In vivo trafficking of nascent H(+)-K(+)-ATPase in rabbit parietal cells

Abstract

Protein metabolic labeling in vivo was used to determine a time course for trafficking of nascent H(+)-K(+)-adenosinetriphosphatase (H(+)-K(+)-ATPase) from endoplasmic reticulum (ER) to mature tubulovesicles in parietal cells. Stomachs of cimetidine-treated rabbits were taken 15-90 min after injection of [35S]methionine/cysteine, and mucosal microsomes were fractionated on sucrose gradients for analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot, and autoradiography. After 15 min, labeled alpha-subunit peaked at approximately 1.14 g/ml, matching the distribution of the high-mannose beta-subunit precursor, "pre-beta." After 30 min, most labeled alpha-subunit was in a peak at approximately 1.10 g/ml, considered to be Golgi. By 90 min, most labeled alpha-subunit was in a light peak, at approximately 1.07 g/ml, aligned with the major peak of total H(+)-K(+)-ATPase previously characterized as mature tubulovesicles. From material enriched in pre-beta, alpha-subunit was coprecipitated with pre-beta by a terminal mannose-specific lectin, Galanthus nivalis agglutinin, in the same ratio as the mature alpha:beta ratio. Thus alpha- and beta-subunits associated early in the ER. This is the first use of protein metabolic labeling to study early trafficking of the H(+)-K(+)-ATPase in vivo. The techniques may be usefully applied to examining changes in H(+)-K(+)-ATPase synthetic rate in response to various pharmacological treatments and studying the divergent pathways for nascent H(+)-K(+)- and Na(+)-K(+)-ATPases.