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Cell-free transport to distinct Golgi cisternae is compartment specific and ARF independent.

Happe S, Weidman P - J. Cell Biol. (1998)

Bottom Line: This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism.The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event.These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.

ABSTRACT
The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide-sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPgammaS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPgammaS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

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Golgi coated bud formation is blocked by ARF depletion and restored by purified ARF. Replicas of Golgi membranes  after a 15-min transport incubation were prepared as described in  Materials and Methods. The transport reaction mixtures contained (A) unfractionated bovine brain cytosol (2.4 mg/ml); (B)  ARF-depleted bovine brain cytosol (2.4 mg/ml); (C) ARF- depleted cytosol plus myr-rARF1 (120 μg/ml, 5.7% myristorylated); and (D) ARF-depleted cytosol plus non–myr-rARF1 (120  μg/ml). Boxed areas are presented at higher magnification in the  panels on the right side of the figure, illustrating the punctate surface coating on Golgi buds in A and C. Buds in B lack this punctate coating and have a granular texture similar to the surrounding tubules. Bars, 0.5 μm.
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Figure 6: Golgi coated bud formation is blocked by ARF depletion and restored by purified ARF. Replicas of Golgi membranes after a 15-min transport incubation were prepared as described in Materials and Methods. The transport reaction mixtures contained (A) unfractionated bovine brain cytosol (2.4 mg/ml); (B) ARF-depleted bovine brain cytosol (2.4 mg/ml); (C) ARF- depleted cytosol plus myr-rARF1 (120 μg/ml, 5.7% myristorylated); and (D) ARF-depleted cytosol plus non–myr-rARF1 (120 μg/ml). Boxed areas are presented at higher magnification in the panels on the right side of the figure, illustrating the punctate surface coating on Golgi buds in A and C. Buds in B lack this punctate coating and have a granular texture similar to the surrounding tubules. Bars, 0.5 μm.

Mentions: We have previously shown that ARF-depleted cytosol prepared by the method of Taylor et al. does not support coated bud and vesicle formation on Golgi membranes (56). Since we have modified that original procedure to retain additional factors required for transport to late Golgi compartments, we determined whether this ARF-depleted cytosol is also deficient in coated bud formation. High-resolution images from replicas of quick-frozen, freeze-dried Golgi membranes reveal that Golgi incubated in vitro with unfractionated cytosol (Fig. 6 A) or reconstituted cytosol (not shown) exhibit abundant buds and vesicles with a punctate surface coating. In contrast, Golgi incubated with ARF-depleted cytosol exhibit few buds or vesicles, and those few that are observed lack the distinctive punctate surface texture of coated buds (Fig. 6 B). As expected, addition of recombinant myr-rARF1 (Fig. 6 C), but not non– myr-rARF1 (Fig. 6 D), to ARF-depleted cytosol restores formation of coated buds on Golgi cisternae. Quantitative analysis demonstrates that coated bud and vesicle density is reduced 50% in incubations containing reconstituted cytosol (Fig. 7 A, black bars), which contains only half as much ARF as the unfractionated cytosol (Table II). Depletion of ARF reduced the coated bud and vesicle density ∼15-fold relative to unfractionated cytosol (Fig. 7 A, black bars), the same level normally observed with unincubated membranes (58). In contrast, there is a low density of uncoated buds and vesicles in all samples that is independent of the ARF concentration (Fig. 7 A, hatched bars). The tips of tubules extending from cisternae make up the majority of these uncoated buds (>85%). These are likely to have been present at the beginning of the incubation since they are also seen on unincubated membranes (56; not shown).


Cell-free transport to distinct Golgi cisternae is compartment specific and ARF independent.

Happe S, Weidman P - J. Cell Biol. (1998)

Golgi coated bud formation is blocked by ARF depletion and restored by purified ARF. Replicas of Golgi membranes  after a 15-min transport incubation were prepared as described in  Materials and Methods. The transport reaction mixtures contained (A) unfractionated bovine brain cytosol (2.4 mg/ml); (B)  ARF-depleted bovine brain cytosol (2.4 mg/ml); (C) ARF- depleted cytosol plus myr-rARF1 (120 μg/ml, 5.7% myristorylated); and (D) ARF-depleted cytosol plus non–myr-rARF1 (120  μg/ml). Boxed areas are presented at higher magnification in the  panels on the right side of the figure, illustrating the punctate surface coating on Golgi buds in A and C. Buds in B lack this punctate coating and have a granular texture similar to the surrounding tubules. Bars, 0.5 μm.
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Figure 6: Golgi coated bud formation is blocked by ARF depletion and restored by purified ARF. Replicas of Golgi membranes after a 15-min transport incubation were prepared as described in Materials and Methods. The transport reaction mixtures contained (A) unfractionated bovine brain cytosol (2.4 mg/ml); (B) ARF-depleted bovine brain cytosol (2.4 mg/ml); (C) ARF- depleted cytosol plus myr-rARF1 (120 μg/ml, 5.7% myristorylated); and (D) ARF-depleted cytosol plus non–myr-rARF1 (120 μg/ml). Boxed areas are presented at higher magnification in the panels on the right side of the figure, illustrating the punctate surface coating on Golgi buds in A and C. Buds in B lack this punctate coating and have a granular texture similar to the surrounding tubules. Bars, 0.5 μm.
Mentions: We have previously shown that ARF-depleted cytosol prepared by the method of Taylor et al. does not support coated bud and vesicle formation on Golgi membranes (56). Since we have modified that original procedure to retain additional factors required for transport to late Golgi compartments, we determined whether this ARF-depleted cytosol is also deficient in coated bud formation. High-resolution images from replicas of quick-frozen, freeze-dried Golgi membranes reveal that Golgi incubated in vitro with unfractionated cytosol (Fig. 6 A) or reconstituted cytosol (not shown) exhibit abundant buds and vesicles with a punctate surface coating. In contrast, Golgi incubated with ARF-depleted cytosol exhibit few buds or vesicles, and those few that are observed lack the distinctive punctate surface texture of coated buds (Fig. 6 B). As expected, addition of recombinant myr-rARF1 (Fig. 6 C), but not non– myr-rARF1 (Fig. 6 D), to ARF-depleted cytosol restores formation of coated buds on Golgi cisternae. Quantitative analysis demonstrates that coated bud and vesicle density is reduced 50% in incubations containing reconstituted cytosol (Fig. 7 A, black bars), which contains only half as much ARF as the unfractionated cytosol (Table II). Depletion of ARF reduced the coated bud and vesicle density ∼15-fold relative to unfractionated cytosol (Fig. 7 A, black bars), the same level normally observed with unincubated membranes (58). In contrast, there is a low density of uncoated buds and vesicles in all samples that is independent of the ARF concentration (Fig. 7 A, hatched bars). The tips of tubules extending from cisternae make up the majority of these uncoated buds (>85%). These are likely to have been present at the beginning of the incubation since they are also seen on unincubated membranes (56; not shown).

Bottom Line: This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism.The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event.These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.

ABSTRACT
The small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide-sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPgammaS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPgammaS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

Show MeSH
Related in: MedlinePlus