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Plasma membrane (PM) H+-ATPases of the P-type family are highly conserved in yeast, other fungi, and plants. Their main role is to establish an H+ gradient driving active transport of small ions and metabolites across the PM and providing the main component of the PM potential. Furthermore, in both yeast and plant cells, conditions have been described under which active H+-ATPases promote activation of TORC1, the rapamycin-sensitive kinase complex controlling cell growth. Fungal and plant PM H+-ATPases are self-inhibited by their respective cytosolic carboxyterminal tails unless this domain is phosphorylated at specific residues. In the yeast H+-ATPase Pma1, neutralization of this autoinhibitory domain depends mostly on phosphorylation of the adjacent Ser911 and Thr912 residues, but the kinase(s) and phosphatase(s) controlling this tandem phosphorylation remain unknown. In this study, we show that S911-T912 phosphorylation in Pma1 is mediated by the largely redundant Ptk1 and Ptk2 kinase paralogs. Dephosphorylation of S911-T912, as occurs under glucose starvation, is dependent on the Glc7 PP1 phosphatase. Furthermore, proper S911-T912 phosphorylation in Pma1 is required for optimal TORC1 activation upon H+ influx coupled amino-acid uptake. We finally show that TORC1 controls S911-T912 phosphorylation in a manner suggesting that activated TORC1 promotes feedback inhibition of Pma1. Our results shed important new light on phosphoregulation of the yeast Pma1 H+- ATPase and on its interconnections with TORC1., SCOPUS: ar.j, info:eu-repo/semantics/published

Plasma membrane H+-ATPases are highly conserved in yeast, other fungi and plants. Their main role is to establish the H+ gradient at the plasma membrane (PM) to maintain the cytosolic pH close to neutral values and fuel the uptake of nutrients through secondary active transport systems. In both plants and yeast, PM H+-ATPases have also been described to promote activation of TORC1, the rapamycin-sensitive kinase complex controlling cell growth. This for instance occurs in yeast upon H+ influx coupled to amino acid uptake. Fungal and plant PM H+-ATPases are self-inhibited by their carboxyterminal tails unless this domain is phosphorylated at specific residues. In the yeast PM H+-ATPase (Pma1), the self-inhibitory action of the carboxyterminal tail is neutralized when the adjacent S911 and T912 are phosphorylated, but the kinase(s) and phosphatase(s) controlling their phosphorylation remain unknown.In this thesis work, with the objective of understanding the molecular events taking place at Pma1`s level upon H+ influx and triggering the signaling cascade towards TORC1, we show that the largely redundant Ptk1 and Ptk2 kinases are responsible for phosphorylating S911-T912 at the carboxyterminal tail of Pma1. We also show that the carboxyterminal tail of Pma1 is not essential to initial TORC1 activation induced by H+-coupled amino acid uptake. However, if not properly phosphorylated by Ptk1 and Ptk2 kinases, the carboxyterminal tail of Pma1 can interfere with TORC1 activation. Additionally, we provide evidence that activated TORC1 exerts negative feedback control on Pma1 by reducing phosphorylation of its carboxyterminal tail.Overall, our results shed important new light on phosphoregulation of the yeast Pma1 H+-ATPase and on its interconnections with TORC1., Doctorat en Sciences, info:eu-repo/semantics/nonPublished

The Target of Rapamycin Complex 1 (TORC1) involved in coordination of cell growth and metabolism is highly conserved among eukaryotes. Yet the signals and mechanisms controlling its activity differ among taxa, according to their biological specificities. A common feature of fungal and plant cells, distinguishing them from animal cells, is that their plasma membrane contains a highly abundant H+-ATPase which establishes an electrochemical H+ gradient driving active nutrient transport. We have previously reported that in yeast, nutrient-uptake-coupled H+ influx elicits transient TORC1 activation and that the plasma-membrane H+-ATPase Pma1 plays an important role in this activation, involving more than just establishment of the H+ gradient. We show here that the PMA2 H+-ATPase from the plant Nicotiana plumbaginifolia can substitute for Pma1 in yeast, to promote H+-elicited TORC1 activation. This H+-ATPase is highly similar to Pma1 but has a longer carboxy-terminal tail binding 14-3-3 proteins. We report that a C-terminally truncated PMA2, which remains fully active, fails to promote H+-elicited TORC1 activation. Activation is also impaired when binding of PMA2 to 14-3-3 s is hindered. Our results show that at least some plant plasma-membrane H+-ATPases share with yeast Pma1 the ability to promote TORC1 activation in yeast upon H+-coupled nutrient uptake., SCOPUS: ar.j, info:eu-repo/semantics/published