Conformational changes of yeast plasma membrane H(+)-ATPase during activation by glucose: role of threonine-912 in the carboxy-terminal tail.
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.
Lecchi
Silvia
S
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Allen
Kenneth E
KE
Pardo
Juan Pablo
JP
Mason
A Brett
AB
Slayman
Carolyn W
CW
D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D005947:Glucose / Q000494:pharmacology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012694:Serine / Q000235:genetics; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014357:Trypsin / Q000737:chemistry
D013912:Threonine; D012694:Serine; D014357:Trypsin; D006180:Proton-Translocating ATPases; D005947:Glucose