Tandem Phosphorylation of Ser-911 and Thr-912 at the C Terminus of Yeast Plasma Membrane H+-ATPase Leads to Glucose-dependent Activation

Abstract

In recent years there has been growing interest in the posttranslational regulation of P-type ATPases by protein kinasemediated phosphorylation. Pma1 H ؉ -ATPase, which is responsible for H ؉ -dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5-10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896 -918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14 N/ 15 N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H ؉ -ATPase. Pma1 H ϩ

Specific Activation of the Plant P-type Plasma Membrane H+-ATPase by Lysophospholipids Depends on the Autoinhibitory N- and C-terminal Domains

“…Like AHA2, Pma1p has a C-terminal regulatory domain and, depending on the phosphorylation status of this domain, exists in either a basal or activated state (45). However, the C-terminal domain of the fungal PM H ϩ -ATPase is considerably shorter than that of the plant pump, and no sequence stretch with homology to Region II in the plant pump is present (Fig.…”

“…Plasmids containing AHA2 under the control of the PMA1 promoter were described previously (17,20,43 ϩ -ATPases in the basal state were prepared as described (45). Plasmids carrying AHA2 with deletions corresponding to 20 or 25 amino acids in the C terminus as well as single or double point mutations were generated by standard procedures using polymerase chain reactions and verified by DNA sequencing.…”

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

“…Both strains had a significantly higher pH in in the presence of glucose, because glucose activates the Pma1 ATPase (Lecchi et al, 2007;Serrano, 1983) and thus the cytosol is alkalinized (MartinezMunoz and Kane, 2008). That shows that even the mutant version of Pma1p can be activated by glucose addition.…”

Symbiosis-dependent gene expression in coral–dinoflagellate association: cloning and characterization of a P-type H+-ATPase gene

“…The Symbiodinium pump possesses the C-terminal peptide present in yeast PMA1 that constitutes the non-essential inhibitory domain involved in the regulation of the enzyme by glucose metabolism (Portillo et al 1989). Indeed, in this peptide, phosphorylation of serine and threonine is clearly related to glucose activation in yeast (Lecchi et al 2007). These residues are present in SspPMA1 (Ser-929 and Thr-930).…”