Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Mutations in membrane and extracytosolic domains of the yeast Pma1 ATPase cause different distribution of polyphosphates
Yeast plasma-membrane Pma1 H+-ATPase is a vital enzyme, which couples energy of ATP hydrolysis to H+ transport and belongs to a physiologically important P-ATPase family. One of the distinguishing features of the enzyme is activation during glucose fermentation, which manifested in increased activity and altered kinetics and accompanied by multiple regulatory phosphorylation during enzyme maturation and trafficking from endoplasmic reticulum to plasma membrane where it resides. Homologous fungal and plant H+ -ATPases have more than 10 such sites of phosphorylation; two of them were identified in the regulatory C-tail (Lecchi et al., 2007). Besides main source of energy, ATP, yeast cell also contains polyphosphates (PolyP), which were earlier believed to be just a phosphate depot. It is clear now that their functions in cells are much more complex and multitargeted. In particular, PolyP could be used to phosphorylate proteins as was found for bacteria; however, there is no such evidence for eukaryotic cells (Kulaev et al., 2005). We have earlier shown that Ala substitution of phosphorylable residues in the enzyme part facing cell envelope and in C-tail led to changes in PolyP distributions in the cell suggesting that these residues could be sites of the enzyme phosphorylation (Tomashevski & Petrov, 2011, 2013, 2015). To test this suggestion, we have undertaken comparative study of replacements at phosphorylable residues in L9-10 loop and residues, which are unphosphorylable due to chemical nature and/or location in the midst of M8 membrane segment. To study this, we have compared changes of ATPase activity in M8 mutant F796A, L801A, and E803A and L9-10 mutant S846A, T850A, and D851A ATPases to changes in distribution of PolyP fractions, which are distinguished by their chain length, method of isolation and location. All but one (S846A) of these mutants had moderately to significantly reduced ATPase activities; however, the PolyP distribution did not directly correlated to ATPase activity. As seen from Fig.1, membrane F796A mutant with the lowest activity had moderate increase in mid-chained PolyP2 (45-70 residues, attributed to nucleus) and PolyP3 (90-115, attributed to vacuole and secretory pathway), while L801A and especially E803A showed increase only for long-chained PolyP4-5 (≥170-200, attributed to cell envelope); these patters of PolyP4-5 were opposite to those in the C-tail mutants (Tomashevski & Petrov, 2015). Of the extracytosolic mutants, E847A with twofold reduced activity showed no changes in PolyP distribution (Tomashevski & Petrov, 2013), while S846A with no change in ATPase activity exhibited 50-60% increase in short-chained PolyP1 (< 25, attributed to cytosole and vacuole) and mid-chained PolyP3 and twofold drop of long-chained PolyP4-5. The S846A patterns were very similar to those of S911A. By contrast, T850A had moderate increase in PolyP1 and dramatic threefold jump in PolyP3; the ATPase activity decreased twofold. ATPase activity of D851A was very similar to T850A; nevertheless, changes in PolyP distribution were less prominent, although they were also found for mid-chained PolyP2 and 3. Changes in PolyP content in F796A could be explained by reduced ATPase activity and/or conformational changes (Petrov, 2010), which may lead to phosphate depositing in a form of PolyP. E803A was implicated in undercoupling between ATP hydrolysis and H+ transport; thus twofold increase in PolyP4-5 may be explained not only in quantitative but also in qualitative way. Results for replacement of the L9-10 loop residues seamed to be more tightly connected to their direct involvement in the enzyme phosphorylation suggesting their stepwise and/or tandem involvement in this process.
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 (2010) Point mutations in Pma1 H+-ATPase of Saccharomyces cerevisiae: influence on its expression and activity. Biochemistry (Moscow) 75, 1055-1063.
Tomashevsky AA & Petrov VV (2011) Point mutation in M9-M10 loop of the yeast Pma1 H+-ATPase affects both ATPase functioning and polyphosphate distribution. J Biomol Struct Dyn 28, 1025-1026.
Tomashevski AA & Petrov VV (2013) Point mutations in the yeast Pma1 H+-ATPase affects polyphosphate (PolyP) distribution. J Biomol Struct Dyn 31, 124-125.
Tomashevski AA & Petrov VV (2015) Mutations in the yeast Pma1 H+-ATPase regulatory domain affect polyphosphate metabolism. J Biomol Struct Dyn 33, http://www.jbsdonline.com/product-p18770.html
Alexandr A. Tomashevski
Skryabin Institute of Biochemistry and Physiology of Microorganisms