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

Abstract

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 function and regulation are tightly connected to glucose metabolism: its fermentation triggers activation of Pma1 function, 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, and 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, and D720 in M5–M6 loop and S846, E847, T850, and 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 1.5–1.7-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.