Use of a fluorescent maleimide to probe structure-function relationships in stalk segments 4 and 5 of the yeast plasma-membrane H+-ATPase.
In the yeast plasma-membrane H(+)-ATPase and other P-type ATPases, conformational changes are transmitted between cytoplasmic and membrane-embedded domains via a stalk region composed of cytoplasmic extensions of membrane segments 2, 3, 4, and 5. The present study has used a fluorescent maleimide (Alexa-488) to probe Cys residues introduced into stalk segments 4 and 5 of the yeast enzyme. In the case of S5, Cys substitutions along one face led to a constitutive, 5- to 10-fold activation of the ATPase in the absence of glucose. Based on homology with SERCA Ca(2+)-ATPase, this face is likely to be buried in the interior of the protein, close to the P domain. Three Cys residues on the opposite face of S5 (A668C, S672C, and D676C) were accessible to Alexa-488 under all conditions tested. In addition, three other Cys residues at or near the boundary between the two faces reacted with Alexa-488 only (V665C, L678C) or preferentially (Y689C) in plasma membranes from glucose-metabolizing cells; this result provides the first direct evidence for a change in conformation of S5 during glucose activation. For stalk segment 4, site-directed mutagenesis gave no sign of a role in glucose-dependent regulation. Rather, substitutions at 13 consecutive positions along S4 caused kinetic changes consistent with a shift in equilibrium from E2 to E1. Four Cys residues along this stretch of S4 (Q357C, K362C, S364C, and S368C) reacted with Alexa-488, indicating that they are exposed to the aqueous medium as predicted in the SERCA-based structural model.
Slayman
Carolyn W
CW
Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510, USA. carolyn.slayman@yale.edu
Miranda
Manuel
M
Pardo
Juan Pablo
JP
Allen
Kenneth E
KE
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002462:Cell Membrane / Q000201:enzymology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006639:Histidine; D007700:Kinetics; D008301:Maleimides / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000737:chemistry* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry
D005456:Fluorescent Dyes; D008301:Maleimides; D006639:Histidine; D006180:Proton-Translocating ATPases; D000254:Sodium-Potassium-Exchanging ATPase