Immunological approaches to the transmembrane topology and conformational changes of the carboxyl-terminal regulatory domain of yeast plasma membrane H(+)-ATPase.
Molecular genetic experiments have suggested that the carboxyl terminus of the Saccharomyces cerevisiae plasma membrane H(+)-ATPase is an inhibitory domain involved in the "in vivo" regulation of the enzyme by glucose metabolism. An antibody prepared against a fusion protein including the last 59 amino acids of the ATPase sequence has been affinity purified to yield a preparation which requires the 18 carboxyl-terminal amino acids for recognition. Antibody binding experiments show that the carboxyl-terminal domain of the ATPase can be selectively exposed by concentrations of the detergent Tween-20 which do not break down the permeability barrier of the plasma membrane to the antibody. Both enzyme-linked immunosorbent assay and immunofluorescence analysis demonstrate that the accessibility of the carboxyl-terminal domain in isolated plasma membranes depends on the physiological state of the cell being increased by glucose metabolism. Immunofluorescence analysis of isolated plasma membrane vesicles, using a dual labeling protocol with concanavalin A and antibody to reveal the orientation of individual vesicles, and colloidal gold immunoelectron microscopy of ultrathin cryosections of whole yeast cells separately demonstrate that the ATPase carboxyl terminus is located in the cytoplasmic compartment. The application of a mutant deleted of the epitope(s) recognized by the affinity purified carboxyl-terminal antibody eliminates the possibility of artifacts arising from nonspecific antibody binding. The accessibility properties and cytoplasmic location of the carboxyl-terminal domain appear to be consistent with its role as a negative regulator of the ATPase.
Monk
B C
BC
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Montesinos
C
C
Ferguson
C
C
Leonard
K
K
Serrano
R
R
D015153:Blotting, Western; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D008854:Microscopy, Electron; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000276:immunology / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000648:ultrastructure
D006180:Proton-Translocating ATPases