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ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

D'Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD, Peters PJ - J. Cell Biol. (1998)

Bottom Line: We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

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Model for the ARF6:GTP/GDP cycle in CHO cells.  Nucleotide exchange on ARF6 drives the transport and subsequent fusion of recycling vesicles with the plasma membrane. Hydrolysis of bound GTP at the cell surface allows the internalization of ARF6 via membrane vesicles inaccessible to HRP or the  release of ARF6 from the membrane into the cytosol. Cytosolic  ARF6 would have to be recruited back onto the recycling compartment (R.C.) for another nucleotide cycle to ensue.
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Figure 9: Model for the ARF6:GTP/GDP cycle in CHO cells. Nucleotide exchange on ARF6 drives the transport and subsequent fusion of recycling vesicles with the plasma membrane. Hydrolysis of bound GTP at the cell surface allows the internalization of ARF6 via membrane vesicles inaccessible to HRP or the release of ARF6 from the membrane into the cytosol. Cytosolic ARF6 would have to be recruited back onto the recycling compartment (R.C.) for another nucleotide cycle to ensue.

Mentions: Based on our observations, it is tempting to speculate how ARF6 could control membrane recycling via its nucleotide cycle (Fig. 9). We propose that nucleotide exchange on ARF6 drives the uncoating/transport and subsequent fusion of ARF6-positive intracellular recycling vesicles with the plasma membrane. At the surface, the hydrolysis of bound GTP would allow either (a) the internalization of membrane-bound ARF6 via the endocytic pathway or (b) the release of ARF6 from the membrane into the cytosol. In the latter case, cytosolic ARF6:GDP would have to be recruited onto the recycling endosomal compartment and with subsequent nucleotide exchange on ARF6, another cycle follows. Although we did not observe any colocalization of ARF6 with HRP loaded early endocytic structures, at this point we cannot rule out the possibility that ARF6 could indeed localize to incoming vesicles that are not accessible to HRP and that later on by the process of endosome fusion become HRP and Tfn-R positive.


ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

D'Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD, Peters PJ - J. Cell Biol. (1998)

Model for the ARF6:GTP/GDP cycle in CHO cells.  Nucleotide exchange on ARF6 drives the transport and subsequent fusion of recycling vesicles with the plasma membrane. Hydrolysis of bound GTP at the cell surface allows the internalization of ARF6 via membrane vesicles inaccessible to HRP or the  release of ARF6 from the membrane into the cytosol. Cytosolic  ARF6 would have to be recruited back onto the recycling compartment (R.C.) for another nucleotide cycle to ensue.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2140168&req=5

Figure 9: Model for the ARF6:GTP/GDP cycle in CHO cells. Nucleotide exchange on ARF6 drives the transport and subsequent fusion of recycling vesicles with the plasma membrane. Hydrolysis of bound GTP at the cell surface allows the internalization of ARF6 via membrane vesicles inaccessible to HRP or the release of ARF6 from the membrane into the cytosol. Cytosolic ARF6 would have to be recruited back onto the recycling compartment (R.C.) for another nucleotide cycle to ensue.
Mentions: Based on our observations, it is tempting to speculate how ARF6 could control membrane recycling via its nucleotide cycle (Fig. 9). We propose that nucleotide exchange on ARF6 drives the uncoating/transport and subsequent fusion of ARF6-positive intracellular recycling vesicles with the plasma membrane. At the surface, the hydrolysis of bound GTP would allow either (a) the internalization of membrane-bound ARF6 via the endocytic pathway or (b) the release of ARF6 from the membrane into the cytosol. In the latter case, cytosolic ARF6:GDP would have to be recruited onto the recycling endosomal compartment and with subsequent nucleotide exchange on ARF6, another cycle follows. Although we did not observe any colocalization of ARF6 with HRP loaded early endocytic structures, at this point we cannot rule out the possibility that ARF6 could indeed localize to incoming vesicles that are not accessible to HRP and that later on by the process of endosome fusion become HRP and Tfn-R positive.

Bottom Line: We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Show MeSH
Related in: MedlinePlus