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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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ARF-depleted cytosol fully supports transport  in all three in vitro assays.  Unfractionated bovine brain  cytosol (open circles), reconstituted cytosol (closed circles), and ARF-depleted cytosol (open squares) were  titrated into the medial (A),  trans (B), and TGN (C) assays. A cytosol equivalent is  the volume of sample equal  to 1 μl of unfractionated control cytosol after taking into  account volume changes after chromatography. The  protein concentration of control cytosol was 12.1 mg/ml.  Data are representative of  two independent ARF- depleted cytosol preparations.
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Figure 3: ARF-depleted cytosol fully supports transport in all three in vitro assays. Unfractionated bovine brain cytosol (open circles), reconstituted cytosol (closed circles), and ARF-depleted cytosol (open squares) were titrated into the medial (A), trans (B), and TGN (C) assays. A cytosol equivalent is the volume of sample equal to 1 μl of unfractionated control cytosol after taking into account volume changes after chromatography. The protein concentration of control cytosol was 12.1 mg/ml. Data are representative of two independent ARF- depleted cytosol preparations.

Mentions: Fig. 3 shows that reconstituted cytosol (recombination of ARF-depleted and ARF-containing pools; closed circles) is able to fully reconstitute transport compared with unfractionated cytosol (open circles) in all three assays. This demonstrates that transport factors were not lost or inactivated by the chromatographic procedure, although factor(s) that inhibit the glycosylation of VSV-G protein at high cytosol concentrations were frequently lost (compare unfractionated versus reconstituted cytosol). Significantly, ARF-depleted cytosol (open squares) is essentially indistinguishable from reconstituted cytosol in all three assays. The slightly greater volume of reconstituted and ARF-depleted cytosols needed to reach the plateau level of transport observed with unfractionated cytosol is likely to be due to the incomplete recovery of protein after chromatography and concentration. These data demonstrate that maximal transport can be achieved in the absence of ARF in all three assays. We conclude that ARF is not required to observe in vitro transport at any level within the Golgi stack.


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

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

ARF-depleted cytosol fully supports transport  in all three in vitro assays.  Unfractionated bovine brain  cytosol (open circles), reconstituted cytosol (closed circles), and ARF-depleted cytosol (open squares) were  titrated into the medial (A),  trans (B), and TGN (C) assays. A cytosol equivalent is  the volume of sample equal  to 1 μl of unfractionated control cytosol after taking into  account volume changes after chromatography. The  protein concentration of control cytosol was 12.1 mg/ml.  Data are representative of  two independent ARF- depleted cytosol preparations.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140162&req=5

Figure 3: ARF-depleted cytosol fully supports transport in all three in vitro assays. Unfractionated bovine brain cytosol (open circles), reconstituted cytosol (closed circles), and ARF-depleted cytosol (open squares) were titrated into the medial (A), trans (B), and TGN (C) assays. A cytosol equivalent is the volume of sample equal to 1 μl of unfractionated control cytosol after taking into account volume changes after chromatography. The protein concentration of control cytosol was 12.1 mg/ml. Data are representative of two independent ARF- depleted cytosol preparations.
Mentions: Fig. 3 shows that reconstituted cytosol (recombination of ARF-depleted and ARF-containing pools; closed circles) is able to fully reconstitute transport compared with unfractionated cytosol (open circles) in all three assays. This demonstrates that transport factors were not lost or inactivated by the chromatographic procedure, although factor(s) that inhibit the glycosylation of VSV-G protein at high cytosol concentrations were frequently lost (compare unfractionated versus reconstituted cytosol). Significantly, ARF-depleted cytosol (open squares) is essentially indistinguishable from reconstituted cytosol in all three assays. The slightly greater volume of reconstituted and ARF-depleted cytosols needed to reach the plateau level of transport observed with unfractionated cytosol is likely to be due to the incomplete recovery of protein after chromatography and concentration. These data demonstrate that maximal transport can be achieved in the absence of ARF in all three assays. We conclude that ARF is not required to observe in vitro transport at any level within the Golgi stack.

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