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Am J Physiol Cell Physiol 270: C1362-C1369, 1996;
0363-6143/96 $5.00
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AJP - Cell Physiology, Vol 270, Issue 5 C1362-C1369, Copyright © 1996 by American Physiological Society

ARTICLES

Role of myristoylation in membrane attachment and function of G alpha i-3 on Golgi membranes

S. H. Brand, E. J. Holtzman, D. A. Scher, D. A. Ausiello and J. L. Stow
Department of Medicine, Massachusetts General Hospital, Charlestown, USA.

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 NH2-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 1-3, 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 beta gamma-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 G beta gamma-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 NH2-terminal myristoylation to bind to Golgi membranes and to maximize its interaction with G beta gamma-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.


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F. Wylie, K. Heimann, T. L. Le, D. Brown, G. Rabnott, and J. L. Stow
GAIP, a Galpha i-3-binding protein, is associated with Golgi-derived vesicles and protein trafficking
Am J Physiol Cell Physiol, February 1, 1999; 276(2): C497 - C506.
[Abstract] [Full Text] [PDF]


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