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ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway.

Radhakrishna H, Donaldson JG - J. Cell Biol. (1997)

Bottom Line: Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively.Thus, the ARF6 GTP cycle regulates this membrane traffic pathway.The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor alpha subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

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The PM protein Tac colocalizes with ARF6 in the tubular compartment during CD treatment and in surface protrusions  during AlF treatment. HeLa cells transfected with plasmids encoding wild-type ARF6 and the PM protein Tac (IL-2 receptor α  subunit) were incubated in the absence (Unt) or presence of 1  μM CD (CD) or AlF (AlF) for 30 min, and they were fixed and  processed for indirect immunofluorescence localization of ARF6  with rabbit polyclonal anti-ARF6 antibodies and of Tac with  monoclonal mouse anti-Tac antibodies, followed by the appropriate secondary antibodies.
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Figure 3: The PM protein Tac colocalizes with ARF6 in the tubular compartment during CD treatment and in surface protrusions during AlF treatment. HeLa cells transfected with plasmids encoding wild-type ARF6 and the PM protein Tac (IL-2 receptor α subunit) were incubated in the absence (Unt) or presence of 1 μM CD (CD) or AlF (AlF) for 30 min, and they were fixed and processed for indirect immunofluorescence localization of ARF6 with rabbit polyclonal anti-ARF6 antibodies and of Tac with monoclonal mouse anti-Tac antibodies, followed by the appropriate secondary antibodies.

Mentions: We used Tac, the IL-2 receptor α subunit, as a PM marker. Tac has been thoroughly studied and used as a generic integral PM protein with no retention or targeting information that moves through the secretory pathway to the PM (Leonard et al., 1984; Weissman et al., 1986; Bonifacino et al., 1990; Subtil et al., 1997). We cotransfected HeLa cells with plasmids encoding Tac and wild-type ARF6, and determined the distributions of the proteins after incubation in the presence or absence of CD or AlF. Tac and ARF6 showed extensive colocalization at the plasma membrane and with an internal, juxtanuclear compartment in untreated cells (Fig. 3). Whereas CD treatment of cells shifted the localization of Tac and ARF6 to the tubular compartment, addition of AlF (obtained by adding 30 mM NaF and 50 μM AlCl3) resulted in colocalization of ARF6 and Tac in surface protrusions (Fig. 3). In HeLa cells coexpressing ARF6 and a Tac chimeric protein containing the cytoplasmic tail of HLA-DM, a sequence specifying clathrin-coated pit localization (Marks et al., 1995), the chimeric protein did not codistribute with ARF6 in either surface protrusions or the tubular compartment (not shown). These results demonstrate that PM proteins lacking known signals for clathrin-coated pit localization colocalize with ARF6 in the tubular compartment of HeLa cells during CD treatment.


ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway.

Radhakrishna H, Donaldson JG - J. Cell Biol. (1997)

The PM protein Tac colocalizes with ARF6 in the tubular compartment during CD treatment and in surface protrusions  during AlF treatment. HeLa cells transfected with plasmids encoding wild-type ARF6 and the PM protein Tac (IL-2 receptor α  subunit) were incubated in the absence (Unt) or presence of 1  μM CD (CD) or AlF (AlF) for 30 min, and they were fixed and  processed for indirect immunofluorescence localization of ARF6  with rabbit polyclonal anti-ARF6 antibodies and of Tac with  monoclonal mouse anti-Tac antibodies, followed by the appropriate secondary antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139810&req=5

Figure 3: The PM protein Tac colocalizes with ARF6 in the tubular compartment during CD treatment and in surface protrusions during AlF treatment. HeLa cells transfected with plasmids encoding wild-type ARF6 and the PM protein Tac (IL-2 receptor α subunit) were incubated in the absence (Unt) or presence of 1 μM CD (CD) or AlF (AlF) for 30 min, and they were fixed and processed for indirect immunofluorescence localization of ARF6 with rabbit polyclonal anti-ARF6 antibodies and of Tac with monoclonal mouse anti-Tac antibodies, followed by the appropriate secondary antibodies.
Mentions: We used Tac, the IL-2 receptor α subunit, as a PM marker. Tac has been thoroughly studied and used as a generic integral PM protein with no retention or targeting information that moves through the secretory pathway to the PM (Leonard et al., 1984; Weissman et al., 1986; Bonifacino et al., 1990; Subtil et al., 1997). We cotransfected HeLa cells with plasmids encoding Tac and wild-type ARF6, and determined the distributions of the proteins after incubation in the presence or absence of CD or AlF. Tac and ARF6 showed extensive colocalization at the plasma membrane and with an internal, juxtanuclear compartment in untreated cells (Fig. 3). Whereas CD treatment of cells shifted the localization of Tac and ARF6 to the tubular compartment, addition of AlF (obtained by adding 30 mM NaF and 50 μM AlCl3) resulted in colocalization of ARF6 and Tac in surface protrusions (Fig. 3). In HeLa cells coexpressing ARF6 and a Tac chimeric protein containing the cytoplasmic tail of HLA-DM, a sequence specifying clathrin-coated pit localization (Marks et al., 1995), the chimeric protein did not codistribute with ARF6 in either surface protrusions or the tubular compartment (not shown). These results demonstrate that PM proteins lacking known signals for clathrin-coated pit localization colocalize with ARF6 in the tubular compartment of HeLa cells during CD treatment.

Bottom Line: Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively.Thus, the ARF6 GTP cycle regulates this membrane traffic pathway.The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, Bethesda, Maryland 20892, USA.

ABSTRACT
ADP-ribosylation factor (ARF) 6 localizes to the plasma membrane (PM) in its GTP state and to a tubulovesicular compartment in its GDP state in HeLa cells that express wild-type or mutant forms of this GTPase. Aluminum fluoride (AlF) treatment of ARF6-transfected cells redistributes ARF6 to the PM and stimulates the formation of actin-rich surface protrusions. Here we show that cytochalasin D (CD) treatment inhibited formation of the AlF-induced protrusions and shifted the distribution of ARF6 to a tubular membrane compartment emanating from the juxtanuclear region of cells, which resembled the compartment where the GTP-binding defective mutant of ARF6 localized. This membrane compartment was distinct from transferrin-positive endosomes, could be detected in the absence of ARF6 overexpression or CD treatment, and was accessible to loading by PM proteins lacking clathrin/AP-2 cytoplasmic targeting sequences, such as the IL-2 receptor alpha subunit Tac. ARF6 and surface Tac moved into this compartment and back out to the PM in the absence of pharmacologic treatment. Whereas AlF treatment blocked internalization, CD treatment blocked the recycling of wild-type ARF6 and Tac back to the PM; these blocks were mimicked by expression of ARF6 mutants Q67L and T27N, which were predicted to be in either the GTP- or GDP-bound state, respectively. Thus, the ARF6 GTP cycle regulates this membrane traffic pathway. The delivery of ARF6 and membrane to defined sites along the PM may provide components necessary for remodeling the cell surface and the underlying actin cytoskeleton.

Show MeSH
Related in: MedlinePlus