In vivo trafficking of nascent h+-k+-atpase in the rabbit parietal
cell.
Crothers, James M., Jr, Dar C. Chow, Michelle L. Scalley, and John G.
Forte.
Department of Molecular and Cell Biology, Division of Cell and
Developmental Biology, University of California, Berkeley, CA
94720
APStracts 2:0112G, 1995.
Protein metabolic labeling in vivo was used to determine a time course
for trafficking of nascent H+-K+-ATPase from endoplasmic reticulum
(ER) to mature tubulovesicles in parietal cells. Stomachs of
cimetidine-treated rabbits were taken from 15 to 90 min after
injection of 35S-methionine/cysteine and mucosal microsomes were
fractionated on sucrose gradients for analysis by SDS-PAGE, Western
blot, and autoradiography. After 15 min labeled [alpha]-subunit
peaked at 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 1.10 g/ml, considered to be
Golgi. By 90 min, most labeled [alpha]-subunit was in a light peak,
at 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 co-precipitated with pre
-[beta] by a terminal mannose-specific lectin, GNA, 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.
Received 3 March 1995; accepted in final form 3 June 1995.
APS Manuscript Number G99-5.
Article publication pending Am. J. Physiol. (Gastrointest. Liver
Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 6 July 1995.