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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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Transport and formation of detergent-resistant membranes is insensitive to BFA treatment. IMR-32 cells were stimulated for 48 h with 50 U/ml IFN-γ. Cells were pulsed in the presence and absence of 10 μg/ml brefeldin A for 10 min with 500 μCi  [35S]methionine and chased (+ and −10 μg/ml BFA) for the time  indicated. Cells were extracted as described in Fig. 7 and separated into a soluble and a TX-100–resistant fraction. Resolubilization of the pellet was done with octylglucoside containing lysis  buffer. ARC9 was used to recover β subunits (+0.2% SDS) (A),  and MHC class I heavy chains were immunoprecipitated with  W6/32.HL (B and C). Endo H digestions of W6/32.HL precipitates were done to demonstrate the effects of anterograde ER to  Golgi transport block. BFA renders MHC class I molecules endo  H sensitive even after 60 min of chase.
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Figure 8: Transport and formation of detergent-resistant membranes is insensitive to BFA treatment. IMR-32 cells were stimulated for 48 h with 50 U/ml IFN-γ. Cells were pulsed in the presence and absence of 10 μg/ml brefeldin A for 10 min with 500 μCi [35S]methionine and chased (+ and −10 μg/ml BFA) for the time indicated. Cells were extracted as described in Fig. 7 and separated into a soluble and a TX-100–resistant fraction. Resolubilization of the pellet was done with octylglucoside containing lysis buffer. ARC9 was used to recover β subunits (+0.2% SDS) (A), and MHC class I heavy chains were immunoprecipitated with W6/32.HL (B and C). Endo H digestions of W6/32.HL precipitates were done to demonstrate the effects of anterograde ER to Golgi transport block. BFA renders MHC class I molecules endo H sensitive even after 60 min of chase.

Mentions: To test for the transport pathway taken by cytosolderived βγ subunits to rapidly formed detergent-resistant domains, ER to Golgi transport was blocked by BFA. We scored for inhibition of intracellular glycoprotein transport by analyzing persistence of sensitivity of MHC class I molecules to Endo H (Fig. 8, B and C). The association of βγ subunits with detergent-resistant membranes was not affected by the BFA-imposed block of ER to Golgi transfer (Fig. 8 A). We conclude that cytosolic βγ subunits are inserted into detergent-resistant membranes at a site distal from the BFA block, probably at the plasma membrane itself.


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

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

Transport and formation of detergent-resistant membranes is insensitive to BFA treatment. IMR-32 cells were stimulated for 48 h with 50 U/ml IFN-γ. Cells were pulsed in the presence and absence of 10 μg/ml brefeldin A for 10 min with 500 μCi  [35S]methionine and chased (+ and −10 μg/ml BFA) for the time  indicated. Cells were extracted as described in Fig. 7 and separated into a soluble and a TX-100–resistant fraction. Resolubilization of the pellet was done with octylglucoside containing lysis  buffer. ARC9 was used to recover β subunits (+0.2% SDS) (A),  and MHC class I heavy chains were immunoprecipitated with  W6/32.HL (B and C). Endo H digestions of W6/32.HL precipitates were done to demonstrate the effects of anterograde ER to  Golgi transport block. BFA renders MHC class I molecules endo  H sensitive even after 60 min of chase.
© Copyright Policy
Related In: Results  -  Collection

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Figure 8: Transport and formation of detergent-resistant membranes is insensitive to BFA treatment. IMR-32 cells were stimulated for 48 h with 50 U/ml IFN-γ. Cells were pulsed in the presence and absence of 10 μg/ml brefeldin A for 10 min with 500 μCi [35S]methionine and chased (+ and −10 μg/ml BFA) for the time indicated. Cells were extracted as described in Fig. 7 and separated into a soluble and a TX-100–resistant fraction. Resolubilization of the pellet was done with octylglucoside containing lysis buffer. ARC9 was used to recover β subunits (+0.2% SDS) (A), and MHC class I heavy chains were immunoprecipitated with W6/32.HL (B and C). Endo H digestions of W6/32.HL precipitates were done to demonstrate the effects of anterograde ER to Golgi transport block. BFA renders MHC class I molecules endo H sensitive even after 60 min of chase.
Mentions: To test for the transport pathway taken by cytosolderived βγ subunits to rapidly formed detergent-resistant domains, ER to Golgi transport was blocked by BFA. We scored for inhibition of intracellular glycoprotein transport by analyzing persistence of sensitivity of MHC class I molecules to Endo H (Fig. 8, B and C). The association of βγ subunits with detergent-resistant membranes was not affected by the BFA-imposed block of ER to Golgi transfer (Fig. 8 A). We conclude that cytosolic βγ subunits are inserted into detergent-resistant membranes at a site distal from the BFA block, probably at the plasma membrane itself.

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