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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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Depletion of ARF  does not change the rate of  transport. The rate of transport (independent of glycosylation) was assessed in the  medial (A), trans (B), and  TGN (C) assays in the presence of ARF (unfractionated  cytosol, open circles) or the  absence of ARF (closed circles) in two-stage assays, as  described in Materials and  Methods. Each point in B  and C represents the mean  value of two or five independent experiments, respectively, using 3–7.5-μl equivalents of cytosol per assay.  The maximum amounts of  3H incorporated into VSV-G  protein were 9,735, 11,682 ±  2,159, and 3,985 ± 980 cpm in  for the medial, trans, and  TGN assays, respectively.
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Figure 8: Depletion of ARF does not change the rate of transport. The rate of transport (independent of glycosylation) was assessed in the medial (A), trans (B), and TGN (C) assays in the presence of ARF (unfractionated cytosol, open circles) or the absence of ARF (closed circles) in two-stage assays, as described in Materials and Methods. Each point in B and C represents the mean value of two or five independent experiments, respectively, using 3–7.5-μl equivalents of cytosol per assay. The maximum amounts of 3H incorporated into VSV-G protein were 9,735, 11,682 ± 2,159, and 3,985 ± 980 cpm in for the medial, trans, and TGN assays, respectively.

Mentions: Since depletion of ARF not only blocks coated vesicle formation but increases the tendency for elongated tubules and fenestrae to form on cisternae, it is possible that the removal of ARF causes the mechanism of transport to shift from vesicular to nonphysiological uncoupled fusion of Golgi membranes, as others have suggested (18). In this case, it would be expected that the distinctive properties of each transport assay would converge on a common value that is characteristic of uncoupled fusion. We therefore examined the effect of removing ARF on the unique properties of each assay, the rate of transport and the sensitivity of the donor membranes to inactivation by NEM. As shown in Fig. 8, when the rate of transport was measured independently of the rate of glycosylation using a two-stage incubation, each assay retained its characteristic kinetics of transport upon depletion of ARF. In addition, the donor membranes in the medial assay remained significantly more sensitive to NEM pretreatment than the acceptor membranes even in the absence of ARF (Fig. 9 A). Moreover, the NEM sensitivity of the donor and acceptor membranes in the trans and TGN assays was also unchanged by removing ARF (Fig. 9, B and C). Thus, the distinctive properties of each assay remain unchanged upon ARF depletion, suggesting that there is no change in the mechanism of transport. It therefore seems likely that vesicles are not required for in vitro transport even when they are formed.


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

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

Depletion of ARF  does not change the rate of  transport. The rate of transport (independent of glycosylation) was assessed in the  medial (A), trans (B), and  TGN (C) assays in the presence of ARF (unfractionated  cytosol, open circles) or the  absence of ARF (closed circles) in two-stage assays, as  described in Materials and  Methods. Each point in B  and C represents the mean  value of two or five independent experiments, respectively, using 3–7.5-μl equivalents of cytosol per assay.  The maximum amounts of  3H incorporated into VSV-G  protein were 9,735, 11,682 ±  2,159, and 3,985 ± 980 cpm in  for the medial, trans, and  TGN assays, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2140162&req=5

Figure 8: Depletion of ARF does not change the rate of transport. The rate of transport (independent of glycosylation) was assessed in the medial (A), trans (B), and TGN (C) assays in the presence of ARF (unfractionated cytosol, open circles) or the absence of ARF (closed circles) in two-stage assays, as described in Materials and Methods. Each point in B and C represents the mean value of two or five independent experiments, respectively, using 3–7.5-μl equivalents of cytosol per assay. The maximum amounts of 3H incorporated into VSV-G protein were 9,735, 11,682 ± 2,159, and 3,985 ± 980 cpm in for the medial, trans, and TGN assays, respectively.
Mentions: Since depletion of ARF not only blocks coated vesicle formation but increases the tendency for elongated tubules and fenestrae to form on cisternae, it is possible that the removal of ARF causes the mechanism of transport to shift from vesicular to nonphysiological uncoupled fusion of Golgi membranes, as others have suggested (18). In this case, it would be expected that the distinctive properties of each transport assay would converge on a common value that is characteristic of uncoupled fusion. We therefore examined the effect of removing ARF on the unique properties of each assay, the rate of transport and the sensitivity of the donor membranes to inactivation by NEM. As shown in Fig. 8, when the rate of transport was measured independently of the rate of glycosylation using a two-stage incubation, each assay retained its characteristic kinetics of transport upon depletion of ARF. In addition, the donor membranes in the medial assay remained significantly more sensitive to NEM pretreatment than the acceptor membranes even in the absence of ARF (Fig. 9 A). Moreover, the NEM sensitivity of the donor and acceptor membranes in the trans and TGN assays was also unchanged by removing ARF (Fig. 9, B and C). Thus, the distinctive properties of each assay remain unchanged upon ARF depletion, suggesting that there is no change in the mechanism of transport. It therefore seems likely that vesicles are not required for in vitro transport even when they are formed.

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