Epitope mapping and accessibility of immunodominant regions of yeast plasma membrane H(+)-ATPase.
Immunodominant regions of yeast plasma membrane H(+)-ATPase have been mapped by two different approaches. A rabbit polyclonal antibody was used to screen a library of random fragments of the ATPase gene in a bacterial expression plasmid. In addition, the epitopes recognized by a panel of mouse monoclonal antibodies against the ATPase were mapped by reactions with defined fragments of the enzyme expressed in Escherichia coli. Both methodologies indicated that two regions within the amino-terminal part of the ATPase (at amino acid positions 5-105 and 168-255) contain most of the antigenic determinants. The accessibility of the monoclonal antibodies to their epitopes in native and solvent-perturbed ATPase preparations was investigated by immunofluorescence studies on yeast protoplasts. Cells fixed and permeabilized with formaldehyde were either treated with or without detergents and organic solvents. ELISA competition tests with plasma membrane vesicles and with detergent-purified ATPase incubated in solution with the monoclonal antibodies gave similar results. All the epitopes were accessible in detergent-treated ATPase preparations. In contrast, only the epitopes at amino acids 24-56 were accessible in ATPase preparations not treated with detergents or organic solvents. These epitopes were cytoplasmic because protoplast permeabilization was required for decoration by the reactive monoclonal antibodies.
Serrano
R
R
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Monk
B C
BC
Villalba
J M
JM
Montesinos
C
C
Weiler
E W
EW
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D000918:Antibody Specificity; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D000939:Epitopes / Q000032:analysis*; D004926:Escherichia coli / Q000235:genetics; D005455:Fluorescent Antibody Technique; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology; D006180:Proton-Translocating ATPases / Q000032:analysis / Q000235:genetics / Q000276:immunology*; D011817:Rabbits / Q000276:immunology; D011994:Recombinant Proteins / Q000032:analysis / Q000276:immunology; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D000906:Antibodies; D000911:Antibodies, Monoclonal; D000939:Epitopes; D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; D011994:Recombinant Proteins; D006180:Proton-Translocating ATPases