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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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Constitutively inactive Arf1 or constitutively active Sar1 mutants that disrupt ER–Golgi trafficking also cause the relocation of Golgi components to ER exit sites. NRK cells stably expressing Sec13–YFP were either transfected with Arf1[T31N] or Sar1[H79G] alone and then fixed and stained for GM130, or were cotransfected with the GTPase mutant together with p58–CFP or GalT–CFP and then visualized directly. Arrows indicate colocalizing puncta. Bars, 5 μm.
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fig5: Constitutively inactive Arf1 or constitutively active Sar1 mutants that disrupt ER–Golgi trafficking also cause the relocation of Golgi components to ER exit sites. NRK cells stably expressing Sec13–YFP were either transfected with Arf1[T31N] or Sar1[H79G] alone and then fixed and stained for GM130, or were cotransfected with the GTPase mutant together with p58–CFP or GalT–CFP and then visualized directly. Arrows indicate colocalizing puncta. Bars, 5 μm.

Mentions: We next examined how the steady-state association of Golgi proteins was affected by expression of the dominant inactive mutant of Arf1, Arf1[T31N]. In Arf1[T31N]-expressing cells, Arf1 is unable to exchange GDP for GTP, an effect similar to that of BFA on Arf1 (Klausner et al., 1992; Dascher and Balch, 1994). Since activated Arf1 on Golgi membranes is required for the recruitment of the COPI coat (Donaldson et al., 1992), which is necessary for Golgi function, Golgi organization is profoundly affected in cells expressing this mutant (Dascher and Balch, 1994). We found that Golgi structures disappeared in cells expressing Arf1[T31N], with Golgi components redistributing in a pattern that was similar to the observed effect in BFA-treated cells: Golgi enzymes were localized to the ER, COPI was released into the cytosol (unpublished data), and p58 and matrix proteins were found in punctate peripheral structures (Fig. 5). Importantly, the peripheral structures containing p58 and Golgi matrix proteins were found to colocalize with Sec13-labeled ER exit sites (Fig. 5, arrows), whose distribution was unaffected by Arf1[T31N] expression.


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)

Constitutively inactive Arf1 or constitutively active Sar1 mutants that disrupt ER–Golgi trafficking also cause the relocation of Golgi components to ER exit sites. NRK cells stably expressing Sec13–YFP were either transfected with Arf1[T31N] or Sar1[H79G] alone and then fixed and stained for GM130, or were cotransfected with the GTPase mutant together with p58–CFP or GalT–CFP and then visualized directly. Arrows indicate colocalizing puncta. Bars, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2198855&req=5

fig5: Constitutively inactive Arf1 or constitutively active Sar1 mutants that disrupt ER–Golgi trafficking also cause the relocation of Golgi components to ER exit sites. NRK cells stably expressing Sec13–YFP were either transfected with Arf1[T31N] or Sar1[H79G] alone and then fixed and stained for GM130, or were cotransfected with the GTPase mutant together with p58–CFP or GalT–CFP and then visualized directly. Arrows indicate colocalizing puncta. Bars, 5 μm.
Mentions: We next examined how the steady-state association of Golgi proteins was affected by expression of the dominant inactive mutant of Arf1, Arf1[T31N]. In Arf1[T31N]-expressing cells, Arf1 is unable to exchange GDP for GTP, an effect similar to that of BFA on Arf1 (Klausner et al., 1992; Dascher and Balch, 1994). Since activated Arf1 on Golgi membranes is required for the recruitment of the COPI coat (Donaldson et al., 1992), which is necessary for Golgi function, Golgi organization is profoundly affected in cells expressing this mutant (Dascher and Balch, 1994). We found that Golgi structures disappeared in cells expressing Arf1[T31N], with Golgi components redistributing in a pattern that was similar to the observed effect in BFA-treated cells: Golgi enzymes were localized to the ER, COPI was released into the cytosol (unpublished data), and p58 and matrix proteins were found in punctate peripheral structures (Fig. 5). Importantly, the peripheral structures containing p58 and Golgi matrix proteins were found to colocalize with Sec13-labeled ER exit sites (Fig. 5, arrows), whose distribution was unaffected by Arf1[T31N] expression.

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