Albany 2013: Book of Abstracts

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Conversation 18
June 11-15 2013
©Adenine Press (2012)

Point mutations in the yeast Pma1 H+-ATPase affect polyphosphate (PolyP) distribution

Yeast plasma membrane Н+-ATPase (Pma1) is a key enzyme of the yeast cell metabolism. It generates electrochemical Н+ gradient providing energy for operating the secondary solute transport systems and maintaining intracellular pH and ion homeostasis. Most of the enzyme molecule anchored in the plasma membrane by M1-M10 segments is located in cytosole and membrane; less than 5 % of the Pma1 face extracellular space. Membrane domain contains amino acid residues, which form H+ transport pathway; cytosole parts house the enzyme active center and cytosolic C-terminal tail has regulatory function. The enzyme functioning and regulation are tightly connected to glucose metabolism: its fermentation triggers activation of Pma1 functioning, structurally accompanied by the enzyme multiple phosphorylation during intracellular traffic on route to plasma membrane. There are ca. 10 phosphorylation sites; only 3 of them are identified: one single and two tandemly located sites are in the C-terminal tail. Both ATP and PolyP can be used to phosphorylate amino acid residues; however, there are little data on the interactive metabolism of ATP and PolyP. Most of phosphorylable Ser, Thr, Asp, Glu, Tyr residues are located in the inner parts of the enzyme; however, there are several such residues in the Pma1 outer parts: D714, S716, D718, D720 in M5-M6 loop and S846, E847, T850, D851 in M9-M10 loop, 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 enzyme extracytosolic part. The M5-M6 loop phosphorylable residues, except D714, were found to be unimportant for the enzyme structure-function relationship; D714A mutant was poorly expressed and inactive (Petrov, 2011). However, D714N did not disturb the enzyme functioning, thus excluding the role of D714 in the enzyme phosphorylation. Therefore, we choose to study further residues in the M9-M10 loop by replacing them with Ala. The ATPase activity of these mutants ranged from the wild-type level (S846A) to 2- (E847A) to 3-fold (T850A) drop. Changes of activity were accompanied by changes in PolyP fractions (Figure 1), which were most significant for S846A and T850A. S846A had 1.5- to 1.7-fold increase in PolyP1 (found mostly in cytosole and vacuoles) and PolyP3 (localized near the cell surface) and dramatic 3-fold decrease in PolyP4-5 fractions (associated with cell wall), while T850A had stable 1.5-fold increase in all PolyP but PolyP4-5 fractions. Both mutants also had 20% (S846A) to 37% (T850A) increase in total PolyP. These data may point to lack of one or more phosphorylation sites and/or participation of PolyP in the Pma1 ATPase phosphorylation. Possibly, the sites at S846 and T850 act jointly, similarly to tandemly located and acting S911 and T912 in the regulatory C-tail (Lecchi et al., 2007). Further study of these mutants, although methodologically challenging, seems certain to yield more useful insights into functioning and regulation of the Pma1 ATPase as well as into the interactive mechanisms of ATP and PolyP metabolism.

Figure 1. Effect of the Pma1 point mutations on the ATPase activity and PolyP distribution.


The study was supported in part by Russian Foundation for Basic Research (RFBR) grant 13-04-02031a.


    Lecchi S, Nelson CJ, Allen KE, Swaney DL, Thompson KL, Coon JJ, Sussman MR & Slayman CW. (2007). Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation. J. Biol. Chem. 282, 35471-35481.

    Petrov VV (2011). Role of M5-M6 loop in the biogenesis and function of the yeast Pma1 H+-ATPase. J. Biomol. Struct. Dynam. 28, 1024-1025.

Alexandr A. Tomashevski
Valery V. Petrov

Institute of Biochemistry and Physiology of Microorganisms
142290 Pushchino, Russia

Ph.: +7 496 731 8698
Fax: +7 495 956 3370
vpetrov07@gmail.com tomashevskialexandr25@gmail.com