Conformational Changes of Yeast Plasma Membrane H⁺-ATPase during Activation by Glucose:  Role of Threonine-912 in the Carboxy-Terminal Tail

Abstract

Yeast Pma1 H(+)-ATPase, which belongs to the P-type family of cation-transporting ATPases, is activated up to 10-fold by growth on glucose, and indirect evidence has linked the activation to Ser/Thr phosphorylation within the C-terminal tail. We have now used limited trypsinolysis to map glucose-induced conformational changes throughout the 100 kDa ATPase. In the wild-type enzyme, trypsin cleaves first at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavages occur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6-M7 cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At the C-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes from glucose-starved cells (GS) but becomes accessible in membranes from glucose-metabolizing cells (GM). In the presence of orthovanadate, Lys-174 at the boundary between M2 and the A domain also becomes open to cleavage in GM but not GS samples (site T5). Significantly, this global conformational change can be suppressed by mutations at Thr-912, a consensus phosphorylation site near the C-terminus. Substitution by Ala at position 912 leads to a GS-like (trypsin-resistant) state, while substitution by Asp leads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecular movements that result in glucose activation.

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

“…Removal of the last 18 amino acids by mutagenesis led to constitutive activation of the ATPase even in the absence of glucose (8,9). Consistent with this observation, immuno-blotting with anti-C-terminal antibody revealed that the C terminus was relatively inaccessible during carbon starvation but could be rapidly digested by trypsin upon the addition of glucose (10). Of added significance was the fact that amino acid substitutions within the tail, especially at Thr-912, reduced glucose activation in a manner that could be suppressed by second-site mutations elsewhere in the 100-kDa protein (11,12).…”

“…Significantly, the C-terminal tail of Pma1 ATPase was relatively inaccessible to trypsin in samples from carbon-starved cells but rapidly degraded in samples from glucose-metabolizing cells. Substitution of Thr-912 by Ala led to arrest of the ATPase in the former state, whereas substitution by Asp led to arrest in the latter state (10). Fig.…”

“…Of added significance was the fact that amino acid substitutions within the tail, especially at Thr-912, reduced glucose activation in a manner that could be suppressed by second-site mutations elsewhere in the 100-kDa protein (11,12). Replacing Thr-912 with Ala (to prevent phosphorylation) arrested the ATPase in a trypsin-resistant state characteristic of carbon-starved wild-type cells, whereas replacing it with Asp (to mimic phosphorylation) locked the ATPase in a trypsinsensitive state typical of glucose-activated wild-type cells (10). Taken together, these results pointed to a key regulatory role of the C-terminal tail, which appears to act in an autoinhibitory fashion during glucose starvation, binding tightly to regions of the N and/or P domains to inhibit ATPase activity.…”

Time-resolved Measurements of Intracellular ATP in the Yeast Saccharomyces cerevisiae using a New Type of Nanobiosensor

“…Otherwise the ATP concentration should decrease to zero. The mechanism controlling the activity of these ATPases is presently a matter of speculation, but for PMA1 it has been shown that the activity is controlled by glucose (40,41). The activation (and presumably also the deactivation) involves a threonine and a serine residue at the C terminus, which are rapidly phosphorylated on activation (42) (and presumably dephosphorylated on deactivation).…”

A heritable switch in carbon source utilization driven by an unusual yeast prion

“…Carbon sources regulate Pma1's phosphorylation state (Lecchi et al 2005), its ATPase activity (Serrano 1983), and its conformation (Miranda et al 2002) through residues S899, S911, and T912 in the C-terminal tail, which faces the cytosol (Eraso et al 2006;Lecchi et al 2007). We mutated S899, S911, and T912 to alanine, which cannot be phosphorylated, or to aspartic acid, which mimics constitutive phosphorylation.…”