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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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Inhibition of GTP hydrolysis by Sar1 leads to the formation of COPII-coated vesicles that fail to detach from the ER. NRK cells were permeabilized and incubated (200 μl final volume) in the presence of Sar1-GTP (5 μg, a–d) or GTP-γ-S (100 μM, e and f) and cytosol as described previously (Bannykh et al., 1996). At the end of incubation the cells were fixed and incubated with primary antibodies to Sar1, Sec23, Sec13, and Sec31 and protein A (5 nm, a, b, and d) or (10 nm, c, e, and f) conjugates. The cells were postfixed, stained, and embedded in Epon. 70 nm sections were analyzed by transmission EM. Clusters of 60 nm vesicles were formed in the presence of Sar1-GTP and cytosol. These vesicles contained both inner (Sar1 in a and b and Sec23 in c) and outer (Sec13 in d and Sec31 in e and f) COPII layers. Arrows indicated membrane continuity between the vesicles themselves and between vesicles and ER membranes. Bars, 100 nm.
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fig4: Inhibition of GTP hydrolysis by Sar1 leads to the formation of COPII-coated vesicles that fail to detach from the ER. NRK cells were permeabilized and incubated (200 μl final volume) in the presence of Sar1-GTP (5 μg, a–d) or GTP-γ-S (100 μM, e and f) and cytosol as described previously (Bannykh et al., 1996). At the end of incubation the cells were fixed and incubated with primary antibodies to Sar1, Sec23, Sec13, and Sec31 and protein A (5 nm, a, b, and d) or (10 nm, c, e, and f) conjugates. The cells were postfixed, stained, and embedded in Epon. 70 nm sections were analyzed by transmission EM. Clusters of 60 nm vesicles were formed in the presence of Sar1-GTP and cytosol. These vesicles contained both inner (Sar1 in a and b and Sec23 in c) and outer (Sec13 in d and Sec31 in e and f) COPII layers. Arrows indicated membrane continuity between the vesicles themselves and between vesicles and ER membranes. Bars, 100 nm.

Mentions: By following the mobilization of cargo proteins we demonstrate that when Sar1 is stabilized in its active state, coated COPII vesicles fail to detach from the ER. We visualized COPII vesicles generated in the presence of Sar1-GTP (Fig. 4, a–d) or GTP-γ-S (Fig. 4, e and f) and cytosol in permeabilized NRK cells using EM (Fig. 4, a–f). COPII vesicles were identified using gold immunolabeling to detect both the inner (Sar1 and Sec23; Fig. 4, a–c) and outer (Sec13 and Sec31; Fig. 4, d–f) layers of the COPII coat. We observed beads on a string like 60–80 nm vesicles generated at ER exit sites (Bannykh et al., 1996). We now demonstrate that these vesicles are COPII-coated vesicles that fail to detach from the ER. Mammalian COPII vesicles seem similar to yeast COPII vesicles (Barlowe et al., 1994). The vesicles appeared coated and assembled yet clear constricted membrane connections between the coated vesicles themselves and the ER membrane were visible (Fig. 4, a–f, see arrows).


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)

Inhibition of GTP hydrolysis by Sar1 leads to the formation of COPII-coated vesicles that fail to detach from the ER. NRK cells were permeabilized and incubated (200 μl final volume) in the presence of Sar1-GTP (5 μg, a–d) or GTP-γ-S (100 μM, e and f) and cytosol as described previously (Bannykh et al., 1996). At the end of incubation the cells were fixed and incubated with primary antibodies to Sar1, Sec23, Sec13, and Sec31 and protein A (5 nm, a, b, and d) or (10 nm, c, e, and f) conjugates. The cells were postfixed, stained, and embedded in Epon. 70 nm sections were analyzed by transmission EM. Clusters of 60 nm vesicles were formed in the presence of Sar1-GTP and cytosol. These vesicles contained both inner (Sar1 in a and b and Sec23 in c) and outer (Sec13 in d and Sec31 in e and f) COPII layers. Arrows indicated membrane continuity between the vesicles themselves and between vesicles and ER membranes. Bars, 100 nm.
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Related In: Results  -  Collection

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fig4: Inhibition of GTP hydrolysis by Sar1 leads to the formation of COPII-coated vesicles that fail to detach from the ER. NRK cells were permeabilized and incubated (200 μl final volume) in the presence of Sar1-GTP (5 μg, a–d) or GTP-γ-S (100 μM, e and f) and cytosol as described previously (Bannykh et al., 1996). At the end of incubation the cells were fixed and incubated with primary antibodies to Sar1, Sec23, Sec13, and Sec31 and protein A (5 nm, a, b, and d) or (10 nm, c, e, and f) conjugates. The cells were postfixed, stained, and embedded in Epon. 70 nm sections were analyzed by transmission EM. Clusters of 60 nm vesicles were formed in the presence of Sar1-GTP and cytosol. These vesicles contained both inner (Sar1 in a and b and Sec23 in c) and outer (Sec13 in d and Sec31 in e and f) COPII layers. Arrows indicated membrane continuity between the vesicles themselves and between vesicles and ER membranes. Bars, 100 nm.
Mentions: By following the mobilization of cargo proteins we demonstrate that when Sar1 is stabilized in its active state, coated COPII vesicles fail to detach from the ER. We visualized COPII vesicles generated in the presence of Sar1-GTP (Fig. 4, a–d) or GTP-γ-S (Fig. 4, e and f) and cytosol in permeabilized NRK cells using EM (Fig. 4, a–f). COPII vesicles were identified using gold immunolabeling to detect both the inner (Sar1 and Sec23; Fig. 4, a–c) and outer (Sec13 and Sec31; Fig. 4, d–f) layers of the COPII coat. We observed beads on a string like 60–80 nm vesicles generated at ER exit sites (Bannykh et al., 1996). We now demonstrate that these vesicles are COPII-coated vesicles that fail to detach from the ER. Mammalian COPII vesicles seem similar to yeast COPII vesicles (Barlowe et al., 1994). The vesicles appeared coated and assembled yet clear constricted membrane connections between the coated vesicles themselves and the ER membrane were visible (Fig. 4, a–f, see arrows).

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