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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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Preexisting pre-Golgi intermediates do not tether and fuse with COPII vesicles released from permeabilized cells. (A) NRK cells were transfected with VSV-G-CFP and incubated at 40°C. Cells were incubated for the indicated times at 32°C before fixation and fluorescence microscopy. (B and C) Two-stage coisolation or heterotrimer assays were conducted using the usual VSV-G-myc vesicles together with VSV-G* vesicles from cells that had been preincubated at 32°C for the indicated times before permeabilization. The first-stage incubation of the chased cells was conducted with or without sar1 T39N. To differentiate the potential involvement of preexisting VTCs from a role for COPI vesicles as defined in Fig. 3, these assays were conducted under COPI-free conditions.
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fig4: Preexisting pre-Golgi intermediates do not tether and fuse with COPII vesicles released from permeabilized cells. (A) NRK cells were transfected with VSV-G-CFP and incubated at 40°C. Cells were incubated for the indicated times at 32°C before fixation and fluorescence microscopy. (B and C) Two-stage coisolation or heterotrimer assays were conducted using the usual VSV-G-myc vesicles together with VSV-G* vesicles from cells that had been preincubated at 32°C for the indicated times before permeabilization. The first-stage incubation of the chased cells was conducted with or without sar1 T39N. To differentiate the potential involvement of preexisting VTCs from a role for COPI vesicles as defined in Fig. 3, these assays were conducted under COPI-free conditions.

Mentions: Another possibility would be that the two populations of COPII vesicles fused in common with preexisting VTCs or another pre-Golgi structure. The putative intervening membrane would also be released from the cells during the first stage of incubation and act as a bridge between COPII vesicles. Thus, if this compartment were first loaded with one of the differently tagged VSV-G, we should be able to detect tethering and/or fusion of this membrane with COPII vesicles containing the other tagged VSV-G. As shown in Fig. 4 A, CFP-tagged VSV-G that is initially locked in the ER moves into VTCs and then to the Golgi during a 0–15-min preincubation of intact cells at the permissive temperature. Importantly, Fig. 4 (B and C) shows that as VSV-G*–containing cells are similarly preincubated before permeabilization, the capacity for coisolation and heterotrimerization with VSV-G-myc–containing COPII vesicles is rapidly lost. No assay signal is obtained when the progressively chased cell population is incubated with sar1 T39N during the vesicle release stage. Thus, only VSV-G* in COPII vesicles directly released from the ER can tether and fuse with VSV-G-myc in COPII vesicles; putative post-ER secretory intermediates such as preexisting VTCs are either not released during the incubation or cannot interact with COPII vesicles. Based on Figs. 3 (A and B) and 4, it would appear that pre-Golgi intermediates, at least in vitro, are built primarily by homotypic COPII vesicle fusion. On the other hand, a formal possibility remains that an unrelated membrane intervenes to mediate fusion between COPII vesicles.


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

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

Preexisting pre-Golgi intermediates do not tether and fuse with COPII vesicles released from permeabilized cells. (A) NRK cells were transfected with VSV-G-CFP and incubated at 40°C. Cells were incubated for the indicated times at 32°C before fixation and fluorescence microscopy. (B and C) Two-stage coisolation or heterotrimer assays were conducted using the usual VSV-G-myc vesicles together with VSV-G* vesicles from cells that had been preincubated at 32°C for the indicated times before permeabilization. The first-stage incubation of the chased cells was conducted with or without sar1 T39N. To differentiate the potential involvement of preexisting VTCs from a role for COPI vesicles as defined in Fig. 3, these assays were conducted under COPI-free conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172600&req=5

fig4: Preexisting pre-Golgi intermediates do not tether and fuse with COPII vesicles released from permeabilized cells. (A) NRK cells were transfected with VSV-G-CFP and incubated at 40°C. Cells were incubated for the indicated times at 32°C before fixation and fluorescence microscopy. (B and C) Two-stage coisolation or heterotrimer assays were conducted using the usual VSV-G-myc vesicles together with VSV-G* vesicles from cells that had been preincubated at 32°C for the indicated times before permeabilization. The first-stage incubation of the chased cells was conducted with or without sar1 T39N. To differentiate the potential involvement of preexisting VTCs from a role for COPI vesicles as defined in Fig. 3, these assays were conducted under COPI-free conditions.
Mentions: Another possibility would be that the two populations of COPII vesicles fused in common with preexisting VTCs or another pre-Golgi structure. The putative intervening membrane would also be released from the cells during the first stage of incubation and act as a bridge between COPII vesicles. Thus, if this compartment were first loaded with one of the differently tagged VSV-G, we should be able to detect tethering and/or fusion of this membrane with COPII vesicles containing the other tagged VSV-G. As shown in Fig. 4 A, CFP-tagged VSV-G that is initially locked in the ER moves into VTCs and then to the Golgi during a 0–15-min preincubation of intact cells at the permissive temperature. Importantly, Fig. 4 (B and C) shows that as VSV-G*–containing cells are similarly preincubated before permeabilization, the capacity for coisolation and heterotrimerization with VSV-G-myc–containing COPII vesicles is rapidly lost. No assay signal is obtained when the progressively chased cell population is incubated with sar1 T39N during the vesicle release stage. Thus, only VSV-G* in COPII vesicles directly released from the ER can tether and fuse with VSV-G-myc in COPII vesicles; putative post-ER secretory intermediates such as preexisting VTCs are either not released during the incubation or cannot interact with COPII vesicles. Based on Figs. 3 (A and B) and 4, it would appear that pre-Golgi intermediates, at least in vitro, are built primarily by homotypic COPII vesicle fusion. On the other hand, a formal possibility remains that an unrelated membrane intervenes to mediate fusion between 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