Limits...
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.

Show MeSH

Related in: MedlinePlus

Domain localization of newly synthesized Gα and Gβ  subunits. IMR-32 cells were pulse-labeled for 5 min with 500 μCi  [35S]methionine and chased for the designated times. Cells were  homogenized as shown (Fig. 3) and resolved on a discontinuous  sucrose gradient. 0.5-ml fractions were collected from the top of  each gradient, lysed in an equal volume of 2× NP-40/Lubrol lysis  buffer, and aliquots from each fraction were immunoprecipitated  with ARC9 (+ 0.2% SDS) (A) or with a mixture of anti-Gα antibodies (3E7, 3C2) (B). The interfaces between different sucrose  densities are designated A–E, whereas the sucrose density steps  are represented in percent sucrose. The distribution of radioactivity along the gradients is shown in C. Data shown are representative for at least three experiments performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139779&req=5

Figure 4: Domain localization of newly synthesized Gα and Gβ subunits. IMR-32 cells were pulse-labeled for 5 min with 500 μCi [35S]methionine and chased for the designated times. Cells were homogenized as shown (Fig. 3) and resolved on a discontinuous sucrose gradient. 0.5-ml fractions were collected from the top of each gradient, lysed in an equal volume of 2× NP-40/Lubrol lysis buffer, and aliquots from each fraction were immunoprecipitated with ARC9 (+ 0.2% SDS) (A) or with a mixture of anti-Gα antibodies (3E7, 3C2) (B). The interfaces between different sucrose densities are designated A–E, whereas the sucrose density steps are represented in percent sucrose. The distribution of radioactivity along the gradients is shown in C. Data shown are representative for at least three experiments performed in duplicate.

Mentions: A homogenate prepared from pulse-labeled IMR-32 cells was loaded on a sucrose step gradient and centrifuged at 85,000 g for 16 h. Fractions were analyzed individually by immunoprecipitation with a mixture of anti-Gα mAbs (3C2/ 3E7) or an anti-Gβ mAb (ARC9, plus 0.2% SDS). The distribution of both radiolabeled α or β in these gradients changed with time (Fig. 4, A and B). At the early time of chase (0 min), α and β were recovered from the denser region of the gradient, reflecting a status where β subunits, shortly after synthesis, are devoid of γ subunits, but they still fractionate with large dense particles, such as the cytosolic ribosomes on which they are synthesized (7). At the 20-min chase point we noted a movement of β and α subunits towards lower density fractions (B, C, and D interface). This result is consistent with the observation that after 20 min of chase most β subunits are complexed with γ subunits, and require SDS for efficient recovery in immunoprecipitation (Figs. 1 and 3). The assembly of β with the γ subunit is likely to provide the βγ complex with a membrane anchor, in the form of the COOH-terminal prenyl modification of the γ subunit. This anchor modification would allow part of the complexes to associate with lower density membranes, which are found in the B and C interface. A fraction of β subunits was still detectable in the D and E interface, where both plasma membranes (D interface), large particles and lysosomal membranes, fractionate (E interface) (7). A similar result was obtained for the α subunit, which at the 0-min chase point was found in the high density region of the gradient and after a 20-min chase moved to the low density fraction. At 60 min of chase we observed that Gα exhibited a more prominent bimodal distribution as compared to β. The peaks of this broad distribution were centered around the C and D interface. This result is consistent with the observation that the C interface accumulates light density membranes with the buoyant density of Golgi membranes, whereas plasma membranes characteristically float to the high density region (D interface) of the sucrose gradient (7). At 60 min of chase we recovered little material from the dense region of the gradient with the anti-Gβ mAb ARC9, indicating that formation of trimers in domains fractionating at the D interface might not be stoichiometric. We performed chases up to 240 min to reveal progressive changes of G protein subunit distribution as a function of time (data not shown), but no obvious alterations in comparison to the 60-min chase interval were discernible. This observation suggests that movement of G protein subunits from their site of synthesis to their final destination is essentially complete at 60 min of chase.


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

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

Domain localization of newly synthesized Gα and Gβ  subunits. IMR-32 cells were pulse-labeled for 5 min with 500 μCi  [35S]methionine and chased for the designated times. Cells were  homogenized as shown (Fig. 3) and resolved on a discontinuous  sucrose gradient. 0.5-ml fractions were collected from the top of  each gradient, lysed in an equal volume of 2× NP-40/Lubrol lysis  buffer, and aliquots from each fraction were immunoprecipitated  with ARC9 (+ 0.2% SDS) (A) or with a mixture of anti-Gα antibodies (3E7, 3C2) (B). The interfaces between different sucrose  densities are designated A–E, whereas the sucrose density steps  are represented in percent sucrose. The distribution of radioactivity along the gradients is shown in C. Data shown are representative for at least three experiments performed in duplicate.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Domain localization of newly synthesized Gα and Gβ subunits. IMR-32 cells were pulse-labeled for 5 min with 500 μCi [35S]methionine and chased for the designated times. Cells were homogenized as shown (Fig. 3) and resolved on a discontinuous sucrose gradient. 0.5-ml fractions were collected from the top of each gradient, lysed in an equal volume of 2× NP-40/Lubrol lysis buffer, and aliquots from each fraction were immunoprecipitated with ARC9 (+ 0.2% SDS) (A) or with a mixture of anti-Gα antibodies (3E7, 3C2) (B). The interfaces between different sucrose densities are designated A–E, whereas the sucrose density steps are represented in percent sucrose. The distribution of radioactivity along the gradients is shown in C. Data shown are representative for at least three experiments performed in duplicate.
Mentions: A homogenate prepared from pulse-labeled IMR-32 cells was loaded on a sucrose step gradient and centrifuged at 85,000 g for 16 h. Fractions were analyzed individually by immunoprecipitation with a mixture of anti-Gα mAbs (3C2/ 3E7) or an anti-Gβ mAb (ARC9, plus 0.2% SDS). The distribution of both radiolabeled α or β in these gradients changed with time (Fig. 4, A and B). At the early time of chase (0 min), α and β were recovered from the denser region of the gradient, reflecting a status where β subunits, shortly after synthesis, are devoid of γ subunits, but they still fractionate with large dense particles, such as the cytosolic ribosomes on which they are synthesized (7). At the 20-min chase point we noted a movement of β and α subunits towards lower density fractions (B, C, and D interface). This result is consistent with the observation that after 20 min of chase most β subunits are complexed with γ subunits, and require SDS for efficient recovery in immunoprecipitation (Figs. 1 and 3). The assembly of β with the γ subunit is likely to provide the βγ complex with a membrane anchor, in the form of the COOH-terminal prenyl modification of the γ subunit. This anchor modification would allow part of the complexes to associate with lower density membranes, which are found in the B and C interface. A fraction of β subunits was still detectable in the D and E interface, where both plasma membranes (D interface), large particles and lysosomal membranes, fractionate (E interface) (7). A similar result was obtained for the α subunit, which at the 0-min chase point was found in the high density region of the gradient and after a 20-min chase moved to the low density fraction. At 60 min of chase we observed that Gα exhibited a more prominent bimodal distribution as compared to β. The peaks of this broad distribution were centered around the C and D interface. This result is consistent with the observation that the C interface accumulates light density membranes with the buoyant density of Golgi membranes, whereas plasma membranes characteristically float to the high density region (D interface) of the sucrose gradient (7). At 60 min of chase we recovered little material from the dense region of the gradient with the anti-Gβ mAb ARC9, indicating that formation of trimers in domains fractionating at the D interface might not be stoichiometric. We performed chases up to 240 min to reveal progressive changes of G protein subunit distribution as a function of time (data not shown), but no obvious alterations in comparison to the 60-min chase interval were discernible. This observation suggests that movement of G protein subunits from their site of synthesis to their final destination is essentially complete at 60 min of chase.

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