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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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Golgi-derived COPI vesicle fusion with pre-Golgi intermediates stimulates, but is not required for assembly. (A) Immunoblot before and after immunodepletion or mock immunodepletion of liver cytosol (left) and before and after 0.75 M KCl extraction of semi-intact NRK cells (right). (B) Single-stage vesicle coisolation (open bars) and heterotrimer (shaded bars) assays were performed using the indicated combinations of cells and cytosol. The double-headed arrow represents the 40% decrease specifically caused by deletion of COPI from the cytosol. (C) KCl-extracted VSV-G-myc–transfected NRK cells were incubated in the presence of regular-, mock-, or β-COP–depleted cytosol, or mock-depleted cytosol containing sar1 T39N, or 1.25 μM of purified 18C8. Released vesicles were immunoisolated using anti-myc antibodies and immunoblotted using anti–VSV-G and antigiantin antibodies. (D) ER-to-Golgi transport of VSV-G* monitored by endo H resistance in the presence or absence of 1.25 μM 18C8. Error bars in B and D represent the SEM of duplicate determinations.
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fig3: Golgi-derived COPI vesicle fusion with pre-Golgi intermediates stimulates, but is not required for assembly. (A) Immunoblot before and after immunodepletion or mock immunodepletion of liver cytosol (left) and before and after 0.75 M KCl extraction of semi-intact NRK cells (right). (B) Single-stage vesicle coisolation (open bars) and heterotrimer (shaded bars) assays were performed using the indicated combinations of cells and cytosol. The double-headed arrow represents the 40% decrease specifically caused by deletion of COPI from the cytosol. (C) KCl-extracted VSV-G-myc–transfected NRK cells were incubated in the presence of regular-, mock-, or β-COP–depleted cytosol, or mock-depleted cytosol containing sar1 T39N, or 1.25 μM of purified 18C8. Released vesicles were immunoisolated using anti-myc antibodies and immunoblotted using anti–VSV-G and antigiantin antibodies. (D) ER-to-Golgi transport of VSV-G* monitored by endo H resistance in the presence or absence of 1.25 μM 18C8. Error bars in B and D represent the SEM of duplicate determinations.

Mentions: The data presented so far are consistent with VTC formation occurring by homotypic fusion of COPII vesicles, but does not exclude involvement of other vesicles. Because only COPII and COPI vesicles are known to function in ER-to-Golgi transport, we wanted to investigate whether COPI vesicles contributed to in vitro VTC formation. Anti–β-COP antibody was used to deplete this essential COPI subunit from rat liver cytosol (Fig. 3 A, left lanes). Washed semi-intact cells contained significant β-COP that could be extracted completely with 0.75 M KCl (Fig. 3 A, right lanes). Thus, we were able to completely deplete our system of COPI and examine the effects on pre-Golgi vesicle events. Salt-stripped semi-intact cells were fully functional for vesicle coisolation in the presence of COPI-containing cytosol (Fig. 3 B, first two open bars). However, these cells were 37% less active in the heterotrimer assay (Fig. 3 B, first two shaded bars), indicating that additional components may have been damaged or extracted. Hence, 63% of the control value should be considered maximal for the heterotrimer assay with salt-stripped cells. When the salt-stripped cells were combined with COPI-depleted cytosol, the coisolation assay was marginally affected and the heterotrimer assay was 60% active relative to maximal for salt-stripped cells (Fig. 3 B, third and fourth bars vs. fifth and sixth bars). Thus, COPI-dependent processes do contribute to pre-Golgi fusion events, perhaps by provision of fusion machinery limiting on COPII vesicles. However, they are not an essential component of in vitro VTC formation.


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

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

Golgi-derived COPI vesicle fusion with pre-Golgi intermediates stimulates, but is not required for assembly. (A) Immunoblot before and after immunodepletion or mock immunodepletion of liver cytosol (left) and before and after 0.75 M KCl extraction of semi-intact NRK cells (right). (B) Single-stage vesicle coisolation (open bars) and heterotrimer (shaded bars) assays were performed using the indicated combinations of cells and cytosol. The double-headed arrow represents the 40% decrease specifically caused by deletion of COPI from the cytosol. (C) KCl-extracted VSV-G-myc–transfected NRK cells were incubated in the presence of regular-, mock-, or β-COP–depleted cytosol, or mock-depleted cytosol containing sar1 T39N, or 1.25 μM of purified 18C8. Released vesicles were immunoisolated using anti-myc antibodies and immunoblotted using anti–VSV-G and antigiantin antibodies. (D) ER-to-Golgi transport of VSV-G* monitored by endo H resistance in the presence or absence of 1.25 μM 18C8. Error bars in B and D represent the SEM of duplicate determinations.
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Related In: Results  -  Collection

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fig3: Golgi-derived COPI vesicle fusion with pre-Golgi intermediates stimulates, but is not required for assembly. (A) Immunoblot before and after immunodepletion or mock immunodepletion of liver cytosol (left) and before and after 0.75 M KCl extraction of semi-intact NRK cells (right). (B) Single-stage vesicle coisolation (open bars) and heterotrimer (shaded bars) assays were performed using the indicated combinations of cells and cytosol. The double-headed arrow represents the 40% decrease specifically caused by deletion of COPI from the cytosol. (C) KCl-extracted VSV-G-myc–transfected NRK cells were incubated in the presence of regular-, mock-, or β-COP–depleted cytosol, or mock-depleted cytosol containing sar1 T39N, or 1.25 μM of purified 18C8. Released vesicles were immunoisolated using anti-myc antibodies and immunoblotted using anti–VSV-G and antigiantin antibodies. (D) ER-to-Golgi transport of VSV-G* monitored by endo H resistance in the presence or absence of 1.25 μM 18C8. Error bars in B and D represent the SEM of duplicate determinations.
Mentions: The data presented so far are consistent with VTC formation occurring by homotypic fusion of COPII vesicles, but does not exclude involvement of other vesicles. Because only COPII and COPI vesicles are known to function in ER-to-Golgi transport, we wanted to investigate whether COPI vesicles contributed to in vitro VTC formation. Anti–β-COP antibody was used to deplete this essential COPI subunit from rat liver cytosol (Fig. 3 A, left lanes). Washed semi-intact cells contained significant β-COP that could be extracted completely with 0.75 M KCl (Fig. 3 A, right lanes). Thus, we were able to completely deplete our system of COPI and examine the effects on pre-Golgi vesicle events. Salt-stripped semi-intact cells were fully functional for vesicle coisolation in the presence of COPI-containing cytosol (Fig. 3 B, first two open bars). However, these cells were 37% less active in the heterotrimer assay (Fig. 3 B, first two shaded bars), indicating that additional components may have been damaged or extracted. Hence, 63% of the control value should be considered maximal for the heterotrimer assay with salt-stripped cells. When the salt-stripped cells were combined with COPI-depleted cytosol, the coisolation assay was marginally affected and the heterotrimer assay was 60% active relative to maximal for salt-stripped cells (Fig. 3 B, third and fourth bars vs. fifth and sixth bars). Thus, COPI-dependent processes do contribute to pre-Golgi fusion events, perhaps by provision of fusion machinery limiting on COPII vesicles. However, they are not an essential component of in vitro VTC formation.

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