Limits...
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.

Show MeSH

Related in: MedlinePlus

Internalization of surface Tac into the tubular compartment is blocked in cells expressing the GTPase-defective ARF6/ Q67L mutant. HeLa cells expressing Tac and ARF6/Q67L were  incubated with anti-Tac antibodies at 4°C and either fixed at 4°C  (4°C Binding) or warmed to 37°C for 30 min in the presence of 1  μM CD. The cells were then fixed either before (CD Total) or after rinsing with low pH buffer (CD Internal) and processed for indirect immunofluorescence.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139810&req=5

Figure 8: Internalization of surface Tac into the tubular compartment is blocked in cells expressing the GTPase-defective ARF6/ Q67L mutant. HeLa cells expressing Tac and ARF6/Q67L were incubated with anti-Tac antibodies at 4°C and either fixed at 4°C (4°C Binding) or warmed to 37°C for 30 min in the presence of 1 μM CD. The cells were then fixed either before (CD Total) or after rinsing with low pH buffer (CD Internal) and processed for indirect immunofluorescence.

Mentions: We first examined the uptake of Tac antibody into cells cotransfected with plasmids encoding Tac and ARF6/ Q67L, following the protocol used in Fig. 6. After the binding at 4°C, Tac antibody was uniformly localized along the PM (Fig. 8, Binding). Upon warming to 37°C in the presence of CD, no change in the distribution of Tac antibody was observed (Fig. 8, CD Total). Washing these cells with low pH buffer before fixation removed all of the Tac antibody (Fig. 8, CD Internal), indicating that the Tac antibody remained at the cell surface during the 37°C incubation. The same results were observed when these cells were warmed in the absence of CD (not shown). This indicates that expression of the active ARF6/Q67L alone blocks the internalization of surface Tac, suggesting that GTP hydrolysis by ARF6 is required for Tac internalization.


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

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

Internalization of surface Tac into the tubular compartment is blocked in cells expressing the GTPase-defective ARF6/ Q67L mutant. HeLa cells expressing Tac and ARF6/Q67L were  incubated with anti-Tac antibodies at 4°C and either fixed at 4°C  (4°C Binding) or warmed to 37°C for 30 min in the presence of 1  μM CD. The cells were then fixed either before (CD Total) or after rinsing with low pH buffer (CD Internal) and processed for indirect immunofluorescence.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 8: Internalization of surface Tac into the tubular compartment is blocked in cells expressing the GTPase-defective ARF6/ Q67L mutant. HeLa cells expressing Tac and ARF6/Q67L were incubated with anti-Tac antibodies at 4°C and either fixed at 4°C (4°C Binding) or warmed to 37°C for 30 min in the presence of 1 μM CD. The cells were then fixed either before (CD Total) or after rinsing with low pH buffer (CD Internal) and processed for indirect immunofluorescence.
Mentions: We first examined the uptake of Tac antibody into cells cotransfected with plasmids encoding Tac and ARF6/ Q67L, following the protocol used in Fig. 6. After the binding at 4°C, Tac antibody was uniformly localized along the PM (Fig. 8, Binding). Upon warming to 37°C in the presence of CD, no change in the distribution of Tac antibody was observed (Fig. 8, CD Total). Washing these cells with low pH buffer before fixation removed all of the Tac antibody (Fig. 8, CD Internal), indicating that the Tac antibody remained at the cell surface during the 37°C incubation. The same results were observed when these cells were warmed in the absence of CD (not shown). This indicates that expression of the active ARF6/Q67L alone blocks the internalization of surface Tac, suggesting that GTP hydrolysis by ARF6 is required for Tac internalization.

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