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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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Sar1-GTP (H79G) inhibits vesicle release. (A) tsO45-VSV-G–containing membranes were incubated (40 μl final volume) in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–6) in the absence (lanes 1 and 2) or presence of Sar1-GTP (lanes 3–5) or Sar1-GDP (lane 6). At the end of incubation, membranes were subjected to a physical trituration step. The vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation and analyzed by Western blotting with antibodies against VSV-G and the SNARE protein Bet1. (B and C) The budding assay was performed as above without physical trituration. The mobilization of VSV-G and Bet1 to the vesicular fraction was analyzed in the presence of increasing concentrations of either Sar1-GTP (B, lanes 3–5) or Sar1 wt (C, lanes 3–6) as indicated. (D) Quantitation of the budding efficiency for VSV-G in three independent experiments under the indicated conditions is shown (means ± SEM).
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fig2: Sar1-GTP (H79G) inhibits vesicle release. (A) tsO45-VSV-G–containing membranes were incubated (40 μl final volume) in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–6) in the absence (lanes 1 and 2) or presence of Sar1-GTP (lanes 3–5) or Sar1-GDP (lane 6). At the end of incubation, membranes were subjected to a physical trituration step. The vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation and analyzed by Western blotting with antibodies against VSV-G and the SNARE protein Bet1. (B and C) The budding assay was performed as above without physical trituration. The mobilization of VSV-G and Bet1 to the vesicular fraction was analyzed in the presence of increasing concentrations of either Sar1-GTP (B, lanes 3–5) or Sar1 wt (C, lanes 3–6) as indicated. (D) Quantitation of the budding efficiency for VSV-G in three independent experiments under the indicated conditions is shown (means ± SEM).

Mentions: Having established that Sar1 can use its tail not only for membrane attachment but also as a GTP-regulated membrane–deforming machine, we investigated the role of this activity in vesicle fission. In previous studies we demonstrated that activation of Sar1 in permeabilized cells, in the absence of COPII subunits led to Sar1-induced tubulation and constriction of ER exit sites (Aridor et al., 2001). These tubules were reminiscent of intermediates in vesicle fission. COPII subunits, which subsequently bind Sar1 may organize the vesicle cage to restrict membranes for fission, and provide the GTPase-activating protein (GAP) activity that controls the interaction of Sar1 amphipathic domain with constricted membranes. Thus GTP hydrolysis may facilitate the progression from Sar1-induced membrane constriction to vesicle fission. We tested the role of GTP hydrolysis in regulating vesicle release. When added together with cytosol to permeabilized cells, Sar1-GTP inhibited the synchronous export of the model cargo protein vesicular stomatitis virus glycoprotein (VSV-G) tsO45 from the ER (as analyzed by immunofluorescence). The resulting morphology was suggestive of intermediates in vesicle formation that are arrested at the membrane constriction and fission stage (Fig. S 1, h, i, q, and r available at http://www.jcb.org/cgi/content/full/jcb.200509095/DC1). To focus on vesicle release, we used a biochemical assay that reconstitutes COPII vesicle formation from ER microsomes expressing ts045 VSV-G. ER membranes were separated from the vesicular fraction using differential centrifugation to monitor Sar1-dependent mobilization of cargo proteins from the ER. As previously characterized, ER membranes incubated with cytosol in the presence of increasing concentrations of Sar1-GTP produced normal COPII-coated vesicles selectively enriched in VSV-G and an endogenous cargo, the SNARE protein Bet1 (Fig. 2 A, lanes 3–5; Rowe et al., 1996; Aridor et al., 1998). Budding was abolished in incubations with cytosol and Sar1-GDP (Fig. 2 A, lane 6, see quantitation of all panels in Fig. 2 D). Although this assay did not reproduce the budding arrest observed by immunofluorescence, the assay used a trituration step originally included to facilitate the separation of vesicles from donor membranes. This step might physically promote the shearing of vesicles connected by membranes already constricted by Sar1-GTP (Fig. 1). Omitting the trituration step from the budding assay enhanced the efficiency of the budding reaction under control conditions. Incubation of VSV-G–containing membranes with cytosol led to efficient mobilization of VSV-G and Bet1 from the ER (Fig. 2, lanes 2 in B and C, and compare with lane 2 in A) and budding was inhibited by Sar1-GDP (Fig. 2 B [lane 6] and C [lane 7]). However, incubating membranes in the presence of Sar1-GTP now blocked budding of VSV-G and Bet1 from the ER (Fig. 2 B, lanes 3–5). Incubating membranes with similar concentrations of wild-type Sar1 did not affect budding (Fig. 2 C, lanes 3–6). Therefore, we could reproduce the Sar1-GTP–induced ER export arrest observed by immunofluorescence in a vesicle formation assay suggesting that inhibition is exerted at the level of vesicle release.


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)

Sar1-GTP (H79G) inhibits vesicle release. (A) tsO45-VSV-G–containing membranes were incubated (40 μl final volume) in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–6) in the absence (lanes 1 and 2) or presence of Sar1-GTP (lanes 3–5) or Sar1-GDP (lane 6). At the end of incubation, membranes were subjected to a physical trituration step. The vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation and analyzed by Western blotting with antibodies against VSV-G and the SNARE protein Bet1. (B and C) The budding assay was performed as above without physical trituration. The mobilization of VSV-G and Bet1 to the vesicular fraction was analyzed in the presence of increasing concentrations of either Sar1-GTP (B, lanes 3–5) or Sar1 wt (C, lanes 3–6) as indicated. (D) Quantitation of the budding efficiency for VSV-G in three independent experiments under the indicated conditions is shown (means ± SEM).
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Related In: Results  -  Collection

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fig2: Sar1-GTP (H79G) inhibits vesicle release. (A) tsO45-VSV-G–containing membranes were incubated (40 μl final volume) in the presence of cytosol for 30 min on ice (lane 1) or at 32°C (lanes 2–6) in the absence (lanes 1 and 2) or presence of Sar1-GTP (lanes 3–5) or Sar1-GDP (lane 6). At the end of incubation, membranes were subjected to a physical trituration step. The vesicle fraction (H) was separated from the donor membranes (M) by differential centrifugation and analyzed by Western blotting with antibodies against VSV-G and the SNARE protein Bet1. (B and C) The budding assay was performed as above without physical trituration. The mobilization of VSV-G and Bet1 to the vesicular fraction was analyzed in the presence of increasing concentrations of either Sar1-GTP (B, lanes 3–5) or Sar1 wt (C, lanes 3–6) as indicated. (D) Quantitation of the budding efficiency for VSV-G in three independent experiments under the indicated conditions is shown (means ± SEM).
Mentions: Having established that Sar1 can use its tail not only for membrane attachment but also as a GTP-regulated membrane–deforming machine, we investigated the role of this activity in vesicle fission. In previous studies we demonstrated that activation of Sar1 in permeabilized cells, in the absence of COPII subunits led to Sar1-induced tubulation and constriction of ER exit sites (Aridor et al., 2001). These tubules were reminiscent of intermediates in vesicle fission. COPII subunits, which subsequently bind Sar1 may organize the vesicle cage to restrict membranes for fission, and provide the GTPase-activating protein (GAP) activity that controls the interaction of Sar1 amphipathic domain with constricted membranes. Thus GTP hydrolysis may facilitate the progression from Sar1-induced membrane constriction to vesicle fission. We tested the role of GTP hydrolysis in regulating vesicle release. When added together with cytosol to permeabilized cells, Sar1-GTP inhibited the synchronous export of the model cargo protein vesicular stomatitis virus glycoprotein (VSV-G) tsO45 from the ER (as analyzed by immunofluorescence). The resulting morphology was suggestive of intermediates in vesicle formation that are arrested at the membrane constriction and fission stage (Fig. S 1, h, i, q, and r available at http://www.jcb.org/cgi/content/full/jcb.200509095/DC1). To focus on vesicle release, we used a biochemical assay that reconstitutes COPII vesicle formation from ER microsomes expressing ts045 VSV-G. ER membranes were separated from the vesicular fraction using differential centrifugation to monitor Sar1-dependent mobilization of cargo proteins from the ER. As previously characterized, ER membranes incubated with cytosol in the presence of increasing concentrations of Sar1-GTP produced normal COPII-coated vesicles selectively enriched in VSV-G and an endogenous cargo, the SNARE protein Bet1 (Fig. 2 A, lanes 3–5; Rowe et al., 1996; Aridor et al., 1998). Budding was abolished in incubations with cytosol and Sar1-GDP (Fig. 2 A, lane 6, see quantitation of all panels in Fig. 2 D). Although this assay did not reproduce the budding arrest observed by immunofluorescence, the assay used a trituration step originally included to facilitate the separation of vesicles from donor membranes. This step might physically promote the shearing of vesicles connected by membranes already constricted by Sar1-GTP (Fig. 1). Omitting the trituration step from the budding assay enhanced the efficiency of the budding reaction under control conditions. Incubation of VSV-G–containing membranes with cytosol led to efficient mobilization of VSV-G and Bet1 from the ER (Fig. 2, lanes 2 in B and C, and compare with lane 2 in A) and budding was inhibited by Sar1-GDP (Fig. 2 B [lane 6] and C [lane 7]). However, incubating membranes in the presence of Sar1-GTP now blocked budding of VSV-G and Bet1 from the ER (Fig. 2 B, lanes 3–5). Incubating membranes with similar concentrations of wild-type Sar1 did not affect budding (Fig. 2 C, lanes 3–6). Therefore, we could reproduce the Sar1-GTP–induced ER export arrest observed by immunofluorescence in a vesicle formation assay suggesting that inhibition is exerted at the level of vesicle release.

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