr pool serves as a temporal energy buffer that can replenish ATP rapidly during phases of high energy demand, according to the following reaction (2): MgADP Ϫ ϩ PCr 2Ϫ ϩ H ϩ i MgATP 2Ϫ ϩ Cr.

Identification and Characterization of a Putative Arginine Kinase Homolog from Myxococcus xanthus Required for Fruiting Body Formation and Cell Differentiation

“…The role of the AK in any bacterial species is unknown, but it is tempting to speculate that the addition of an AK gene to a genome can confer a selective advantage immediately. This is supported by work conducted by Sauer and colleagues, who demonstrated that the introduction of an AK gene into both eukaryotic and prokaryotic genomes conferred an increased capacity to withstand environmental stress (3,4,25). This is supported by our work in M. xanthus, and thus it is plausible that the introduction of the AK conferred an enhanced ability to tolerate some forms of environmental stress, such as pH, salt, and osmotic stress.…”

“…Phosphagen kinases have been shown to have protective effects against a variety of stressors in many organisms and could potentially affect vegetative growth of M. xanthus under stress conditions, including salt, osmotic, pH, and oxidative stress (3,4,25). To determine if M. xanthus ark plays a role in the response to any of these stressors, we constructed an in-frame deletion that Numbering is relative to that of the rabbit muscle CK (RmCK) sequence.…”

“…One possibility is that ark creates a pool of high-energy phosphoarginine that is metabolically inert but able to be converted rapidly back into a useable form for the energyintensive processes of development and response to stress. A role in direct pH buffering inside the cell cannot be excluded; expression of arginine kinase in other systems (3) has been shown to have such effects and could also play a role in M. xanthus. Finally, it is also possible that phosphoarginine acts as a messenger molecule, affecting gene expression or the activity of another protein in the cell, though this would be a function not previously reported for this molecule in the literature.…”

Involvement of the Nonhomologous Region of Subunit A of the Yeast V-ATPase in Coupling and in Vivo Dissociation

“…The previous studies were conducted at a single ATP concentration (0.5 mM) that is below the typical cytoplasmic ATP concentration measured in yeast cells (44). In addition, it had previously been reported for the V-ATPase from bovine brain clathrin-coated vesicles that the tightness of coupling of proton transport and ATPase activity by the V-ATPase varied as a function of ATP concentration, with higher concentrations of ATP leading to a progressive uncoupling of these activities (42).…”

Comparative Proteome Analysis of Silkworm in Its Susceptibility and Resistance Responses to <i>Bombyx mori </i>Densonucleosis Virus

“…AK plays an important role as a regulator of energetic reserves and cell growth by catalyzing the reversible transfer of the phosphate group from MgATP to arginine, leading to production of phosphoarginine and MgADP [29] . Canonaco et al [30] reported that recombinant yeast expressing AK showed improved resistance under stress challenges that drain cellular energy, which were transient pH reduction and starvation. Probably the increase in the level of AK in the resistant insects is required to replenish the energy in the resistant larval midgut cells that have a relatively higher utilization to adjust to the extreme conditions than the susceptible strain.…”