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Maintenance of Golgi structure and function depends on the integrity of ER export.

Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J - J. Cell Biol. (2001)

Bottom Line: We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model.A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region.These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.

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Golgi components are actively recruited to ER exit sites in cells with disrupted ER–Golgi trafficking. Using NRK cells stably expressing p58–GFP or GRASP65–GFP, untreated cells were incubated on ice for 15 min to depolymerize microtubules. 1 μg/ml nocodazole was added to prevent motion of preGolgi intermediates and cells were imaged immediately. BFA-treated cells were incubated with 5 μg/ml BFA for 30 min and then prepared in the same way before imaging. Sar1[H79G]-expressing cells were transiently transfected with the mutant Sar1 construct 16 h before imaging. The marked area was photobleached and fluorescence recovery was subsequently monitored. The arrows point to puncta before bleaching and following recovery. Bars, 5 μm.
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fig7: Golgi components are actively recruited to ER exit sites in cells with disrupted ER–Golgi trafficking. Using NRK cells stably expressing p58–GFP or GRASP65–GFP, untreated cells were incubated on ice for 15 min to depolymerize microtubules. 1 μg/ml nocodazole was added to prevent motion of preGolgi intermediates and cells were imaged immediately. BFA-treated cells were incubated with 5 μg/ml BFA for 30 min and then prepared in the same way before imaging. Sar1[H79G]-expressing cells were transiently transfected with the mutant Sar1 construct 16 h before imaging. The marked area was photobleached and fluorescence recovery was subsequently monitored. The arrows point to puncta before bleaching and following recovery. Bars, 5 μm.

Mentions: The dynamic association of the COPII coat at ER exit sites has been interpreted as evidence for the constitutive function of COPII vesicles in ER export (Antonny et al., 2001). We therefore asked whether cargo proteins could be actively recruited into COPII-coated structures in BFA-treated or Sar1[H79G]-expressing cells. FRAP was used to assess whether p58–GFP, which colocalized with COPII-containing structures in BFA-treated or Sar1[H79G]-expressing cells (Figs. 4 B and 5), was stably associated with these membranes or was still undergoing flux. Fluorescence was found to rapidly recover in p58–GFP-containing ER exit sites after photobleaching in BFA-treated or Sar1[H79G]-expressing cells, similar to the rate of p58–GFP recovery after photobleaching these sites in untreated cells (Fig. 7). In these experiments, we depolymerized microtubules with nocodazole before photobleaching to prevent translocation of the peripheral structures containing p58–GFP. This allowed us to rule out the possibility that recovery occurred by the movement of unbleached peripheral structures into the FRAP box. Thereby, we were able to demonstrate that recovery occurred by the exchange of bleached p58–GFP molecules with unbleached p58 molecules within the surrounding ER. Thus, in BFA-treated or Sar1[H79G]-expressing cells, p58–GFP is constitutively cycling between the ER and ER exit sites.


Maintenance of Golgi structure and function depends on the integrity of ER export.

Ward TH, Polishchuk RS, Caplan S, Hirschberg K, Lippincott-Schwartz J - J. Cell Biol. (2001)

Golgi components are actively recruited to ER exit sites in cells with disrupted ER–Golgi trafficking. Using NRK cells stably expressing p58–GFP or GRASP65–GFP, untreated cells were incubated on ice for 15 min to depolymerize microtubules. 1 μg/ml nocodazole was added to prevent motion of preGolgi intermediates and cells were imaged immediately. BFA-treated cells were incubated with 5 μg/ml BFA for 30 min and then prepared in the same way before imaging. Sar1[H79G]-expressing cells were transiently transfected with the mutant Sar1 construct 16 h before imaging. The marked area was photobleached and fluorescence recovery was subsequently monitored. The arrows point to puncta before bleaching and following recovery. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2198855&req=5

fig7: Golgi components are actively recruited to ER exit sites in cells with disrupted ER–Golgi trafficking. Using NRK cells stably expressing p58–GFP or GRASP65–GFP, untreated cells were incubated on ice for 15 min to depolymerize microtubules. 1 μg/ml nocodazole was added to prevent motion of preGolgi intermediates and cells were imaged immediately. BFA-treated cells were incubated with 5 μg/ml BFA for 30 min and then prepared in the same way before imaging. Sar1[H79G]-expressing cells were transiently transfected with the mutant Sar1 construct 16 h before imaging. The marked area was photobleached and fluorescence recovery was subsequently monitored. The arrows point to puncta before bleaching and following recovery. Bars, 5 μm.
Mentions: The dynamic association of the COPII coat at ER exit sites has been interpreted as evidence for the constitutive function of COPII vesicles in ER export (Antonny et al., 2001). We therefore asked whether cargo proteins could be actively recruited into COPII-coated structures in BFA-treated or Sar1[H79G]-expressing cells. FRAP was used to assess whether p58–GFP, which colocalized with COPII-containing structures in BFA-treated or Sar1[H79G]-expressing cells (Figs. 4 B and 5), was stably associated with these membranes or was still undergoing flux. Fluorescence was found to rapidly recover in p58–GFP-containing ER exit sites after photobleaching in BFA-treated or Sar1[H79G]-expressing cells, similar to the rate of p58–GFP recovery after photobleaching these sites in untreated cells (Fig. 7). In these experiments, we depolymerized microtubules with nocodazole before photobleaching to prevent translocation of the peripheral structures containing p58–GFP. This allowed us to rule out the possibility that recovery occurred by the movement of unbleached peripheral structures into the FRAP box. Thereby, we were able to demonstrate that recovery occurred by the exchange of bleached p58–GFP molecules with unbleached p58 molecules within the surrounding ER. Thus, in BFA-treated or Sar1[H79G]-expressing cells, p58–GFP is constitutively cycling between the ER and ER exit sites.

Bottom Line: We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model.A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region.These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National institutes of Health, Bethesda, MD 20892, USA.

ABSTRACT
The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.

Show MeSH