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Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment.

Xu D, Hay JC - J. Cell Biol. (2004)

Bottom Line: In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack.The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function.However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

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NSF, syntaxin 5, and rsly1 are required for homotypic fusion of COPII vesicles. (A) Two-stage VSV-G* coisolation (top) and heterotrimer (bottom) assays were conducted in the presence of increasing concentrations of wild-type or α-SNAP L294A. (B) Two-stage heterotrimer assay was conducted under COPI-free conditions in the presence of 130 nM of control rabbit IgG or affinity-purified polyclonal anti–syntaxin 5 antibodies, either native or boiled. (inset) Two-stage coisolation assay using anti–syntaxin 5 antibody at the same concentrations. (B and C) Error bars represent the SEM of duplicate determinations. (C) Two-stage coisolation (open bars) and heterotrimer (shaded bars) assays were conducted under COPI-free conditions in the presence of GST-rsly1–purified protein and 267 nM anti-rsly1 antibodies. (D) Model of in vitro VTC biogenesis and maturation (Results and discussion).
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fig5: NSF, syntaxin 5, and rsly1 are required for homotypic fusion of COPII vesicles. (A) Two-stage VSV-G* coisolation (top) and heterotrimer (bottom) assays were conducted in the presence of increasing concentrations of wild-type or α-SNAP L294A. (B) Two-stage heterotrimer assay was conducted under COPI-free conditions in the presence of 130 nM of control rabbit IgG or affinity-purified polyclonal anti–syntaxin 5 antibodies, either native or boiled. (inset) Two-stage coisolation assay using anti–syntaxin 5 antibody at the same concentrations. (B and C) Error bars represent the SEM of duplicate determinations. (C) Two-stage coisolation (open bars) and heterotrimer (shaded bars) assays were conducted under COPI-free conditions in the presence of GST-rsly1–purified protein and 267 nM anti-rsly1 antibodies. (D) Model of in vitro VTC biogenesis and maturation (Results and discussion).

Mentions: A mammalian ER–Golgi SNARE complex has been well-characterized and documented to function in transport (Xu et al., 2000; Williams et al., 2004), but the precise fusion event it catalyzes is unresolved. SNAREs were required for assembly of nascent VTCs because dominant-negative α-SNAP L294A (Barnard et al., 1997) inhibited both vesicle coisolation and heterotrimer formation, with a more potent and complete inhibition of the latter (Fig. 5 A). Significant inhibition of coisolation was unexpected because SNAREs are not known to function in tethering. One potential explanation is that dissociation of cis-SNARE complexes by NSF may be required to recruit or activate the tethering machinery. Fig. 5 B shows that polyclonal antibodies against syntaxin 5, the QA-SNARE for the ER–Golgi quaternary complex, inhibited the two-stage heterotrimer formation assay. Unlike the 18C8 monoclonal antibody used in Fig. 3, this anti–syntaxin 5 antiserum recognizes free as well as the cis-complexed syntaxin 5 (Williams et al., 2004) and inhibits a broader range of its functions. This experiment was conducted under COPI-free conditions, thus the results demonstrate that syntaxin 5 is required for the homotypic fusion of COPII vesicles to form early VTCs. As expected for a SNARE involved specifically in membrane fusion, syntaxin 5 inhibition had only minor effects on the vesicle coisolation assay performed in parallel (inset). Fig. 5 C shows that polyclonal antibodies against rsly1, the SM protein inextricably linked to syntaxin 5 function (Williams et al., 2004), specifically inhibited the two-stage heterotrimer assay under COPI-free conditions without significant effect on vesicle coisolation, suggesting a post-docking action close to membrane fusion. These results establish that the syntaxin 5–SNARE complex and rsly1 function in the earliest pre-Golgi membrane fusion event that appears to involve homotypic fusion of COPII vesicles.


Reconstitution of COPII vesicle fusion to generate a pre-Golgi intermediate compartment.

Xu D, Hay JC - J. Cell Biol. (2004)

NSF, syntaxin 5, and rsly1 are required for homotypic fusion of COPII vesicles. (A) Two-stage VSV-G* coisolation (top) and heterotrimer (bottom) assays were conducted in the presence of increasing concentrations of wild-type or α-SNAP L294A. (B) Two-stage heterotrimer assay was conducted under COPI-free conditions in the presence of 130 nM of control rabbit IgG or affinity-purified polyclonal anti–syntaxin 5 antibodies, either native or boiled. (inset) Two-stage coisolation assay using anti–syntaxin 5 antibody at the same concentrations. (B and C) Error bars represent the SEM of duplicate determinations. (C) Two-stage coisolation (open bars) and heterotrimer (shaded bars) assays were conducted under COPI-free conditions in the presence of GST-rsly1–purified protein and 267 nM anti-rsly1 antibodies. (D) Model of in vitro VTC biogenesis and maturation (Results and discussion).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172600&req=5

fig5: NSF, syntaxin 5, and rsly1 are required for homotypic fusion of COPII vesicles. (A) Two-stage VSV-G* coisolation (top) and heterotrimer (bottom) assays were conducted in the presence of increasing concentrations of wild-type or α-SNAP L294A. (B) Two-stage heterotrimer assay was conducted under COPI-free conditions in the presence of 130 nM of control rabbit IgG or affinity-purified polyclonal anti–syntaxin 5 antibodies, either native or boiled. (inset) Two-stage coisolation assay using anti–syntaxin 5 antibody at the same concentrations. (B and C) Error bars represent the SEM of duplicate determinations. (C) Two-stage coisolation (open bars) and heterotrimer (shaded bars) assays were conducted under COPI-free conditions in the presence of GST-rsly1–purified protein and 267 nM anti-rsly1 antibodies. (D) Model of in vitro VTC biogenesis and maturation (Results and discussion).
Mentions: A mammalian ER–Golgi SNARE complex has been well-characterized and documented to function in transport (Xu et al., 2000; Williams et al., 2004), but the precise fusion event it catalyzes is unresolved. SNAREs were required for assembly of nascent VTCs because dominant-negative α-SNAP L294A (Barnard et al., 1997) inhibited both vesicle coisolation and heterotrimer formation, with a more potent and complete inhibition of the latter (Fig. 5 A). Significant inhibition of coisolation was unexpected because SNAREs are not known to function in tethering. One potential explanation is that dissociation of cis-SNARE complexes by NSF may be required to recruit or activate the tethering machinery. Fig. 5 B shows that polyclonal antibodies against syntaxin 5, the QA-SNARE for the ER–Golgi quaternary complex, inhibited the two-stage heterotrimer formation assay. Unlike the 18C8 monoclonal antibody used in Fig. 3, this anti–syntaxin 5 antiserum recognizes free as well as the cis-complexed syntaxin 5 (Williams et al., 2004) and inhibits a broader range of its functions. This experiment was conducted under COPI-free conditions, thus the results demonstrate that syntaxin 5 is required for the homotypic fusion of COPII vesicles to form early VTCs. As expected for a SNARE involved specifically in membrane fusion, syntaxin 5 inhibition had only minor effects on the vesicle coisolation assay performed in parallel (inset). Fig. 5 C shows that polyclonal antibodies against rsly1, the SM protein inextricably linked to syntaxin 5 function (Williams et al., 2004), specifically inhibited the two-stage heterotrimer assay under COPI-free conditions without significant effect on vesicle coisolation, suggesting a post-docking action close to membrane fusion. These results establish that the syntaxin 5–SNARE complex and rsly1 function in the earliest pre-Golgi membrane fusion event that appears to involve homotypic fusion of COPII vesicles.

Bottom Line: In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack.The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function.However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109, USA.

ABSTRACT
What is the first membrane fusion step in the secretory pathway? In mammals, transport vesicles coated with coat complex (COP) II deliver secretory cargo to vesicular tubular clusters (VTCs) that ferry cargo from endoplasmic reticulum exit sites to the Golgi stack. However, the precise origin of VTCs and the membrane fusion step(s) involved have remained experimentally intractable. Here, we document in vitro direct tethering and SNARE-dependent fusion of endoplasmic reticulum-derived COPII transport vesicles to form larger cargo containers. The assembly did not require detectable Golgi membranes, preexisting VTCs, or COPI function. Therefore, COPII vesicles appear to contain all of the machinery to initiate VTC biogenesis via homotypic fusion. However, COPI function enhanced VTC assembly, and early VTCs acquired specific Golgi components by heterotypic fusion with Golgi-derived COPI vesicles.

Show MeSH
Related in: MedlinePlus