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Albany 2019: 20th Conversation - Abstracts

category image Albany 2019
Conversation 20
June 11-15 2019
Adenine Press (2019)

Change in the yeast Pma1 H+-АТРаse regulation causes redistribution of polyphosphates (PolyP)

Plasma membrane Pma1 H+-АТРаse is a key enzyme of yeast metabolism. The enzyme functioning is regulated by glucose, which fermentation leads to producing ATP being used for phosphorylation of proteins and other substrates and maintaning energy metabolism. Glucose-induced activation of Pma1 is structurally accompanied by the enzyme multiple and reversible phosphorylation during intracellular traffic via secretory pathway on route to plasma membrane. There is evidence that the Pma1 activation is linked to Ser/Thr phosphorylation within the regulatory C-terminus and it leads to essential conformational changes; S911 and T912 were identified as two major phosphosites, which phosphorylation goes tandemly (Lecchi et al., 2007). The location of all sites of phosphorylation is still unclear; however, it seems logical that phosphorylation of Pma1 goes step by step and such sites could be located both in the enzyme intra- and extracellular parts. Both ATP and PolyP can phosphorylate Ser and Thr like in the case of Lys (Azevedo et al., 2015); we have earlier shown that Ala replacement of phosphorylable residues in the extracellular L9-10 loop, which is close to the C-tail, has affected PolyP distribution (Tomashevsky & Petrov, 2011, 2013). Other extracellular phosphorylable residues were found not to be important for the enzyme structure-function relationship (Petrov, 2011, 2015). We have also shown that mutations at S911 and T912 affected PolyP metabolism and phosphate distribution among PolyP1-5 fractions (Tomashevski & Petrov, 2015). Results described here extend our study towards interaction of PolyP metabolism and the enzyme regulation at S911 and T912 phosphorylation sites. Replacement by Ala led to removing phosphosite of phosphoester type and by Asp, to altering site to acylphosphate type. Yeast cells carrying wild type (NY13) and mutant ATPases (S911A, S911D, and T912D) were grown until mid-log phase, harvested and incubated with or without 2% glucose. Then PolyP were extracted and plasma membranes were obtained to assay ATPase activity. Wild-type ATPase underwent glucose-dependent activation by 9-fold, while mutant enzymes exhibited already elevated basal activity by 1.6- to 10.2-fold. S911D ATPase was constituvely activated by ca. 10-fold regardless presence of glucose, whilst S911A and T912D exposed ability to be additionally activated by 1.7-5.0-fold upon addition of glucose. During glucose fermentation, orthophosphate (Pi) content dropped 3-4-times in all strains as compared to starvation mode pointing to its use for phosphorylation; however, S911D mutant with changed type of phosphorylation had significantly evevated Pi content both in the absence and presence of glucose (Fig. 1). This points either to inability of altered acylphosphate site at D911 to be phosphorylated or, more likely, to irreversible phosphorylation at this site, which should disturb cascade of reactions ignited by glucose fermentation. PolyP content in the wild-type strain did not differ much in the presence and absence of glucose; in mutant strains, there was a difference seen with the most pronounced effect for the shortest PolyP1 fraction, which decreased in S911A and T912D and completely dissapeared in S911D in the absence of glucose; glucose addition brought PolyP1 amount up to control level and higher in the case of S911D. This strain had also the most noticible changes in PolyP2-5 fractions, which were higher comparing to the wild type. This points to involvement of PolyP directly or indirectly – through Pi pool – in phosphorylation. Presented data allow to suggest interaction between ATPase functioning and PolyP metabolism; removal of phosphosite at S911 or altering its type causes changing in Pi content and redistribution between PolyP fractions, which may point to preventing or altering Pi usage for phosphorylation both of the Pma1 and other proteins and substances at different steps. Further study is needed to get helpful insights into fine mechanisms of functioning and regulating of the Pma1 ATPase.

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Figure 1. Effect of point mutations in C-terminal end of the Pma1 ATPase on the distribution of PolyP fractions during glucose-dependent activation.

References
    Azevedo C, Livermore T, & Saiardi A (2015) Protein polyphosphorylation of lysine residues by inorganic polyphosphate. Molecular Cell 58, 71–82.
    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 Dyn 28, 1024-1025.
    Petrov VV (2015) Point mutations in the extracytosolic loop between transmembrane segments M5 and M6 of the yeast Pma1 H+-ATPase: alanine-scanning mutagenesis. J Biomol Struct Dyn 33, 70-84.
    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 affects polyphosphate metabolism. J Biomol Struct Dyn 33:sup1, 105-106.

Аlexandr А. Tomashevsky and
Valery V. Petrov*

Skryabin Institute of Biochemistry and Physiology of Microorganisms,
Russian Academy of Sciences,
142290 Pushchino, Russia,

Email* vpetrov07@gmail.com.
Ph.: +7 4967318698.
Fax: +7 4959563370