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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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Gα and Gβ are associated with non-ionic detergent-insoluble cell pellets. (A) IMR-32 cells were labeled overnight with 100  μCi of [35S]methionine. Cells were lysed in NP-40/Lubrol lysis buffer and separated into supernatant (soluble) and insoluble pellet. The  pellet was solubilized in 1% SDS, DNA was sheared by 15 passages through a 21-gauge needle and the extract was boiled twice at 100°C  for 3 min. The SDS concentration in the pellet fraction was adjusted to 0.2% with NP-40/Lubrol lysis buffer. Immunoprecipitations from  the supernatant were done either in the presence (+) or absence (−) of 0.2% SDS. A mixture of 3C2 and 3E7 were used to recover α  subunits and β was precipitated with ARC9. Samples were analyzed on a 12.5% SDS-PAGE (A–C). Arrows indicate the positions of β  and α subunits. (B) IMR-32 cells were labeled for 2 h with 250 μCi [35S]methionine followed by solubilization in NP-40/ Lubrol lysis  buffer. Insoluble pellets were extracted twice with non-ionic detergent buffer and subsequently treated as shown in A. (C) Kinetics of  Gβ subunit association with detergent-insoluble cell pellets. IMR-32 cells were pulsed for 2.5 min with 150 μCi [35S]methionine and  chased for the time intervals indicated. Cells were separated into supernatant (soluble) and a detergent-insoluble pellet. The pellet was  re-extracted as in A, followed by immunoprecipitation with ARC 9. (D) Detection of endogenous GPI-anchored proteins in non-ionic  detergent-resistant membrane domains. IMR-32 cells were labeled overnight with 100 μCi [35S]methionine and lysed in buffer containing TX-100. After centrifugation, the resulting pellet was re-extracted with TX-114–containing lysis buffer for 20 min at 37°C. After centrifugation, the supernatant was subjected to temperature-induced phase separation. Samples were treated in the absence (−) and presence (+) of 5 U/ml PI-PLC. This treatment induces the transition from a hydrophobic to a hydrophilic state of GPI-anchored proteins.  Polypeptides released into the aqueous phase were recovered by acetone precipitation and analyzed on a 12.5% SDS-PAGE. Arrows  on the right indicate the appearance of some of the proteins specifically released by PI-PLC.
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Figure 5: Gα and Gβ are associated with non-ionic detergent-insoluble cell pellets. (A) IMR-32 cells were labeled overnight with 100 μCi of [35S]methionine. Cells were lysed in NP-40/Lubrol lysis buffer and separated into supernatant (soluble) and insoluble pellet. The pellet was solubilized in 1% SDS, DNA was sheared by 15 passages through a 21-gauge needle and the extract was boiled twice at 100°C for 3 min. The SDS concentration in the pellet fraction was adjusted to 0.2% with NP-40/Lubrol lysis buffer. Immunoprecipitations from the supernatant were done either in the presence (+) or absence (−) of 0.2% SDS. A mixture of 3C2 and 3E7 were used to recover α subunits and β was precipitated with ARC9. Samples were analyzed on a 12.5% SDS-PAGE (A–C). Arrows indicate the positions of β and α subunits. (B) IMR-32 cells were labeled for 2 h with 250 μCi [35S]methionine followed by solubilization in NP-40/ Lubrol lysis buffer. Insoluble pellets were extracted twice with non-ionic detergent buffer and subsequently treated as shown in A. (C) Kinetics of Gβ subunit association with detergent-insoluble cell pellets. IMR-32 cells were pulsed for 2.5 min with 150 μCi [35S]methionine and chased for the time intervals indicated. Cells were separated into supernatant (soluble) and a detergent-insoluble pellet. The pellet was re-extracted as in A, followed by immunoprecipitation with ARC 9. (D) Detection of endogenous GPI-anchored proteins in non-ionic detergent-resistant membrane domains. IMR-32 cells were labeled overnight with 100 μCi [35S]methionine and lysed in buffer containing TX-100. After centrifugation, the resulting pellet was re-extracted with TX-114–containing lysis buffer for 20 min at 37°C. After centrifugation, the supernatant was subjected to temperature-induced phase separation. Samples were treated in the absence (−) and presence (+) of 5 U/ml PI-PLC. This treatment induces the transition from a hydrophobic to a hydrophilic state of GPI-anchored proteins. Polypeptides released into the aqueous phase were recovered by acetone precipitation and analyzed on a 12.5% SDS-PAGE. Arrows on the right indicate the appearance of some of the proteins specifically released by PI-PLC.

Mentions: When non-ionic detergent insoluble material from labeled IMR-32 cells was analyzed for the presence of β subunits, we found substantial amounts of β subunits even after two rounds of non-ionic detergent extraction, although the majority of β subunits was still recovered from the detergent soluble fraction (Fig. 5, A and B). Whereas the recovery of β from the soluble extract required SDS, the efficiency of α recovery was independent of SDS, an indication that the epitopes recognized on α are freely accessible. To determine the time point when β associates with the insoluble pellet, IMR-32 cells were pulse-labeled for 2.5 min, chased for the times indicated, and extracted with NP-40/Lubrol. The β subunit was found in the pellet beginning at 1 min of chase (Fig. 5 C).


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

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

Gα and Gβ are associated with non-ionic detergent-insoluble cell pellets. (A) IMR-32 cells were labeled overnight with 100  μCi of [35S]methionine. Cells were lysed in NP-40/Lubrol lysis buffer and separated into supernatant (soluble) and insoluble pellet. The  pellet was solubilized in 1% SDS, DNA was sheared by 15 passages through a 21-gauge needle and the extract was boiled twice at 100°C  for 3 min. The SDS concentration in the pellet fraction was adjusted to 0.2% with NP-40/Lubrol lysis buffer. Immunoprecipitations from  the supernatant were done either in the presence (+) or absence (−) of 0.2% SDS. A mixture of 3C2 and 3E7 were used to recover α  subunits and β was precipitated with ARC9. Samples were analyzed on a 12.5% SDS-PAGE (A–C). Arrows indicate the positions of β  and α subunits. (B) IMR-32 cells were labeled for 2 h with 250 μCi [35S]methionine followed by solubilization in NP-40/ Lubrol lysis  buffer. Insoluble pellets were extracted twice with non-ionic detergent buffer and subsequently treated as shown in A. (C) Kinetics of  Gβ subunit association with detergent-insoluble cell pellets. IMR-32 cells were pulsed for 2.5 min with 150 μCi [35S]methionine and  chased for the time intervals indicated. Cells were separated into supernatant (soluble) and a detergent-insoluble pellet. The pellet was  re-extracted as in A, followed by immunoprecipitation with ARC 9. (D) Detection of endogenous GPI-anchored proteins in non-ionic  detergent-resistant membrane domains. IMR-32 cells were labeled overnight with 100 μCi [35S]methionine and lysed in buffer containing TX-100. After centrifugation, the resulting pellet was re-extracted with TX-114–containing lysis buffer for 20 min at 37°C. After centrifugation, the supernatant was subjected to temperature-induced phase separation. Samples were treated in the absence (−) and presence (+) of 5 U/ml PI-PLC. This treatment induces the transition from a hydrophobic to a hydrophilic state of GPI-anchored proteins.  Polypeptides released into the aqueous phase were recovered by acetone precipitation and analyzed on a 12.5% SDS-PAGE. Arrows  on the right indicate the appearance of some of the proteins specifically released by PI-PLC.
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Figure 5: Gα and Gβ are associated with non-ionic detergent-insoluble cell pellets. (A) IMR-32 cells were labeled overnight with 100 μCi of [35S]methionine. Cells were lysed in NP-40/Lubrol lysis buffer and separated into supernatant (soluble) and insoluble pellet. The pellet was solubilized in 1% SDS, DNA was sheared by 15 passages through a 21-gauge needle and the extract was boiled twice at 100°C for 3 min. The SDS concentration in the pellet fraction was adjusted to 0.2% with NP-40/Lubrol lysis buffer. Immunoprecipitations from the supernatant were done either in the presence (+) or absence (−) of 0.2% SDS. A mixture of 3C2 and 3E7 were used to recover α subunits and β was precipitated with ARC9. Samples were analyzed on a 12.5% SDS-PAGE (A–C). Arrows indicate the positions of β and α subunits. (B) IMR-32 cells were labeled for 2 h with 250 μCi [35S]methionine followed by solubilization in NP-40/ Lubrol lysis buffer. Insoluble pellets were extracted twice with non-ionic detergent buffer and subsequently treated as shown in A. (C) Kinetics of Gβ subunit association with detergent-insoluble cell pellets. IMR-32 cells were pulsed for 2.5 min with 150 μCi [35S]methionine and chased for the time intervals indicated. Cells were separated into supernatant (soluble) and a detergent-insoluble pellet. The pellet was re-extracted as in A, followed by immunoprecipitation with ARC 9. (D) Detection of endogenous GPI-anchored proteins in non-ionic detergent-resistant membrane domains. IMR-32 cells were labeled overnight with 100 μCi [35S]methionine and lysed in buffer containing TX-100. After centrifugation, the resulting pellet was re-extracted with TX-114–containing lysis buffer for 20 min at 37°C. After centrifugation, the supernatant was subjected to temperature-induced phase separation. Samples were treated in the absence (−) and presence (+) of 5 U/ml PI-PLC. This treatment induces the transition from a hydrophobic to a hydrophilic state of GPI-anchored proteins. Polypeptides released into the aqueous phase were recovered by acetone precipitation and analyzed on a 12.5% SDS-PAGE. Arrows on the right indicate the appearance of some of the proteins specifically released by PI-PLC.
Mentions: When non-ionic detergent insoluble material from labeled IMR-32 cells was analyzed for the presence of β subunits, we found substantial amounts of β subunits even after two rounds of non-ionic detergent extraction, although the majority of β subunits was still recovered from the detergent soluble fraction (Fig. 5, A and B). Whereas the recovery of β from the soluble extract required SDS, the efficiency of α recovery was independent of SDS, an indication that the epitopes recognized on α are freely accessible. To determine the time point when β associates with the insoluble pellet, IMR-32 cells were pulse-labeled for 2.5 min, chased for the times indicated, and extracted with NP-40/Lubrol. The β subunit was found in the pellet beginning at 1 min of chase (Fig. 5 C).

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