Albany 2015:Book of Abstracts
June 9-13 2015
©Adenine Press (2012)
Mutations in the yeast Pma1 H+-ATPase regulatory domain affect polyphosphate metabolism
Yeast plasma membrane Pma1 H+-ATPase is an H+ pump, which provides energy for operating secondary solute transport systems and maintaining intracellular pH and ion homeostasis. It is activated up to 5-10-fold when carbon-starved cells are exposed to glucose or other fermented sugars; at the same time, ATP is synthesized in mitochondria. Activation of Pma1 is structurally accompanied by the enzyme multiple phosphorylation, which occurs during intracellular traffic on route to plasma membrane. There are ca. 10 phosphorylation sites in fungal and plant H+-ATPases and there is evidence that the Pma1 activation is linked to Ser/Thr phosphorylation within the regulatory C-terminus and leads to essential conformational changes (Lecchi et al., 2007). The location of all sites of phosphorylation is still unclear; however, 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. Both ATP and polyphosphates (PolyP) can phosphorylate Ser and Thr residues; we have earlier shown that Ala replacement of phosphorylable residues in L9-10 loop, which is close to the C-tail, has affected PolyP intracellular distribution and have suggested that residues S846 and T850 could be phosphorylation sites (Tomashevsky & Petrov, 2011, 2013). Other extracellular phosphorylable residues, except for D714, were found not to be important for the enzyme structure-function relationship; however, D714 can be substituted by Asn without significant disturbing the enzyme functioning, thus ruling out role of D714 in the enzyme phosphorylation (Petrov, 2011, 2015). The results described here extend our study of ATP and PolyP metabolism interconnection and its involvement in the enzyme regulation by focusing on the C-terminal phosphorylation sites at tandemly positioned S911 and T912, which are implicated in stepwise phosphorylation (Lecchi et al., 2007). We have used mutants, which could and could not be phosphorylated: S911A, S911D, T912D, and S911D/T912A. These replacements did not seriously affect mutant ATPase activity but changed the enzyme ability to be regulated by glucose. Non-phosphorylable S911A showed 40-60% increase of short-chained PolyP1 widely spread in the cell and mid-chained PolyP3 fraction found in vacuoles and near cell wall and decrease by one third for short-chained PolyP2 associated with nucleus or even twofold for long-chained PolyP4-5 found in the cell envelope (Fig. 1). At the same time phosphorylable S911D exhibited twofold decrease in PolyP1 and PolyP4-5 and 40% increase of PolyP2 and 3. Similar to S911D patterns displayed T912D, although changes in PolyP distribution were less prominent. Double mutant S911D/T912A with one site of phosphorylation removed (T912A) while the other (S911D) changed its type from O- to A-phosphorylation has patterns both of S911A showing significant increase of PolyP3 and S911D (T912D) with decrease of PolyP1 and increase of PolyP2. Remarkably, PolyP4-5 fraction was decreased in all cases. Since phosphorylation sites at S911 and T912 are always exposed to cytosole, one might suggest that ATPase trafficking and PolyP synthesis (chain elongation) are coordinated and occurs on route from endoplasmic reticulum where Pma1 is synthesized and brought to plasmalemma by secretory vesicle fusion. The removal of one or more phosphorylation sites in C-terminus may also explain preferable accumulation of short- and mid-chained PolyP fractions (and their direct or indirect involvement in phosphorylation) along with long-chained PolyP4-5 decrease in cell envelope. Further study of these mutants will help to yield useful insights into functioning and regulating of the Pma1 ATPase as well as into mechanisms of ATP and PolyP interactive metabolisms.
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.
Alexandr A. Tomashevski
Skryabin Institute of Biochemistry and Physiology of Microorganisms