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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 is capable of constricting lipid bilayers. (A) Helical wheel representation of amino acids 1–18 of the NH2 terminus of hamster Sar1a. (B) The NH2-terminal tail of Sar1 destabilizes lipid membranes. Liposomes (100–120 nm, DOPC/DLPA, 80/20 mol percent) were incubated with 10 μM GST (a) or with 10 μM GST-Sar1-N-Tail (b) at 37°C for 1 h. At the end of incubations, liposomes were absorbed on charged grids and negatively stained with 1% phosphotungstic acid for EM analysis. (C) Sar1-GTP is capable of constricting liposome membranes. Liposomes (80–100 nm, DOPC and DLPA, 80/20 mol percent) were incubated in buffer (control; a), 10 μM Sar1-GDP (Sar1-T39N, b), 15 μM Sar1-GTP (Sar1-H79G, c), or 10 μM Δ9-Sar1 (d) mutants with GTP or GDP for 1 h at 37°C. Liposomes were stained and analyzed by EM. (D) A gallery of Sar1-GTP–induced tubulating (a–d) and fused (e and f) DOPC/DLPA liposomes. (E) Cholesterol/DOPC/DLPA (20/75/5 mol percent) liposomes (100–120 nm) were incubated as described above in the absence or presence of Sar1-GTP as indicated. (F) A gallery of tubulating cholesterol/DOPC/DLPA liposomes deformed during incubations with Sar1-GTP. Bars, 100 nm. (G) Sar1 uses its amphipathic NH2 terminus to bind liposomes in a GTP-dependent manner. Sar1 wt (1 μg) or Δ9-Sar1 (1 μg) was incubated with GTP or GDP in the absence or presence of liposomes as indicated. Liposomes were floated into a sucrose gradient and fractions collected from the top were numbered sequentially and analyzed by Western blot with Sar1 antibody. Fractions 1–4 contain liposome-associated Sar1, whereas fractions 5–12 contain unbound Sar1. (H) Liposome-bound Sar1 GTP can form lateral protein interactions. Liposome-bound Sar1 wt (1 μg) was cross-linked with DTSSP (100 μM) and loaded onto sucrose gradients as in G. Floated fractions were analyzed on nonreducing gels by Western blotting. When indicated, DTT (50 mM) was added to reverse cross-linking.
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fig1: Sar1 is capable of constricting lipid bilayers. (A) Helical wheel representation of amino acids 1–18 of the NH2 terminus of hamster Sar1a. (B) The NH2-terminal tail of Sar1 destabilizes lipid membranes. Liposomes (100–120 nm, DOPC/DLPA, 80/20 mol percent) were incubated with 10 μM GST (a) or with 10 μM GST-Sar1-N-Tail (b) at 37°C for 1 h. At the end of incubations, liposomes were absorbed on charged grids and negatively stained with 1% phosphotungstic acid for EM analysis. (C) Sar1-GTP is capable of constricting liposome membranes. Liposomes (80–100 nm, DOPC and DLPA, 80/20 mol percent) were incubated in buffer (control; a), 10 μM Sar1-GDP (Sar1-T39N, b), 15 μM Sar1-GTP (Sar1-H79G, c), or 10 μM Δ9-Sar1 (d) mutants with GTP or GDP for 1 h at 37°C. Liposomes were stained and analyzed by EM. (D) A gallery of Sar1-GTP–induced tubulating (a–d) and fused (e and f) DOPC/DLPA liposomes. (E) Cholesterol/DOPC/DLPA (20/75/5 mol percent) liposomes (100–120 nm) were incubated as described above in the absence or presence of Sar1-GTP as indicated. (F) A gallery of tubulating cholesterol/DOPC/DLPA liposomes deformed during incubations with Sar1-GTP. Bars, 100 nm. (G) Sar1 uses its amphipathic NH2 terminus to bind liposomes in a GTP-dependent manner. Sar1 wt (1 μg) or Δ9-Sar1 (1 μg) was incubated with GTP or GDP in the absence or presence of liposomes as indicated. Liposomes were floated into a sucrose gradient and fractions collected from the top were numbered sequentially and analyzed by Western blot with Sar1 antibody. Fractions 1–4 contain liposome-associated Sar1, whereas fractions 5–12 contain unbound Sar1. (H) Liposome-bound Sar1 GTP can form lateral protein interactions. Liposome-bound Sar1 wt (1 μg) was cross-linked with DTSSP (100 μM) and loaded onto sucrose gradients as in G. Floated fractions were analyzed on nonreducing gels by Western blotting. When indicated, DTT (50 mM) was added to reverse cross-linking.

Mentions: Fig. 1 A shows the first 18 amino acids of the amphipathic tail of hamster Sar1a when presented on a helical wheel diagram. The charge distribution (12–6) with five of the polar residues clustered at the same helical plane, suggests that the tail can function as membrane destabilizer. To test the effects of Sar1 amphipathic domain on membrane stability, we incubated GST protein, or a GST protein fused to Sar1 amphipathic domain encompassing amino acid 1 to 26 (GST-Sar1-N-Tail) with spherical and uniform liposomes, sized at 100 nm by filter extrusion and verified by light scattering and EM. Protein effects on membrane stability were reported by changes in liposome morphology, as visualized by EM. Although GST protein alone did not affect liposome morphology, incubations with GST-Sar1-N-Tail destabilized the liposomes such that no ovoid membranes remained and fragmented disorganized membranes were visible (Fig. 1 B, a and b). Therefore, the NH2 terminus of Sar1 is capable of destabilizing lipid bilayers. We next analyzed whether GTP loading on Sar1 can regulate the membrane-destabilizing activity of the Sar1 NH2 terminus. In the absence of Sar1, liposomes appeared as uniformly sized spheres as observed in incubations with GST (Fig. 1 C, a). This morphology was unchanged when liposomes were incubated with Sar1 mutant that is deficient in GTP binding, (Sar1-GDP [T39N]; Fig. 1 C, b). In contrast, liposome morphology was markedly modified when liposomes were incubated with a constitutively active mutant of Sar1 that cannot hydrolyze GTP (Sar1-GTP [H79G]; Aridor et al., 1995) (Fig. 1, C [c], E, and gallery in D and F). Short tubules were seen emanating from the liposomes (Fig. 1 D, a–d), as well as longer tubular structures (Fig. 1, D [e] and F), which sometimes developed to form extended networks of tubular membranes (Fig. 1 D, f). Formation of elongated structures with extended membrane surface is likely due to fusion of smaller ovoid liposome membranes (refer to Fig. 1 D, e, for examples of intermediates in liposome fusion). Initiation of liposome tubulation was detected with Sar1-GTP at 4 μM (unpublished data). Therefore, the activity of Sar1 NH2 terminus depends on Sar1 activation. Furthermore, this activity is dependent on the presence of intact NH2 terminus. A GTP-loaded truncated form of Sar1 with the proximal nine amino acids of the NH2 terminus removed (Δ9-Sar1) failed to destabilize or tubulate liposomes (Fig. 1 C, d). Δ9-Sar1 can bind and hydrolyze GTP but cannot interact efficiently with membranes (Huang et al., 2001). We separated liposome bound Sar1 from unbound protein on sucrose density gradients to analyze Sar1 interaction with liposomes. As observed morphologically, Sar1 interactions with liposomes were dependent on GTP binding and intact NH2 terminus amphipathic domain (Fig. 1 G). High molecular weight products of Sar1 suggestive of oligomerization were observed when liposome bound Sar1 was cross-linked with reversible cross-linker (3,3′-dithio-bis-sulfosuccinimidyl propionate [DTSSP]; Fig. 1 H), and these products were reduced to mostly monomeric (∼25 kD) protein by DTT. It is possible that the high concentrations of membrane-recruited Sar1 supported lateral protein interactions on the liposome surface, and these interactions may have further promoted local perturbation of lipid packing and membrane constriction. High concentrations of Sar1 are observed on tubules formed on ER membranes (Aridor et al., 2001).


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 is capable of constricting lipid bilayers. (A) Helical wheel representation of amino acids 1–18 of the NH2 terminus of hamster Sar1a. (B) The NH2-terminal tail of Sar1 destabilizes lipid membranes. Liposomes (100–120 nm, DOPC/DLPA, 80/20 mol percent) were incubated with 10 μM GST (a) or with 10 μM GST-Sar1-N-Tail (b) at 37°C for 1 h. At the end of incubations, liposomes were absorbed on charged grids and negatively stained with 1% phosphotungstic acid for EM analysis. (C) Sar1-GTP is capable of constricting liposome membranes. Liposomes (80–100 nm, DOPC and DLPA, 80/20 mol percent) were incubated in buffer (control; a), 10 μM Sar1-GDP (Sar1-T39N, b), 15 μM Sar1-GTP (Sar1-H79G, c), or 10 μM Δ9-Sar1 (d) mutants with GTP or GDP for 1 h at 37°C. Liposomes were stained and analyzed by EM. (D) A gallery of Sar1-GTP–induced tubulating (a–d) and fused (e and f) DOPC/DLPA liposomes. (E) Cholesterol/DOPC/DLPA (20/75/5 mol percent) liposomes (100–120 nm) were incubated as described above in the absence or presence of Sar1-GTP as indicated. (F) A gallery of tubulating cholesterol/DOPC/DLPA liposomes deformed during incubations with Sar1-GTP. Bars, 100 nm. (G) Sar1 uses its amphipathic NH2 terminus to bind liposomes in a GTP-dependent manner. Sar1 wt (1 μg) or Δ9-Sar1 (1 μg) was incubated with GTP or GDP in the absence or presence of liposomes as indicated. Liposomes were floated into a sucrose gradient and fractions collected from the top were numbered sequentially and analyzed by Western blot with Sar1 antibody. Fractions 1–4 contain liposome-associated Sar1, whereas fractions 5–12 contain unbound Sar1. (H) Liposome-bound Sar1 GTP can form lateral protein interactions. Liposome-bound Sar1 wt (1 μg) was cross-linked with DTSSP (100 μM) and loaded onto sucrose gradients as in G. Floated fractions were analyzed on nonreducing gels by Western blotting. When indicated, DTT (50 mM) was added to reverse cross-linking.
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fig1: Sar1 is capable of constricting lipid bilayers. (A) Helical wheel representation of amino acids 1–18 of the NH2 terminus of hamster Sar1a. (B) The NH2-terminal tail of Sar1 destabilizes lipid membranes. Liposomes (100–120 nm, DOPC/DLPA, 80/20 mol percent) were incubated with 10 μM GST (a) or with 10 μM GST-Sar1-N-Tail (b) at 37°C for 1 h. At the end of incubations, liposomes were absorbed on charged grids and negatively stained with 1% phosphotungstic acid for EM analysis. (C) Sar1-GTP is capable of constricting liposome membranes. Liposomes (80–100 nm, DOPC and DLPA, 80/20 mol percent) were incubated in buffer (control; a), 10 μM Sar1-GDP (Sar1-T39N, b), 15 μM Sar1-GTP (Sar1-H79G, c), or 10 μM Δ9-Sar1 (d) mutants with GTP or GDP for 1 h at 37°C. Liposomes were stained and analyzed by EM. (D) A gallery of Sar1-GTP–induced tubulating (a–d) and fused (e and f) DOPC/DLPA liposomes. (E) Cholesterol/DOPC/DLPA (20/75/5 mol percent) liposomes (100–120 nm) were incubated as described above in the absence or presence of Sar1-GTP as indicated. (F) A gallery of tubulating cholesterol/DOPC/DLPA liposomes deformed during incubations with Sar1-GTP. Bars, 100 nm. (G) Sar1 uses its amphipathic NH2 terminus to bind liposomes in a GTP-dependent manner. Sar1 wt (1 μg) or Δ9-Sar1 (1 μg) was incubated with GTP or GDP in the absence or presence of liposomes as indicated. Liposomes were floated into a sucrose gradient and fractions collected from the top were numbered sequentially and analyzed by Western blot with Sar1 antibody. Fractions 1–4 contain liposome-associated Sar1, whereas fractions 5–12 contain unbound Sar1. (H) Liposome-bound Sar1 GTP can form lateral protein interactions. Liposome-bound Sar1 wt (1 μg) was cross-linked with DTSSP (100 μM) and loaded onto sucrose gradients as in G. Floated fractions were analyzed on nonreducing gels by Western blotting. When indicated, DTT (50 mM) was added to reverse cross-linking.
Mentions: Fig. 1 A shows the first 18 amino acids of the amphipathic tail of hamster Sar1a when presented on a helical wheel diagram. The charge distribution (12–6) with five of the polar residues clustered at the same helical plane, suggests that the tail can function as membrane destabilizer. To test the effects of Sar1 amphipathic domain on membrane stability, we incubated GST protein, or a GST protein fused to Sar1 amphipathic domain encompassing amino acid 1 to 26 (GST-Sar1-N-Tail) with spherical and uniform liposomes, sized at 100 nm by filter extrusion and verified by light scattering and EM. Protein effects on membrane stability were reported by changes in liposome morphology, as visualized by EM. Although GST protein alone did not affect liposome morphology, incubations with GST-Sar1-N-Tail destabilized the liposomes such that no ovoid membranes remained and fragmented disorganized membranes were visible (Fig. 1 B, a and b). Therefore, the NH2 terminus of Sar1 is capable of destabilizing lipid bilayers. We next analyzed whether GTP loading on Sar1 can regulate the membrane-destabilizing activity of the Sar1 NH2 terminus. In the absence of Sar1, liposomes appeared as uniformly sized spheres as observed in incubations with GST (Fig. 1 C, a). This morphology was unchanged when liposomes were incubated with Sar1 mutant that is deficient in GTP binding, (Sar1-GDP [T39N]; Fig. 1 C, b). In contrast, liposome morphology was markedly modified when liposomes were incubated with a constitutively active mutant of Sar1 that cannot hydrolyze GTP (Sar1-GTP [H79G]; Aridor et al., 1995) (Fig. 1, C [c], E, and gallery in D and F). Short tubules were seen emanating from the liposomes (Fig. 1 D, a–d), as well as longer tubular structures (Fig. 1, D [e] and F), which sometimes developed to form extended networks of tubular membranes (Fig. 1 D, f). Formation of elongated structures with extended membrane surface is likely due to fusion of smaller ovoid liposome membranes (refer to Fig. 1 D, e, for examples of intermediates in liposome fusion). Initiation of liposome tubulation was detected with Sar1-GTP at 4 μM (unpublished data). Therefore, the activity of Sar1 NH2 terminus depends on Sar1 activation. Furthermore, this activity is dependent on the presence of intact NH2 terminus. A GTP-loaded truncated form of Sar1 with the proximal nine amino acids of the NH2 terminus removed (Δ9-Sar1) failed to destabilize or tubulate liposomes (Fig. 1 C, d). Δ9-Sar1 can bind and hydrolyze GTP but cannot interact efficiently with membranes (Huang et al., 2001). We separated liposome bound Sar1 from unbound protein on sucrose density gradients to analyze Sar1 interaction with liposomes. As observed morphologically, Sar1 interactions with liposomes were dependent on GTP binding and intact NH2 terminus amphipathic domain (Fig. 1 G). High molecular weight products of Sar1 suggestive of oligomerization were observed when liposome bound Sar1 was cross-linked with reversible cross-linker (3,3′-dithio-bis-sulfosuccinimidyl propionate [DTSSP]; Fig. 1 H), and these products were reduced to mostly monomeric (∼25 kD) protein by DTT. It is possible that the high concentrations of membrane-recruited Sar1 supported lateral protein interactions on the liposome surface, and these interactions may have further promoted local perturbation of lipid packing and membrane constriction. High concentrations of Sar1 are observed on tubules formed on ER membranes (Aridor et al., 2001).

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