Stalk segment 5 of the yeast plasma membrane H(+)-ATPase. Labeling with a fluorescent maleimide reveals a conformational change during glucose activation.
Glucose is well known to cause a rapid, reversible activation of the yeast plasma membrane H(+)-ATPase, very likely mediated by phosphorylation of two or more Ser/Thr residues near the C terminus. Recent mutagenesis studies have shown that glucose-dependent activation can be mimicked constitutively by amino acid substitutions in stalk segment 5 (S5), an alpha-helical stretch connecting the catalytic part of the ATPase with transmembrane segment 5 (Miranda, M., Allen, K. E., Pardo, J. P., and Slayman, C. W. (2001) J. Biol. Chem. 276, 22485-22490). In the present work, the fluorescent maleimide Alexa-488 has served as a probe for glucose-dependent changes in the conformation of S5. Experiments were carried out in a "3C" version of the ATPase, from which six of nine native cysteines had been removed by site-directed mutagenesis to eliminate background labeling by Alexa-488. In this construct, three of twelve cysteines introduced at various positions along S5 (A668C, S672C, and D676C) reacted with the Alexa dye in a glucose-independent manner, as shown by fluorescent labeling of the 100 kDa Pma1 polypeptide and by isolation and identification of the corresponding tryptic peptides. Especially significant was the fact that three additional cysteines reacted with Alexa-488 more rapidly (Y689C) or only (V665C and L678C) in plasma membranes from glucose-metabolizing cells. The results support a model in which the S5 alpha-helix undergoes a significant change in conformation to expose positions 665, 678, and 689 during glucose-dependent activation of the ATPase.
Miranda
Manuel
M
Department of Genetics, Yale University School of Medicine, New Haven, CT 06510, USA.
Pardo
Juan Pablo
JP
Allen
Kenneth E
KE
Slayman
Carolyn W
CW
D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D005456:Fluorescent Dyes / Q000737:chemistry*; D005947:Glucose / Q000378:metabolism*; D008301:Maleimides / Q000737:chemistry*; D008958:Models, Molecular; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D005456:Fluorescent Dyes; D008301:Maleimides; C043592:maleimide; D006180:Proton-Translocating ATPases; D005947:Glucose