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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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Low pH buffer removes surface-bound Tac antibodies,  but not the internalized Tac antibodies in the tubular compartment. Tac- and ARF6-transfected HeLa cells were chilled to 4°C  and incubated with mouse anti-Tac antibodies for 30 min. One  set of cells were fixed at 4°C either before (4 °C Total) or after  (4 °C Internal) rinsing with low pH buffer (0.5% acetic acid, 0.5 M  NaCl, pH 3.0). Another set of cells were warmed to 37°C for 30  min in the presence of 1 μM CD and then fixed before (37 °C +  CD Total) or after (37 °C +CD Internal) rinsing with low pH  buffer. Fixed cells were then processed for indirect immunofluorescence.
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Figure 6: Low pH buffer removes surface-bound Tac antibodies, but not the internalized Tac antibodies in the tubular compartment. Tac- and ARF6-transfected HeLa cells were chilled to 4°C and incubated with mouse anti-Tac antibodies for 30 min. One set of cells were fixed at 4°C either before (4 °C Total) or after (4 °C Internal) rinsing with low pH buffer (0.5% acetic acid, 0.5 M NaCl, pH 3.0). Another set of cells were warmed to 37°C for 30 min in the presence of 1 μM CD and then fixed before (37 °C + CD Total) or after (37 °C +CD Internal) rinsing with low pH buffer. Fixed cells were then processed for indirect immunofluorescence.

Mentions: To determine the efficiency of removing surface-bound antibody with low pH buffer, cells expressing ARF6 and Tac were incubated with Tac antibody at 4°C to label surface Tac. Some cells were warmed to 37°C to allow internalization in the presence of CD. The cells were then either fixed immediately (Fig. 6, Total) or rinsed briefly with low pH buffer (0.5% acetic acid, 0.5 M NaCl, pH 3.0; Fig.6, Internal) before fixation and immunolabeling for ARF6 and Tac antibody. The Tac antibody labeled only the cell surface at 4°C, whereas ARF6 localized both to the PM and to internal structures (Fig. 6, 4°C Total). Rinsing with low pH buffer completely removed the surface-bound Tac antibody, but did not alter the distribution of ARF6 (Fig. 6, Internal). Incubation of cells at 37°C for 30 min in the presence of CD resulted in the internalization of Tac antibody into the tubular compartment (Fig. 6, 37 °C +CD Total). Washing with low pH buffer before fixation removed the surface Tac antibody, but did not remove the internalized Tac antibody that colocalized with ARF6 in the tubular compartment (Fig. 6, 37 °C +CD Internal). The inability of the low pH wash to remove Tac antibody lends additional support to the argument that this compartment is internal and not continuous with the PM. The tubular compartment had a beaded appearance possibly because of the low pH wash. Nevertheless, ARF6 and Tac remained colocalized in this compartment.


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

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

Low pH buffer removes surface-bound Tac antibodies,  but not the internalized Tac antibodies in the tubular compartment. Tac- and ARF6-transfected HeLa cells were chilled to 4°C  and incubated with mouse anti-Tac antibodies for 30 min. One  set of cells were fixed at 4°C either before (4 °C Total) or after  (4 °C Internal) rinsing with low pH buffer (0.5% acetic acid, 0.5 M  NaCl, pH 3.0). Another set of cells were warmed to 37°C for 30  min in the presence of 1 μM CD and then fixed before (37 °C +  CD Total) or after (37 °C +CD Internal) rinsing with low pH  buffer. Fixed cells were then processed for indirect immunofluorescence.
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Related In: Results  -  Collection

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

Figure 6: Low pH buffer removes surface-bound Tac antibodies, but not the internalized Tac antibodies in the tubular compartment. Tac- and ARF6-transfected HeLa cells were chilled to 4°C and incubated with mouse anti-Tac antibodies for 30 min. One set of cells were fixed at 4°C either before (4 °C Total) or after (4 °C Internal) rinsing with low pH buffer (0.5% acetic acid, 0.5 M NaCl, pH 3.0). Another set of cells were warmed to 37°C for 30 min in the presence of 1 μM CD and then fixed before (37 °C + CD Total) or after (37 °C +CD Internal) rinsing with low pH buffer. Fixed cells were then processed for indirect immunofluorescence.
Mentions: To determine the efficiency of removing surface-bound antibody with low pH buffer, cells expressing ARF6 and Tac were incubated with Tac antibody at 4°C to label surface Tac. Some cells were warmed to 37°C to allow internalization in the presence of CD. The cells were then either fixed immediately (Fig. 6, Total) or rinsed briefly with low pH buffer (0.5% acetic acid, 0.5 M NaCl, pH 3.0; Fig.6, Internal) before fixation and immunolabeling for ARF6 and Tac antibody. The Tac antibody labeled only the cell surface at 4°C, whereas ARF6 localized both to the PM and to internal structures (Fig. 6, 4°C Total). Rinsing with low pH buffer completely removed the surface-bound Tac antibody, but did not alter the distribution of ARF6 (Fig. 6, Internal). Incubation of cells at 37°C for 30 min in the presence of CD resulted in the internalization of Tac antibody into the tubular compartment (Fig. 6, 37 °C +CD Total). Washing with low pH buffer before fixation removed the surface Tac antibody, but did not remove the internalized Tac antibody that colocalized with ARF6 in the tubular compartment (Fig. 6, 37 °C +CD Internal). The inability of the low pH wash to remove Tac antibody lends additional support to the argument that this compartment is internal and not continuous with the PM. The tubular compartment had a beaded appearance possibly because of the low pH wash. Nevertheless, ARF6 and Tac remained colocalized in 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