Book of Abstracts: Albany 2011
June 14-18 2011
©Adenine Press (2010)
Point mutation in M9-M10 loop of the yeast Pma1 H+-ATPase affects both ATPase functioning and polyphosphate (PolyP) distribution
Both ATP and linear polymers of inorganic phosphate (Pi) PolyP are involved in cell energetics and metabolism. Role of ATP and ATPases is well established, while less is known about PolyP. However, it is clear that PolyP are involved in storage of Pi, membrane channel formation, cation binding, regulation of enzyme activities, gene expression (1). There are very little data on the interaction between ATP and PolyP metabolism. Yeast plasma membrane (Pma1) Н+-ATPase generates electrochemical Н+ gradient providing energy for operating the secondary solute transport systems. The enzyme is embedded in the membrane by 10 segments (M1-M10) with most of the molecule located in cytosole or in the membrane; only 5 % of the molecule face extracellular space. Yeast Pma1 Н+-АТРase is regulated by glucose: during glucose consumption ATP is produced, this triggers activation of Pma1 functioning manifested in 3-10-fold increase of Vmax, and decrease of Km and Ki. ATPase activation is structurally accompanied by the enzyme multiple phosphorylation (2); two tandemly positioned sites are located in the enzyme C-terminal tail (3).
Most of the plausible residues for the ATPase phosphorylation (Ser, Thr, Asp, and Glu) are located in the inner parts of the enzyme; however, there are several phosphorylable residues located in the Pma1 outer parts: D714, S716, D718, and D720 in the M5-M6 loop and S846, E847, T850, and D851 in the M9-M10 loop which is close to the enzyme regulatory C-tail. It seems reasonable that multiple phosphorylation of Pma1 goes subsequently, and first of such sites could be located in the extracellular part of the enzyme. The M5-M6 loop residues, except D714, were found not to be important for the enzyme structure-function relationship; the D714A mutant activity was unessential to be studied further (4). Therefore, we choose to replace with Ala one of the residues in the M9-M10 loop of the enzyme which could be phosphorylated – T850. When T850 is replaced with Ala, the mutated enzyme activity dropped significantly; at the same time the ability of the mutated enzyme to be activated by glucose was strongly impaired (Fig. 1, left). In parallel with ATPase activity assay, distribution of PolyP fractions (PolyP1-PolyP5) was analyzed. No significant changes were found between most PolyP fractions in logarithmic and stationary phases of the wild type (WT) and T850A mutant. However, PolyP3 fraction stood out of the rest displaying almost twofold increase of PolyP amount in the mutant during stationary phase compare with only a quarter in the WT (Fig. 1, right). Since ATPase is more active during logarithmic phase (similar to GM and CS conditions in Fig. 1, left), it points to a connection between ATP and Poly metabolisms. Significant increase of PolyP3 amount in the T850A mutant in stationary phase may point to the lack of one of the phosphorylation sites. Further study of this and similar mutants, although methodologically challenging, seems certain to yield useful insights into the fundamental mechanisms of ATP and PolyP interactive metabolisms.
Fig. 1. Left: Pma1 ATPase activity in the WT and T850A mutant strains under carbon-starved (CS) and glucose-metabolizing (GM) conditions (%). Right: PolyP3 content in the WT and T850A strains in the logarithmic (log) and stationary (stat) phases (μmol P/g wet weight).
Alexander A. Tomashevsky
Institute of Biochemistry and Physiology of Microorganisms, RAS, 142290 Pushchino, Russia