Abstract
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.
Highlights
Molecular genetic experiments have suggested that the enzyme [1].The amount of H’-ATPase antigen found in the carboxyl terminusof the Saccharomyces cerevisiae plasma membranes is fairly constant and only demonstrates plasma membrane H+-ATPaseis aninhibitory domain a small decrease in the absence of glucose [6]
Concanavalin A and antibody to reveal the orientation ATPase gene has provided some clues about the mechanism of individual vesicles, and colloidal gold immunoelec- of regulation
Tron microscopy of ultrathin cryosections of whole or a point mutation at the ATP-binding sit(e11)give a fully tepycepaearriarmbttsomotipnxeecayne(llstl.l)tsareTenrschmteioebpignoaanudrpiaszypteleiidslceyballdiyotmiecotmahinnteeoaodntfeasisftanrfaimptntthheoiuetesytsactinbhyptitaulotidrptteyihlflaeoeiseftmedadAirctcToiaffcParoatbcmhsotees-xylatsr-heucgagtiugtvleeagstletousnrcyztohysamedtoemmtwehatieiatncbhaoowrlbuihotsixmctyhhlseoitnenmrteemeerhdaionfcwoutrssmwgcloiuodtchniofstishteeiestmuattcehettseiavbaeeonnslziisyitmnemha.einbTdaihtnoidsry arising from nonspecific antibody binding
Summary
ExperimentalOral Biology Unit,Faculty of Dentistry,University of Otago, Dunedin, New Zealand. Plasma Membrane H+-ATPase-Plasmid pRS-60 is a pUC18 deriv- ELISA-Plasma membranes at 30 pg/ml in PBS were treated with ative with a 5-kilobase Hind111 fragment which includes the yeast 25 pg/ml of the indicated detergent for 5 min at room temperature plasma membrane H'-ATPase gene [26] It was cut with the restric- and diluted 30-fold in 50 mM NaHCO?, p H 8.5. The slides were washed four times (2 min each) with 35ml of PBSGT, incubated for ' The abbreviationsused are: SDS-PAGE, sodiumdodecyl sulfate- 1h in a dark humid chamber with 10ml of 15 pg/ml pre-centrifuged polyacrylamide gel electrophoresis; ELISA, enzyme-linked immuno- fluorescein conjugated anti-rabbit IgG (Dianova GmbH) in PBSGT, sorbent assay. Our experiencewith immunoelectron microscopv of thin Lowacryl sections [33] indicates that the plasma rnembrane ATPase carboxyl terminus is anextremelvsensitive
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