Role of myristoylation in membrane-attachment and function of
g[alpha]i-3 on golgi membranes.
Brand, Susan H., Eliezer J. Holtzman, Daniel A. Scher, Dennis A.
Ausiello, and Jennifer L. Stow.
Renal Unit, Departments of Medicine and Pathology, Massachusetts
General Hospital and Harvard Medical School, Charlestown MA 02129
APStracts 2:0426C, 1995.
Heterotrimeric G protein [alpha] subunits localized on the cytoplasmic
face of Golgi membranes are involved in regulating vesicle
trafficking and protein secretion. We investigated the role of
myristoylation in attachment of the G[alpha]i-3 subunit to Golgi
membranes. G[alpha]i-3 was epitope-tagged by insertion of a FLAG
sequence at an N-terminal site predicted to interfere with
myristoylation and the resulting NT-[alpha]i-3 construct was stably
-transfected and expressed in polarized epithelial LLC-PK1 cells.
Metabolic labeling confirmed that the translation product of NT
-[alpha]i-3 was not myristoylated. In contrast to endogenous
G[alpha]i-3, which is tightly bound to Golgi membranes, the
unmyristoylated FLAG-tagged NT-[alpha]i-3 did not attach to
membranes; it was localized by immunofluorescence in the cytoplasm of
LLC-PK1 cells and was detected only in the cytosol fraction of cell
homogenates. Pertussis toxin-dependent ADP-ribosylation was used to
test the ability of NT-[alpha]i-3 to interact with membrane-bound bg
subunits. In both in vitro and in vivo assays cytosolic NT-[alpha]i-3
alone was not ADP-ribosylated, although in the presence of membranes
it could interact with Gbg subunits to form heterotrimers. The
expression of NT-[alpha]i-3 in LLC-PK1 cells altered the rate of
basolateral secretion of sulfated proteoglycans, consistent with the
demonstrated function of endogenous G[alpha]i-3. These data are
consistent with a model in which G[alpha]i-3 utilizes N-terminal
myristoylation to bind to Golgi membranes and to maximize its
interaction with Gbg subunits. Furthermore our results show that
stable attachment of G[alpha]i-3 to Golgi membranes is not required
for it to participate as a regulatory element in vesicle trafficking
in the secretory pathway.
Received 8 September 1995; accepted in final form 9 November
1995.
APS Manuscript Number C547-5.
Article publication pending Am. J. Physiol. (Cell Physiology).
ISSN 1080-4757 Copyright 1995 The American Physiological Society.
Published in APStracts on 8 December 95