| 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/Thrphosphorylation 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 cleavesfirst at Lys-28 and Arg-73 in the extended N-terminal segment (sites T1 and T2); subsequent cleavagesoccur at Arg-271 between the A domain and M3 (site T3) and at Lys-749 or Lys-754 in the M6−M7cytoplasmic loop (site T4). Activation by glucose leads to a striking increase in trypsin sensitivity. At theC-terminal end of the protein, the Arg- and Lys-rich tail is shielded from trypsin in membranes fromglucose-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 becomesopen to cleavage in GM but not GS samples (site T5). Significantly, this global conformational changecan 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 Aspleads to a GM-like (trypsin-sensitive) state. Thus, the present results help to dissect the intramolecularmovements that result in glucose activation.
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