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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 of pre-Golgi intermediates in vitro. Two populations of permeabilized cells were prepared containing ER-restricted VSV-G ts045: one was transfected with VSV-G-myc; the other was infected with VSV and pulse radiolabeled. After incubating the cells under various conditions, cells were removed by centrifugation at 15,000 g, and sedimentable vesicles in the supernatant were analyzed. (A) Release of vesicular VSV-G-myc (top, anti-myc immunoblot) and radioactive VSV-G* (bottom, autoradiogram) from separate cell populations. Purified sar1 proteins were added at 0.8–1.0 μM for this and subsequent experiments. Released vesicles were either directly sedimented at 100,000 g (top) or immunoisolated using anti-p24 antibody (bottom). (A–C) White lines indicate that intervening lanes have been spliced out. (B) Sedimentation analysis of VSV-G-myc* and VSV-G* present on released vesicles after coincubation of the two cell populations. Vesicles were solubilized with Triton X-100 before gradient. After sedimentation, gradient fractions were subjected to immunoprecipitation with anti-myc antibody and analyzed by autoradiography. In this and subsequent figures, “NRK” reactions contained nontransfected cells instead of VSV-G-myc* cells, but did contain regular VSV-G* cells. (C) Time course analysis of vesicle coisolation (top), heterotrimer formation (middle), and vesicle release (bottom) by autoradiography. 90*, centrifuged at 100,000 g before immunoisolation; 90 + TX, immunoisolation of vesicles followed by washing of immunobeads with Triton X-100; Sep., the two populations were incubated separately, and then combined before detergent solubilization. (D) Quantitation of the results from C. Vesicle release from semi-intact cells is expressed relative to the total cellular pool of VSV-G*. Coisolation and heterotrimer formation are expressed as a percentage of released vesicular pool.
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fig1: Assembly of pre-Golgi intermediates in vitro. Two populations of permeabilized cells were prepared containing ER-restricted VSV-G ts045: one was transfected with VSV-G-myc; the other was infected with VSV and pulse radiolabeled. After incubating the cells under various conditions, cells were removed by centrifugation at 15,000 g, and sedimentable vesicles in the supernatant were analyzed. (A) Release of vesicular VSV-G-myc (top, anti-myc immunoblot) and radioactive VSV-G* (bottom, autoradiogram) from separate cell populations. Purified sar1 proteins were added at 0.8–1.0 μM for this and subsequent experiments. Released vesicles were either directly sedimented at 100,000 g (top) or immunoisolated using anti-p24 antibody (bottom). (A–C) White lines indicate that intervening lanes have been spliced out. (B) Sedimentation analysis of VSV-G-myc* and VSV-G* present on released vesicles after coincubation of the two cell populations. Vesicles were solubilized with Triton X-100 before gradient. After sedimentation, gradient fractions were subjected to immunoprecipitation with anti-myc antibody and analyzed by autoradiography. In this and subsequent figures, “NRK” reactions contained nontransfected cells instead of VSV-G-myc* cells, but did contain regular VSV-G* cells. (C) Time course analysis of vesicle coisolation (top), heterotrimer formation (middle), and vesicle release (bottom) by autoradiography. 90*, centrifuged at 100,000 g before immunoisolation; 90 + TX, immunoisolation of vesicles followed by washing of immunobeads with Triton X-100; Sep., the two populations were incubated separately, and then combined before detergent solubilization. (D) Quantitation of the results from C. Vesicle release from semi-intact cells is expressed relative to the total cellular pool of VSV-G*. Coisolation and heterotrimer formation are expressed as a percentage of released vesicular pool.

Mentions: ER-to-Golgi transport is often studied in permeabilized mammalian cells (Rowe et al., 1998; Williams et al., 2004). Although a large majority of the model cargo, vesicular stomatitis virus glycoprotein (VSV-G) ts045, is transported to the Golgi under these conditions, the fusion events preceding arrival in the Golgi cannot be monitored. However, a portion of the COPII vesicles are released into the extracellular buffer during transport incubations (Joglekar et al., 2003), and these extracellular vesicles could potentially be monitored as they undergo pre-Golgi fusion events that would normally occur en route to the Golgi. Fig. 1 A demonstrates that this wayward vesicle pool carries 1.3% of epitope-tagged VSV-G-myc and is released from transfected semi-intact normal rat kidney (NRK) cells in a temperature-, energy-, cytosol-, and sar1-dependent process reflecting budding of COPII vesicles from the ER. A similar fraction of non–myc-tagged, radioactive VSV-G (VSV-G*) is released from another population of NRK cells that were infected with VSV and pulse radiolabeled. At the onset of the reaction, all VSV-G-myc and VSV-G* are present in the ER, because high-level expression of the protein (VSV-G-myc) or pulse labeling (VSV-G*) is accomplished at 40°C, a temperature at which VSV-G ts045 is retained in the ER. The initial ER restriction of the cargo, together with the inhibition by GDP-locked sar1 T39N and stimulation by wild-type sar1 indicates that a large majority of the released VSV-G is present in COPII vesicles. This population of vesicles contains four ER–Golgi SNAREs (Xu et al., 2000; Joglekar et al., 2003), as well as the vesicle marker p24 (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200408135/DC1). The cytoplasmically disposed myc epitope of VSV-G-myc allows immunoisolation of cargo-loaded vesicles with ∼25% efficiency (Fig. S1 B).


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

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

Assembly of pre-Golgi intermediates in vitro. Two populations of permeabilized cells were prepared containing ER-restricted VSV-G ts045: one was transfected with VSV-G-myc; the other was infected with VSV and pulse radiolabeled. After incubating the cells under various conditions, cells were removed by centrifugation at 15,000 g, and sedimentable vesicles in the supernatant were analyzed. (A) Release of vesicular VSV-G-myc (top, anti-myc immunoblot) and radioactive VSV-G* (bottom, autoradiogram) from separate cell populations. Purified sar1 proteins were added at 0.8–1.0 μM for this and subsequent experiments. Released vesicles were either directly sedimented at 100,000 g (top) or immunoisolated using anti-p24 antibody (bottom). (A–C) White lines indicate that intervening lanes have been spliced out. (B) Sedimentation analysis of VSV-G-myc* and VSV-G* present on released vesicles after coincubation of the two cell populations. Vesicles were solubilized with Triton X-100 before gradient. After sedimentation, gradient fractions were subjected to immunoprecipitation with anti-myc antibody and analyzed by autoradiography. In this and subsequent figures, “NRK” reactions contained nontransfected cells instead of VSV-G-myc* cells, but did contain regular VSV-G* cells. (C) Time course analysis of vesicle coisolation (top), heterotrimer formation (middle), and vesicle release (bottom) by autoradiography. 90*, centrifuged at 100,000 g before immunoisolation; 90 + TX, immunoisolation of vesicles followed by washing of immunobeads with Triton X-100; Sep., the two populations were incubated separately, and then combined before detergent solubilization. (D) Quantitation of the results from C. Vesicle release from semi-intact cells is expressed relative to the total cellular pool of VSV-G*. Coisolation and heterotrimer formation are expressed as a percentage of released vesicular pool.
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Related In: Results  -  Collection

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fig1: Assembly of pre-Golgi intermediates in vitro. Two populations of permeabilized cells were prepared containing ER-restricted VSV-G ts045: one was transfected with VSV-G-myc; the other was infected with VSV and pulse radiolabeled. After incubating the cells under various conditions, cells were removed by centrifugation at 15,000 g, and sedimentable vesicles in the supernatant were analyzed. (A) Release of vesicular VSV-G-myc (top, anti-myc immunoblot) and radioactive VSV-G* (bottom, autoradiogram) from separate cell populations. Purified sar1 proteins were added at 0.8–1.0 μM for this and subsequent experiments. Released vesicles were either directly sedimented at 100,000 g (top) or immunoisolated using anti-p24 antibody (bottom). (A–C) White lines indicate that intervening lanes have been spliced out. (B) Sedimentation analysis of VSV-G-myc* and VSV-G* present on released vesicles after coincubation of the two cell populations. Vesicles were solubilized with Triton X-100 before gradient. After sedimentation, gradient fractions were subjected to immunoprecipitation with anti-myc antibody and analyzed by autoradiography. In this and subsequent figures, “NRK” reactions contained nontransfected cells instead of VSV-G-myc* cells, but did contain regular VSV-G* cells. (C) Time course analysis of vesicle coisolation (top), heterotrimer formation (middle), and vesicle release (bottom) by autoradiography. 90*, centrifuged at 100,000 g before immunoisolation; 90 + TX, immunoisolation of vesicles followed by washing of immunobeads with Triton X-100; Sep., the two populations were incubated separately, and then combined before detergent solubilization. (D) Quantitation of the results from C. Vesicle release from semi-intact cells is expressed relative to the total cellular pool of VSV-G*. Coisolation and heterotrimer formation are expressed as a percentage of released vesicular pool.
Mentions: ER-to-Golgi transport is often studied in permeabilized mammalian cells (Rowe et al., 1998; Williams et al., 2004). Although a large majority of the model cargo, vesicular stomatitis virus glycoprotein (VSV-G) ts045, is transported to the Golgi under these conditions, the fusion events preceding arrival in the Golgi cannot be monitored. However, a portion of the COPII vesicles are released into the extracellular buffer during transport incubations (Joglekar et al., 2003), and these extracellular vesicles could potentially be monitored as they undergo pre-Golgi fusion events that would normally occur en route to the Golgi. Fig. 1 A demonstrates that this wayward vesicle pool carries 1.3% of epitope-tagged VSV-G-myc and is released from transfected semi-intact normal rat kidney (NRK) cells in a temperature-, energy-, cytosol-, and sar1-dependent process reflecting budding of COPII vesicles from the ER. A similar fraction of non–myc-tagged, radioactive VSV-G (VSV-G*) is released from another population of NRK cells that were infected with VSV and pulse radiolabeled. At the onset of the reaction, all VSV-G-myc and VSV-G* are present in the ER, because high-level expression of the protein (VSV-G-myc) or pulse labeling (VSV-G*) is accomplished at 40°C, a temperature at which VSV-G ts045 is retained in the ER. The initial ER restriction of the cargo, together with the inhibition by GDP-locked sar1 T39N and stimulation by wild-type sar1 indicates that a large majority of the released VSV-G is present in COPII vesicles. This population of vesicles contains four ER–Golgi SNAREs (Xu et al., 2000; Joglekar et al., 2003), as well as the vesicle marker p24 (Fig. S1 A, available at http://www.jcb.org/cgi/content/full/jcb.200408135/DC1). The cytoplasmically disposed myc epitope of VSV-G-myc allows immunoisolation of cargo-loaded vesicles with ∼25% efficiency (Fig. S1 B).

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