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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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Recycling of Tac  from the tubular compartment back out to the PM is  blocked in cells expressing  the GTP-binding–defective  ARF6/T27N mutant. HeLa  cells transfected with Tac and  ARF6/T27N plasmids were  incubated with anti-Tac antibodies 4°C, and were then either fixed (4 °C Binding) or  warmed to 37°C for 30 min.  These cells were then chilled,  rinsed with low pH buffer,  and either fixed (37°C Uptake) to assess the internalization of Tac antibody or  warmed again to 37°C for 30  min before fixation, and surface reappearance of Tac antibody was assessed as in Fig.  7. Note that the anti-Tac antibodies accumulated in the  tubular compartment in the  absence of CD in cells expressing ARF6/T27N.
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Figure 9: Recycling of Tac from the tubular compartment back out to the PM is blocked in cells expressing the GTP-binding–defective ARF6/T27N mutant. HeLa cells transfected with Tac and ARF6/T27N plasmids were incubated with anti-Tac antibodies 4°C, and were then either fixed (4 °C Binding) or warmed to 37°C for 30 min. These cells were then chilled, rinsed with low pH buffer, and either fixed (37°C Uptake) to assess the internalization of Tac antibody or warmed again to 37°C for 30 min before fixation, and surface reappearance of Tac antibody was assessed as in Fig. 7. Note that the anti-Tac antibodies accumulated in the tubular compartment in the absence of CD in cells expressing ARF6/T27N.

Mentions: We next examined both the internalization and recycling of Tac antibody in cells cotransfected with plasmids encoding Tac and ARF6/T27N. After binding at 4°C, Tac antibody localized to the surface of cells and did not colocalize with ARF6/T27N (Fig. 9, 4°C Binding). Cells were then incubated at 37°C in the absence of CD, followed by low pH removal of remaining surface antibody to assess the internalized Tac antibody. The Tac antibody accumulated in the tubular compartment, where it colocalized in part with ARF6/T27N (Fig. 9, Uptake). After the low pH wash, cells that had taken up the Tac antibody were then further incubated at 37°C to allow recycling, and surface reappearance of Tac antibody was determined as described above (Fig. 7). The internalized Tac antibody did not appear on the PM in these cells (Fig. 7, Surface Reappearance). This indicates that expression of the inactive ARF6/T27N mutant (ARF6-GDP) alone, in the absence of cytochalasin treatment, blocks the recycling of internalized Tac from the tubular compartment back to the PM, suggesting that activation of ARF6 is required for exit from this compartment.


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

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

Recycling of Tac  from the tubular compartment back out to the PM is  blocked in cells expressing  the GTP-binding–defective  ARF6/T27N mutant. HeLa  cells transfected with Tac and  ARF6/T27N plasmids were  incubated with anti-Tac antibodies 4°C, and were then either fixed (4 °C Binding) or  warmed to 37°C for 30 min.  These cells were then chilled,  rinsed with low pH buffer,  and either fixed (37°C Uptake) to assess the internalization of Tac antibody or  warmed again to 37°C for 30  min before fixation, and surface reappearance of Tac antibody was assessed as in Fig.  7. Note that the anti-Tac antibodies accumulated in the  tubular compartment in the  absence of CD in cells expressing ARF6/T27N.
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Figure 9: Recycling of Tac from the tubular compartment back out to the PM is blocked in cells expressing the GTP-binding–defective ARF6/T27N mutant. HeLa cells transfected with Tac and ARF6/T27N plasmids were incubated with anti-Tac antibodies 4°C, and were then either fixed (4 °C Binding) or warmed to 37°C for 30 min. These cells were then chilled, rinsed with low pH buffer, and either fixed (37°C Uptake) to assess the internalization of Tac antibody or warmed again to 37°C for 30 min before fixation, and surface reappearance of Tac antibody was assessed as in Fig. 7. Note that the anti-Tac antibodies accumulated in the tubular compartment in the absence of CD in cells expressing ARF6/T27N.
Mentions: We next examined both the internalization and recycling of Tac antibody in cells cotransfected with plasmids encoding Tac and ARF6/T27N. After binding at 4°C, Tac antibody localized to the surface of cells and did not colocalize with ARF6/T27N (Fig. 9, 4°C Binding). Cells were then incubated at 37°C in the absence of CD, followed by low pH removal of remaining surface antibody to assess the internalized Tac antibody. The Tac antibody accumulated in the tubular compartment, where it colocalized in part with ARF6/T27N (Fig. 9, Uptake). After the low pH wash, cells that had taken up the Tac antibody were then further incubated at 37°C to allow recycling, and surface reappearance of Tac antibody was determined as described above (Fig. 7). The internalized Tac antibody did not appear on the PM in these cells (Fig. 7, Surface Reappearance). This indicates that expression of the inactive ARF6/T27N mutant (ARF6-GDP) alone, in the absence of cytochalasin treatment, blocks the recycling of internalized Tac from the tubular compartment back to the PM, suggesting that activation of ARF6 is required for exit from this compartment.

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