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Assembly and intracellular targeting of the betagamma subunits of heterotrimeric G proteins.

Rehm A, Ploegh HL - J. Cell Biol. (1997)

Bottom Line: The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma 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-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes.Brefeldin A treatment does not interfere with delivery of beta gamma 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma 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-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of beta gamma complexes with the plasma membrane fraction starts between 15-30 min of chase. Three pools of beta subunits can be distinguished based on their association with gamma subunits, their localization, and their detergent solubility. Association of beta and alpha 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 beta gamma 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.

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Related in: MedlinePlus

Detection of biosynthetic intermediates, half-life, and  complex formation of Gβ subunits. (A) IMR-32 cells were pulselabeled for 2.5 min with 150 μCi [35S]methionine and chased for  up to 20 h. β subunits were recovered with mAb ARC9 from nonionic detergent lysates, either in the presence (+) or absence (−)  of 0.2% SDS. Samples in A–C were subjected to 12.5% SDSPAGE. (B) Pulse-labeling of IMR-32 cells for 1.5 min with 150  μCi [35S]methionine (see A); chases were done for the designated  intervals. Precipitations with ARC9 were performed as in A. (C)  IMR-32 cells were pulse-labeled for 5 min with 150 μCi [35S]methionine and chased for the times indicated. β subunits were immunoprecipitated with mAb ARC9 in the absence (−) or presence (+) of 0.2% SDS.
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Figure 1: Detection of biosynthetic intermediates, half-life, and complex formation of Gβ subunits. (A) IMR-32 cells were pulselabeled for 2.5 min with 150 μCi [35S]methionine and chased for up to 20 h. β subunits were recovered with mAb ARC9 from nonionic detergent lysates, either in the presence (+) or absence (−) of 0.2% SDS. Samples in A–C were subjected to 12.5% SDSPAGE. (B) Pulse-labeling of IMR-32 cells for 1.5 min with 150 μCi [35S]methionine (see A); chases were done for the designated intervals. Precipitations with ARC9 were performed as in A. (C) IMR-32 cells were pulse-labeled for 5 min with 150 μCi [35S]methionine and chased for the times indicated. β subunits were immunoprecipitated with mAb ARC9 in the absence (−) or presence (+) of 0.2% SDS.

Mentions: Pulse-chased IMR-32 cells were lysed in the non-ionic detergents NP-40/Lubrol and equal aliquots of lysate were immunoprecipitated in the absence (−) or presence (+) of 0.2% SDS (Fig. 1 C). At 0 min of chase, the recovery of β from lysates prepared with and without SDS was identical, whereas at 2.5 min and later time points, we observed a progressive loss of immunoreactivity from lysates not exposed to SDS. After 30 min essentially no β subunits were detected in the absence of SDS (see also Fig. 1 A). Over the chase period examined, there is no loss of β in extracts prepared in the presence of SDS.


Assembly and intracellular targeting of the betagamma subunits of heterotrimeric G proteins.

Rehm A, Ploegh HL - J. Cell Biol. (1997)

Detection of biosynthetic intermediates, half-life, and  complex formation of Gβ subunits. (A) IMR-32 cells were pulselabeled for 2.5 min with 150 μCi [35S]methionine and chased for  up to 20 h. β subunits were recovered with mAb ARC9 from nonionic detergent lysates, either in the presence (+) or absence (−)  of 0.2% SDS. Samples in A–C were subjected to 12.5% SDSPAGE. (B) Pulse-labeling of IMR-32 cells for 1.5 min with 150  μCi [35S]methionine (see A); chases were done for the designated  intervals. Precipitations with ARC9 were performed as in A. (C)  IMR-32 cells were pulse-labeled for 5 min with 150 μCi [35S]methionine and chased for the times indicated. β subunits were immunoprecipitated with mAb ARC9 in the absence (−) or presence (+) of 0.2% SDS.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139779&req=5

Figure 1: Detection of biosynthetic intermediates, half-life, and complex formation of Gβ subunits. (A) IMR-32 cells were pulselabeled for 2.5 min with 150 μCi [35S]methionine and chased for up to 20 h. β subunits were recovered with mAb ARC9 from nonionic detergent lysates, either in the presence (+) or absence (−) of 0.2% SDS. Samples in A–C were subjected to 12.5% SDSPAGE. (B) Pulse-labeling of IMR-32 cells for 1.5 min with 150 μCi [35S]methionine (see A); chases were done for the designated intervals. Precipitations with ARC9 were performed as in A. (C) IMR-32 cells were pulse-labeled for 5 min with 150 μCi [35S]methionine and chased for the times indicated. β subunits were immunoprecipitated with mAb ARC9 in the absence (−) or presence (+) of 0.2% SDS.
Mentions: Pulse-chased IMR-32 cells were lysed in the non-ionic detergents NP-40/Lubrol and equal aliquots of lysate were immunoprecipitated in the absence (−) or presence (+) of 0.2% SDS (Fig. 1 C). At 0 min of chase, the recovery of β from lysates prepared with and without SDS was identical, whereas at 2.5 min and later time points, we observed a progressive loss of immunoreactivity from lysates not exposed to SDS. After 30 min essentially no β subunits were detected in the absence of SDS (see also Fig. 1 A). Over the chase period examined, there is no loss of β in extracts prepared in the presence of SDS.

Bottom Line: The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma 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-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes.Brefeldin A treatment does not interfere with delivery of beta gamma 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.

View Article: PubMed Central - PubMed

Affiliation: Center for Cancer Research, Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.

ABSTRACT
The assembly in living cells of heterotrimeric guanine nucleotide binding proteins from their constituent alpha, beta, and gamma 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-beta antibodies (ARC5 and ARC9), raised against immunoaffinity purified beta gamma complexes, recognize beta subunits when not associated with gamma and can thus be used to monitor assembly of beta gamma complexes. Complex formation starts immediately after synthesis and is complete within 30 min. Assembly occurs predominantly in the cytosol, and association of beta gamma complexes with the plasma membrane fraction starts between 15-30 min of chase. Three pools of beta subunits can be distinguished based on their association with gamma subunits, their localization, and their detergent solubility. Association of beta and alpha 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 beta gamma 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.

Show MeSH
Related in: MedlinePlus