Tandem phosphorylation of Ser-911 and Thr-912 at the C terminus of yeast plasma membrane H+-ATPase leads to glucose-dependent activation.
In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated 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.
Lecchi
Silvia
S
Department of Genetics and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06511, USA.
Nelson
Clark J
CJ
Allen
Kenneth E
KE
Swaney
Danielle L
DL
Thompson
Katie L
KL
Coon
Joshua J
JJ
Sussman
Michael R
MR
Slayman
Carolyn W
CW
D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D005947:Glucose / Q000378:metabolism / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D017434:Protein Structure, Tertiary / Q000502:physiology; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013549:Sweetening Agents / Q000378:metabolism / Q000494:pharmacology*
D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D013549:Sweetening Agents; C092477:PMA1 protein, S cerevisiae; D006180:Proton-Translocating ATPases; D005947:Glucose