AJP - Gastrointestinal and Liver Physiology, Vol 269, Issue 6 883-G891, Copyright © 1995 by American Physiological Society
In vivo trafficking of nascent H(+)-K(+)-ATPase in rabbit parietal cells
J. M. Crothers Jr, D. C. Chow, M. L. Scalley and J. G. Forte
Department of Molecular and Cell Biology, University of California, Berkeley 94720-3200, USA.
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.
