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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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The constitutively inactive Sar1[T39N] mutant causes disruption of ER exit sites and redistribution of all Golgi components to the ER. (A) NRK cells stably expressing Sec13–YFP were transfected with Sar1[T39N]. They were fixed and then stained for either immunofluorescence or immunoEM with the HA epitope on the Sar1 construct. (B) NRK cells stably expressing GalT–CFP were cotransfected with Sar1[T39N] and either p58–YFP or GRASP65–GFP. 16 h after transfection, cells were either visualized directly or fixed and stained for endogenous GM130. Note that in control cells, i.e., those not expressing Sar1[T39N], staining for endogenous GM130 and transiently transfected GRASP65–GFP is restricted to the Golgi and has no ER background (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A, left) 5 μm; (A, right) 650 nm; (B) 5 μm.
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fig8: The constitutively inactive Sar1[T39N] mutant causes disruption of ER exit sites and redistribution of all Golgi components to the ER. (A) NRK cells stably expressing Sec13–YFP were transfected with Sar1[T39N]. They were fixed and then stained for either immunofluorescence or immunoEM with the HA epitope on the Sar1 construct. (B) NRK cells stably expressing GalT–CFP were cotransfected with Sar1[T39N] and either p58–YFP or GRASP65–GFP. 16 h after transfection, cells were either visualized directly or fixed and stained for endogenous GM130. Note that in control cells, i.e., those not expressing Sar1[T39N], staining for endogenous GM130 and transiently transfected GRASP65–GFP is restricted to the Golgi and has no ER background (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A, left) 5 μm; (A, right) 650 nm; (B) 5 μm.

Mentions: In cells expressing the Sar1[T39N] mutant protein, ER exit sites were no longer detectable by Sec13–YFP labeling, suggesting that COPII function was completely disrupted in these cells. Sec13–YFP was now diffusely localized throughout the ER, as was Sar1 (Fig. 8 A). The ER itself, visualized at the EM level using Sar1 immunogold labeling, was more abundant and enlarged, likely due to the buildup of secretory cargo that was unable to enter the secretory pathway in these cells (Bonfanti et al., 1998).


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)

The constitutively inactive Sar1[T39N] mutant causes disruption of ER exit sites and redistribution of all Golgi components to the ER. (A) NRK cells stably expressing Sec13–YFP were transfected with Sar1[T39N]. They were fixed and then stained for either immunofluorescence or immunoEM with the HA epitope on the Sar1 construct. (B) NRK cells stably expressing GalT–CFP were cotransfected with Sar1[T39N] and either p58–YFP or GRASP65–GFP. 16 h after transfection, cells were either visualized directly or fixed and stained for endogenous GM130. Note that in control cells, i.e., those not expressing Sar1[T39N], staining for endogenous GM130 and transiently transfected GRASP65–GFP is restricted to the Golgi and has no ER background (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A, left) 5 μm; (A, right) 650 nm; (B) 5 μm.
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Related In: Results  -  Collection

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fig8: The constitutively inactive Sar1[T39N] mutant causes disruption of ER exit sites and redistribution of all Golgi components to the ER. (A) NRK cells stably expressing Sec13–YFP were transfected with Sar1[T39N]. They were fixed and then stained for either immunofluorescence or immunoEM with the HA epitope on the Sar1 construct. (B) NRK cells stably expressing GalT–CFP were cotransfected with Sar1[T39N] and either p58–YFP or GRASP65–GFP. 16 h after transfection, cells were either visualized directly or fixed and stained for endogenous GM130. Note that in control cells, i.e., those not expressing Sar1[T39N], staining for endogenous GM130 and transiently transfected GRASP65–GFP is restricted to the Golgi and has no ER background (Fig. S2). Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb/200107045/DC1. Bars: (A, left) 5 μm; (A, right) 650 nm; (B) 5 μm.
Mentions: In cells expressing the Sar1[T39N] mutant protein, ER exit sites were no longer detectable by Sec13–YFP labeling, suggesting that COPII function was completely disrupted in these cells. Sec13–YFP was now diffusely localized throughout the ER, as was Sar1 (Fig. 8 A). The ER itself, visualized at the EM level using Sar1 immunogold labeling, was more abundant and enlarged, likely due to the buildup of secretory cargo that was unable to enter the secretory pathway in these cells (Bonfanti et al., 1998).

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