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Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission.

Bielli A, Haney CJ, Gabreski G, Watkins SC, Bannykh SI, Aridor M - J. Cell Biol. (2005)

Bottom Line: Goldberg. 2002.Nature. 419:271-277).Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

ABSTRACT
The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271-277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.

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Vesicle release is regulated by endogenous Sar1 GTPase activity and requires functional NH2-terminal amphipathic domain. (A) Inhibition of endogenous Sar1 GTPase activity inhibits COPII vesicle release. VSV-G–containing membranes were incubated in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–7) in the absence (lanes 1 and 2) or presence of increasing concentrations of GTP-γ-S as indicated. At the end of incubation, the vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation without trituration and analyzed by Western blotting as indicated. (B) COPII components are not limiting under conditions inhibitory to vesicle release. The vesicle formation reaction was carried in the absence or presence of GTP-γ-S (100 μM). At the end of incubation the vesicle fraction was separated from the membrane fraction. The supernatant of the vesicle fraction was collected and analyzed for available COPII component Sec23. Quantitation of the amount of VSV-G in the vesicle fraction (left) and COPII Sec23 subunit remaining in the supernatant of the vesicle fraction (right) averaged from three independent experiments ± SEM. (C) Cargo-free COPII vesicles are not produced when Sar1 GTPase activity is inhibited. Vesicles generated as described in A, in the presence or absence of 100 μM GTP-γ-S, were separated from donor membranes by centrifugation and floated into sucrose gradients. Fractions 1–4 (collected from the top of the gradient) contain floated vesicles (VSV-G– and Bet1-containing fractions). The presence of VSV-G, Sec23, and Bet1 in the vesicle fraction was determined as indicated. (D) Sar1FPF (Y9F, G11P, S14F) does not support efficient vesicle release. In the upper panel, VSV-G–containing membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2.5 μg each) on ice or at 32°C for 30 min as indicated. At the end of incubation the vesicle fraction was prepared without physical trituration and analyzed by Western blotting. For the trituration assay (lower panel), membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2 μg each) for 15 min on ice or at 32°C as indicated. The vesicle fraction was prepared with physical trituration and analyzed by Western blotting.
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fig3: Vesicle release is regulated by endogenous Sar1 GTPase activity and requires functional NH2-terminal amphipathic domain. (A) Inhibition of endogenous Sar1 GTPase activity inhibits COPII vesicle release. VSV-G–containing membranes were incubated in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–7) in the absence (lanes 1 and 2) or presence of increasing concentrations of GTP-γ-S as indicated. At the end of incubation, the vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation without trituration and analyzed by Western blotting as indicated. (B) COPII components are not limiting under conditions inhibitory to vesicle release. The vesicle formation reaction was carried in the absence or presence of GTP-γ-S (100 μM). At the end of incubation the vesicle fraction was separated from the membrane fraction. The supernatant of the vesicle fraction was collected and analyzed for available COPII component Sec23. Quantitation of the amount of VSV-G in the vesicle fraction (left) and COPII Sec23 subunit remaining in the supernatant of the vesicle fraction (right) averaged from three independent experiments ± SEM. (C) Cargo-free COPII vesicles are not produced when Sar1 GTPase activity is inhibited. Vesicles generated as described in A, in the presence or absence of 100 μM GTP-γ-S, were separated from donor membranes by centrifugation and floated into sucrose gradients. Fractions 1–4 (collected from the top of the gradient) contain floated vesicles (VSV-G– and Bet1-containing fractions). The presence of VSV-G, Sec23, and Bet1 in the vesicle fraction was determined as indicated. (D) Sar1FPF (Y9F, G11P, S14F) does not support efficient vesicle release. In the upper panel, VSV-G–containing membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2.5 μg each) on ice or at 32°C for 30 min as indicated. At the end of incubation the vesicle fraction was prepared without physical trituration and analyzed by Western blotting. For the trituration assay (lower panel), membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2 μg each) for 15 min on ice or at 32°C as indicated. The vesicle fraction was prepared with physical trituration and analyzed by Western blotting.

Mentions: Several reasons could account for the inhibition of vesicle release by Sar1-GTP. Addition of exogenous excess Sar1 may create a stoichiometric imbalance between COPII components, inhibiting proper vesicle coating, formation, and release. We therefore tested whether constitutive activation of endogenous cytosolic Sar1 protein affected vesicle release. We used a nonhydrolyzable analogue of GTP, GTP-γ-S, to activate cytosolic Sar1. Similar to the results obtained with Sar1-GTP, GTP-γ-S inhibited vesicle release (Fig. 3 A, lanes 3–7, and B, lane 3). Irreversible binding of Sar1 to membranes could prevent vesicle uncoating depleting coat components needed for budding. However, the abundance of residual-free COPII (as analyzed by Sec23 levels) remained largely unchanged under conditions that inhibited vesicle release (Fig. 3 B). It is possible that inhibition of Sar1-mediated GTP hydrolysis inhibits cargo selection, yet release of vesicles, which remain coated due to the inhibition of GTP hydrolysis, progresses normally. We thus analyzed whether cargo-depleted COPII-coated vesicles are generated in incubations with Sar1-GTP-γ-S by analyzing the vesicular fraction on sucrose gradients. We found VSV-G– and Bet1-containing vesicles that retained coat components (identified using Sec23 antibody) in control (32°C) incubations, but not in similar incubations performed with GTP-γ-S (Fig. 3 C). Therefore cargo-depleted COPII vesicles are not released under these conditions. Indeed, GTP hydrolysis is not required for efficient cargo selection, as observed during the formation of Sar1-GTP–stabilized cargo selective prebudding complexes (Aridor et al., 1998; Kuehn et al., 1998), or in budding assays carried in the presence of Sar1-GTP that included physical trituration (Fig. 2 A).


Regulation of Sar1 NH2 terminus by GTP binding and hydrolysis promotes membrane deformation to control COPII vesicle fission.

Bielli A, Haney CJ, Gabreski G, Watkins SC, Bannykh SI, Aridor M - J. Cell Biol. (2005)

Vesicle release is regulated by endogenous Sar1 GTPase activity and requires functional NH2-terminal amphipathic domain. (A) Inhibition of endogenous Sar1 GTPase activity inhibits COPII vesicle release. VSV-G–containing membranes were incubated in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–7) in the absence (lanes 1 and 2) or presence of increasing concentrations of GTP-γ-S as indicated. At the end of incubation, the vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation without trituration and analyzed by Western blotting as indicated. (B) COPII components are not limiting under conditions inhibitory to vesicle release. The vesicle formation reaction was carried in the absence or presence of GTP-γ-S (100 μM). At the end of incubation the vesicle fraction was separated from the membrane fraction. The supernatant of the vesicle fraction was collected and analyzed for available COPII component Sec23. Quantitation of the amount of VSV-G in the vesicle fraction (left) and COPII Sec23 subunit remaining in the supernatant of the vesicle fraction (right) averaged from three independent experiments ± SEM. (C) Cargo-free COPII vesicles are not produced when Sar1 GTPase activity is inhibited. Vesicles generated as described in A, in the presence or absence of 100 μM GTP-γ-S, were separated from donor membranes by centrifugation and floated into sucrose gradients. Fractions 1–4 (collected from the top of the gradient) contain floated vesicles (VSV-G– and Bet1-containing fractions). The presence of VSV-G, Sec23, and Bet1 in the vesicle fraction was determined as indicated. (D) Sar1FPF (Y9F, G11P, S14F) does not support efficient vesicle release. In the upper panel, VSV-G–containing membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2.5 μg each) on ice or at 32°C for 30 min as indicated. At the end of incubation the vesicle fraction was prepared without physical trituration and analyzed by Western blotting. For the trituration assay (lower panel), membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2 μg each) for 15 min on ice or at 32°C as indicated. The vesicle fraction was prepared with physical trituration and analyzed by Western blotting.
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fig3: Vesicle release is regulated by endogenous Sar1 GTPase activity and requires functional NH2-terminal amphipathic domain. (A) Inhibition of endogenous Sar1 GTPase activity inhibits COPII vesicle release. VSV-G–containing membranes were incubated in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–7) in the absence (lanes 1 and 2) or presence of increasing concentrations of GTP-γ-S as indicated. At the end of incubation, the vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation without trituration and analyzed by Western blotting as indicated. (B) COPII components are not limiting under conditions inhibitory to vesicle release. The vesicle formation reaction was carried in the absence or presence of GTP-γ-S (100 μM). At the end of incubation the vesicle fraction was separated from the membrane fraction. The supernatant of the vesicle fraction was collected and analyzed for available COPII component Sec23. Quantitation of the amount of VSV-G in the vesicle fraction (left) and COPII Sec23 subunit remaining in the supernatant of the vesicle fraction (right) averaged from three independent experiments ± SEM. (C) Cargo-free COPII vesicles are not produced when Sar1 GTPase activity is inhibited. Vesicles generated as described in A, in the presence or absence of 100 μM GTP-γ-S, were separated from donor membranes by centrifugation and floated into sucrose gradients. Fractions 1–4 (collected from the top of the gradient) contain floated vesicles (VSV-G– and Bet1-containing fractions). The presence of VSV-G, Sec23, and Bet1 in the vesicle fraction was determined as indicated. (D) Sar1FPF (Y9F, G11P, S14F) does not support efficient vesicle release. In the upper panel, VSV-G–containing membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2.5 μg each) on ice or at 32°C for 30 min as indicated. At the end of incubation the vesicle fraction was prepared without physical trituration and analyzed by Western blotting. For the trituration assay (lower panel), membranes were incubated (40 μl final volume) with limiting cytosol in the presence of Sar1 wt or Sar1FPF (2 μg each) for 15 min on ice or at 32°C as indicated. The vesicle fraction was prepared with physical trituration and analyzed by Western blotting.
Mentions: Several reasons could account for the inhibition of vesicle release by Sar1-GTP. Addition of exogenous excess Sar1 may create a stoichiometric imbalance between COPII components, inhibiting proper vesicle coating, formation, and release. We therefore tested whether constitutive activation of endogenous cytosolic Sar1 protein affected vesicle release. We used a nonhydrolyzable analogue of GTP, GTP-γ-S, to activate cytosolic Sar1. Similar to the results obtained with Sar1-GTP, GTP-γ-S inhibited vesicle release (Fig. 3 A, lanes 3–7, and B, lane 3). Irreversible binding of Sar1 to membranes could prevent vesicle uncoating depleting coat components needed for budding. However, the abundance of residual-free COPII (as analyzed by Sec23 levels) remained largely unchanged under conditions that inhibited vesicle release (Fig. 3 B). It is possible that inhibition of Sar1-mediated GTP hydrolysis inhibits cargo selection, yet release of vesicles, which remain coated due to the inhibition of GTP hydrolysis, progresses normally. We thus analyzed whether cargo-depleted COPII-coated vesicles are generated in incubations with Sar1-GTP-γ-S by analyzing the vesicular fraction on sucrose gradients. We found VSV-G– and Bet1-containing vesicles that retained coat components (identified using Sec23 antibody) in control (32°C) incubations, but not in similar incubations performed with GTP-γ-S (Fig. 3 C). Therefore cargo-depleted COPII vesicles are not released under these conditions. Indeed, GTP hydrolysis is not required for efficient cargo selection, as observed during the formation of Sar1-GTP–stabilized cargo selective prebudding complexes (Aridor et al., 1998; Kuehn et al., 1998), or in budding assays carried in the presence of Sar1-GTP that included physical trituration (Fig. 2 A).

Bottom Line: Goldberg. 2002.Nature. 419:271-277).Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, USA.

ABSTRACT
The mechanisms by which the coat complex II (COPII) coat mediates membrane deformation and vesicle fission are unknown. Sar1 is a structural component of the membrane-binding inner layer of COPII (Bi, X., R.A. Corpina, and J. Goldberg. 2002. Nature. 419:271-277). Using model liposomes we found that Sar1 uses GTP-regulated exposure of its NH2-terminal tail, an amphipathic peptide domain, to bind, deform, constrict, and destabilize membranes. Although Sar1 activation leads to constriction of endoplasmic reticulum (ER) membranes, progression to effective vesicle fission requires a functional Sar1 NH2 terminus and guanosine triphosphate (GTP) hydrolysis. Inhibition of Sar1 GTP hydrolysis, which stabilizes Sar1 membrane binding, resulted in the formation of coated COPII vesicles that fail to detach from the ER. Thus Sar1-mediated GTP binding and hydrolysis regulates the NH2-terminal tail to perturb membrane packing, promote membrane deformation, and control vesicle fission.

Show MeSH
Related in: MedlinePlus