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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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Intracellular redistribution of newly synthesized Gβ subunits. (A) IMR-32 cells were pulse-labeled with 150 μCi [35S]methionine for 2.5 min and chased for the times indicated. A crude homogenate was separated into microsomes and cytosol by centrifugation  at 100,000 g for 1 h at 4°C. The pellets recovered were considered the M fractions and contained plasma membranes and microsomal  membranes, whereas the supernatant contained the cytosol (fraction C). Solubilization of subcellular fractions in NP-40/Lubrol lysis  buffer was as for Fig. 1. Gβ was recovered in the absence (−) or presence (+) of 0.2% SDS. mAb ARC9 immunoprecipitates were analyzed on a 12.5% SDS-PAGE. (B) IMR-32 cells were pulse-labeled as in A and chased for up to 180 min. Membrane and cytosol fractions were prepared by differential centrifugation, yielding fractions designated plasma membranes (28,000 g pellet); microsomes (100,000 g  pellet); and cytosol (100,000 g supernatant). All immunoprecipitations with ARC9 were done in the presence of 0.2% SDS. Samples  were resolved by 12.5% SDS-PAGE. Although at 15-min chase some of the total cell lysate was inadvertently lost in this experiment,  the ratio between the cytosolic and microsomal β as the relevant parameter can still be evaluated. (C) The plasma membrane marker  α-Na+/K+-ATPase is detectable in the 28,000 g membrane fraction, but not in the 100,000 g pellet. 50-μg aliquots of membrane fractions as obtained in B were subjected to electrophoresis on a 12.5% SDS-PAGE and transferred to nitrocellulose. The blotting membrane was incubated with rabbit anti–α-Na+/K+-ATPase antiserum.
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Figure 3: Intracellular redistribution of newly synthesized Gβ subunits. (A) IMR-32 cells were pulse-labeled with 150 μCi [35S]methionine for 2.5 min and chased for the times indicated. A crude homogenate was separated into microsomes and cytosol by centrifugation at 100,000 g for 1 h at 4°C. The pellets recovered were considered the M fractions and contained plasma membranes and microsomal membranes, whereas the supernatant contained the cytosol (fraction C). Solubilization of subcellular fractions in NP-40/Lubrol lysis buffer was as for Fig. 1. Gβ was recovered in the absence (−) or presence (+) of 0.2% SDS. mAb ARC9 immunoprecipitates were analyzed on a 12.5% SDS-PAGE. (B) IMR-32 cells were pulse-labeled as in A and chased for up to 180 min. Membrane and cytosol fractions were prepared by differential centrifugation, yielding fractions designated plasma membranes (28,000 g pellet); microsomes (100,000 g pellet); and cytosol (100,000 g supernatant). All immunoprecipitations with ARC9 were done in the presence of 0.2% SDS. Samples were resolved by 12.5% SDS-PAGE. Although at 15-min chase some of the total cell lysate was inadvertently lost in this experiment, the ratio between the cytosolic and microsomal β as the relevant parameter can still be evaluated. (C) The plasma membrane marker α-Na+/K+-ATPase is detectable in the 28,000 g membrane fraction, but not in the 100,000 g pellet. 50-μg aliquots of membrane fractions as obtained in B were subjected to electrophoresis on a 12.5% SDS-PAGE and transferred to nitrocellulose. The blotting membrane was incubated with rabbit anti–α-Na+/K+-ATPase antiserum.

Mentions: Pulse-chase experiments in combination with subcellular fractionation were carried out to further resolve β subunit intermediates. When we fractionated cells simply into a particulate or membrane fraction (M) and a cytosolic fraction (C), and immunoprecipitated β in the absence and presence of SDS, we observed three distinct pools of β subunits (Fig. 3 A). At 0 min chase, the majority of β was detected in the C fraction as the free, uncomplexed form. After 2.5 min of chase, the ratio between cytosolic and membrane-associated β was slightly shifted, with β still found predominantly in the cytosol. Starting at 7.5 min of chase, the recovery of β in the absence of SDS was significantly reduced. The loss of free β indicates the formation of complexes that are different from those at 0 and 2.5 min of chase. At the 15-min chase interval, the distribution between cytosolic (C)- and membrane (M)-associated β appeared to be equivalent, whereas their ratio finally shifted towards the M fraction after 35 and 60 min of chase. After the 60-min chase interval, only a minor amount of β remained in the cytosolic compartment.


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

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

Intracellular redistribution of newly synthesized Gβ subunits. (A) IMR-32 cells were pulse-labeled with 150 μCi [35S]methionine for 2.5 min and chased for the times indicated. A crude homogenate was separated into microsomes and cytosol by centrifugation  at 100,000 g for 1 h at 4°C. The pellets recovered were considered the M fractions and contained plasma membranes and microsomal  membranes, whereas the supernatant contained the cytosol (fraction C). Solubilization of subcellular fractions in NP-40/Lubrol lysis  buffer was as for Fig. 1. Gβ was recovered in the absence (−) or presence (+) of 0.2% SDS. mAb ARC9 immunoprecipitates were analyzed on a 12.5% SDS-PAGE. (B) IMR-32 cells were pulse-labeled as in A and chased for up to 180 min. Membrane and cytosol fractions were prepared by differential centrifugation, yielding fractions designated plasma membranes (28,000 g pellet); microsomes (100,000 g  pellet); and cytosol (100,000 g supernatant). All immunoprecipitations with ARC9 were done in the presence of 0.2% SDS. Samples  were resolved by 12.5% SDS-PAGE. Although at 15-min chase some of the total cell lysate was inadvertently lost in this experiment,  the ratio between the cytosolic and microsomal β as the relevant parameter can still be evaluated. (C) The plasma membrane marker  α-Na+/K+-ATPase is detectable in the 28,000 g membrane fraction, but not in the 100,000 g pellet. 50-μg aliquots of membrane fractions as obtained in B were subjected to electrophoresis on a 12.5% SDS-PAGE and transferred to nitrocellulose. The blotting membrane was incubated with rabbit anti–α-Na+/K+-ATPase antiserum.
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Related In: Results  -  Collection

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Figure 3: Intracellular redistribution of newly synthesized Gβ subunits. (A) IMR-32 cells were pulse-labeled with 150 μCi [35S]methionine for 2.5 min and chased for the times indicated. A crude homogenate was separated into microsomes and cytosol by centrifugation at 100,000 g for 1 h at 4°C. The pellets recovered were considered the M fractions and contained plasma membranes and microsomal membranes, whereas the supernatant contained the cytosol (fraction C). Solubilization of subcellular fractions in NP-40/Lubrol lysis buffer was as for Fig. 1. Gβ was recovered in the absence (−) or presence (+) of 0.2% SDS. mAb ARC9 immunoprecipitates were analyzed on a 12.5% SDS-PAGE. (B) IMR-32 cells were pulse-labeled as in A and chased for up to 180 min. Membrane and cytosol fractions were prepared by differential centrifugation, yielding fractions designated plasma membranes (28,000 g pellet); microsomes (100,000 g pellet); and cytosol (100,000 g supernatant). All immunoprecipitations with ARC9 were done in the presence of 0.2% SDS. Samples were resolved by 12.5% SDS-PAGE. Although at 15-min chase some of the total cell lysate was inadvertently lost in this experiment, the ratio between the cytosolic and microsomal β as the relevant parameter can still be evaluated. (C) The plasma membrane marker α-Na+/K+-ATPase is detectable in the 28,000 g membrane fraction, but not in the 100,000 g pellet. 50-μg aliquots of membrane fractions as obtained in B were subjected to electrophoresis on a 12.5% SDS-PAGE and transferred to nitrocellulose. The blotting membrane was incubated with rabbit anti–α-Na+/K+-ATPase antiserum.
Mentions: Pulse-chase experiments in combination with subcellular fractionation were carried out to further resolve β subunit intermediates. When we fractionated cells simply into a particulate or membrane fraction (M) and a cytosolic fraction (C), and immunoprecipitated β in the absence and presence of SDS, we observed three distinct pools of β subunits (Fig. 3 A). At 0 min chase, the majority of β was detected in the C fraction as the free, uncomplexed form. After 2.5 min of chase, the ratio between cytosolic and membrane-associated β was slightly shifted, with β still found predominantly in the cytosol. Starting at 7.5 min of chase, the recovery of β in the absence of SDS was significantly reduced. The loss of free β indicates the formation of complexes that are different from those at 0 and 2.5 min of chase. At the 15-min chase interval, the distribution between cytosolic (C)- and membrane (M)-associated β appeared to be equivalent, whereas their ratio finally shifted towards the M fraction after 35 and 60 min of chase. After the 60-min chase interval, only a minor amount of β remained in the cytosolic compartment.

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