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© The Rockefeller University Press,
0021-9525/1997//305 $5.00
The Journal of Cell Biology, Volume 137, Number 2,
, 1997 305-317
Article |
Assembly and Intracellular Targeting of the β
Subunits of Heterotrimeric G Proteins
The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent
, β, and
subunits is a complex process, compounded by the multiplicity of the genes that encode them, and the diversity of receptors and effectors with which they interact. Monoclonal anti-β antibodies (ARC5 and ARC9), raised against immunoaffinity purified β
complexes, recognize β subunits when not associated with
and can thus be used to monitor assembly of β
complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of β
complexes with the plasma membrane fraction starts between 15–30 min of chase. Three pools of β subunits can be distinguished based on their association with
subunits, their localization, and their detergent solubility. Association of β and
subunits with detergent-insoluble domains occurs within 1 min of chase, and increases to reach a plateau of near complete detergent resistance within 30 min of chase. Brefeldin A treatment does not interfere with delivery of β
subunits to detergent-insoluble domains, suggesting that assembly of G protein subunits with their receptors occurs distally from the BFA-imposed block of intracellular membrane trafficking and may occur directly at the plasma membrane.
1. Abbreviations used in this paper: Endo H, endoglycosidase H; G proteins, heterotrimeric guanine nucleotide-binding proteins; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; IFN-
, interferon-
; Staph. A, Staphylococcus aureus bacteria. A. Rehm was supported by a fellowship from the Deutsche Forschungsgemeinschaft (DFG). This work was supported in part by Amgen.
Please address all correspondence to H.L. Ploegh, Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, 40 Ames Street, E 17-322, Cambridge, MA 02139. Tel.: (617) 253-0520. Fax: (617) 253-9891.
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