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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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Assembly involves symmetric populations of COPII vesicles but not Golgi. (A) Released VSV-G-myc vesicles and VSV-G* vesicles were prepared separately, semi-intact cells were removed, and the vesicles were combined for second-stage incubations. After the second incubation, vesicles were analyzed for coisolation and heterotrimer formation as in Fig. 1. Purified recombinant sar1 T39N was included at different points. (A) Error bars represent the SEM of duplicate determinations. (B) Single-stage coisolation assay was performed as in Fig. 1. The coisolated vesicles, as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo H and analyzed by autoradiography. R and S represent resistant and sensitive bands, respectively. (C) Single-stage coisolation and heterotrimer assays using CHO 15B cells. The α-SNAP L294A reaction contained 5 μM of recombinant protein. (D) Single-stage heterotrimer assay was performed as in C. Immunoprecipitated heterotrimers as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo D and analyzed by autoradiography.
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fig2: Assembly involves symmetric populations of COPII vesicles but not Golgi. (A) Released VSV-G-myc vesicles and VSV-G* vesicles were prepared separately, semi-intact cells were removed, and the vesicles were combined for second-stage incubations. After the second incubation, vesicles were analyzed for coisolation and heterotrimer formation as in Fig. 1. Purified recombinant sar1 T39N was included at different points. (A) Error bars represent the SEM of duplicate determinations. (B) Single-stage coisolation assay was performed as in Fig. 1. The coisolated vesicles, as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo H and analyzed by autoradiography. R and S represent resistant and sensitive bands, respectively. (C) Single-stage coisolation and heterotrimer assays using CHO 15B cells. The α-SNAP L294A reaction contained 5 μM of recombinant protein. (D) Single-stage heterotrimer assay was performed as in C. Immunoprecipitated heterotrimers as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo D and analyzed by autoradiography.

Mentions: The aforementioned experiments involved coincubation of both donor cell populations in one tube and preclude analysis of the individual fusion partners. To overcome this limitation, we used a two-stage protocol in which VSV-G-myc–containing vesicles and VSV-G*–containing vesicles were obtained in separate vesicle release incubations, and then combined to allow tethering and fusion during a second incubation. As shown in Fig. 2 A, both vesicle coisolation (open bars) and heterotrimer formation (shaded bars) displayed symmetric requirements for COPII-packaged cargo, because sar1 T39N fully inhibited tethering and fusion when included during either first-stage incubation. This rules out the possibility that our assays depend on fusion of COPII vesicles with ER fragments released from semi-intact cells. Furthermore, the functional requirement for COPII packaging occurs during the first stage only, because sar1 T39N addition during stage 2 is ineffective. A portion of the coisolation signal is obtained from vesicles held on ice during stage 2; this result is consistent with a tethered, unfused intermediate. Heterotrimer formation was completely inhibited on ice, as expected for membrane fusion.


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

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

Assembly involves symmetric populations of COPII vesicles but not Golgi. (A) Released VSV-G-myc vesicles and VSV-G* vesicles were prepared separately, semi-intact cells were removed, and the vesicles were combined for second-stage incubations. After the second incubation, vesicles were analyzed for coisolation and heterotrimer formation as in Fig. 1. Purified recombinant sar1 T39N was included at different points. (A) Error bars represent the SEM of duplicate determinations. (B) Single-stage coisolation assay was performed as in Fig. 1. The coisolated vesicles, as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo H and analyzed by autoradiography. R and S represent resistant and sensitive bands, respectively. (C) Single-stage coisolation and heterotrimer assays using CHO 15B cells. The α-SNAP L294A reaction contained 5 μM of recombinant protein. (D) Single-stage heterotrimer assay was performed as in C. Immunoprecipitated heterotrimers as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo D and analyzed by autoradiography.
© Copyright Policy
Related In: Results  -  Collection

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

fig2: Assembly involves symmetric populations of COPII vesicles but not Golgi. (A) Released VSV-G-myc vesicles and VSV-G* vesicles were prepared separately, semi-intact cells were removed, and the vesicles were combined for second-stage incubations. After the second incubation, vesicles were analyzed for coisolation and heterotrimer formation as in Fig. 1. Purified recombinant sar1 T39N was included at different points. (A) Error bars represent the SEM of duplicate determinations. (B) Single-stage coisolation assay was performed as in Fig. 1. The coisolated vesicles, as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo H and analyzed by autoradiography. R and S represent resistant and sensitive bands, respectively. (C) Single-stage coisolation and heterotrimer assays using CHO 15B cells. The α-SNAP L294A reaction contained 5 μM of recombinant protein. (D) Single-stage heterotrimer assay was performed as in C. Immunoprecipitated heterotrimers as well as samples of semi-intact cells from before and after the incubation were subjected to digestion with endo D and analyzed by autoradiography.
Mentions: The aforementioned experiments involved coincubation of both donor cell populations in one tube and preclude analysis of the individual fusion partners. To overcome this limitation, we used a two-stage protocol in which VSV-G-myc–containing vesicles and VSV-G*–containing vesicles were obtained in separate vesicle release incubations, and then combined to allow tethering and fusion during a second incubation. As shown in Fig. 2 A, both vesicle coisolation (open bars) and heterotrimer formation (shaded bars) displayed symmetric requirements for COPII-packaged cargo, because sar1 T39N fully inhibited tethering and fusion when included during either first-stage incubation. This rules out the possibility that our assays depend on fusion of COPII vesicles with ER fragments released from semi-intact cells. Furthermore, the functional requirement for COPII packaging occurs during the first stage only, because sar1 T39N addition during stage 2 is ineffective. A portion of the coisolation signal is obtained from vesicles held on ice during stage 2; this result is consistent with a tethered, unfused intermediate. Heterotrimer formation was completely inhibited on ice, as expected for membrane fusion.

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