ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway - PubMed (original) (raw)
ADP-ribosylation factor 6 regulates a novel plasma membrane recycling pathway
H Radhakrishna et al. J Cell Biol. 1997.
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.
Figures
Figure 1
CD redistributes ARF6 to juxtanuclear tubular structures that resemble the compartment where the GTP-binding defective mutant of ARF6, T27N, resides. HeLa cells transfected with wild-type ARF6 plasmid were either untreated (Untreated), or treated with 1 μM CD for 30 min (CD). Other cells were transfected with the ARF6/T27N plasmid. The cells were then fixed in 2% formaldehyde and labeled with ARF6-specific antiserum, followed by rhodamine-labeled donkey anti–rabbit IgG and Oregon green–labeled phalloidin to visualize actin filaments.
Figure 4
Endogenous MHC-I localizes to the tubular compartment with or without ARF6 overexpression. HeLa cells were transfected with ARF6 plasmid and then incubated in the absence (Unt) or presence (CD) of 1 μM CD for 30 min at 37°C, and they were fixed and processed for immunofluorescence localization of MHC-I using mouse anti–MHC-I antibodies and for overexpression of ARF6 using antibody to ARF6. Anti–MHC-I was detected with sequential FITC-labeled goat anti–mouse and FITC-labeled donkey anti–goat antibodies, while anti-ARF6 was detected with rhodamine-conjugated donkey anti–rabbit antibody. The tubular structures (arrowheads) radiating from the perinuclear region, labeled with anti–MHC-I antibodies, become more apparent after CD treatment, and are observed in both ARF6-transfected and untransfected cells.
Figure 5
Surface Tac is internalized into and colocalizes with ARF6 in the tubular compartment, and accumulates in this compartment during CD treatment. HeLa cells expressing both ARF6 and Tac were chilled to 4°C and incubated with mouse anti-Tac antibodies (7G7) for 30 min to label surface Tac. After washing to remove excess antibodies, cells were warmed to 37°C for 30 min in the absence (No drug) or presence of 1 μM CD (CD) or AlF (AlF) to permit internalization of the bound anti-Tac antibodies, and were fixed and processed for indirect immunofluorescence. Tac antibodies were detected with fluorescent donkey anti– mouse secondary antibodies and ARF6 was localized with rabbit anti-ARF6 antiserum. In some cells, after CD treatment, Tac antibodies were localized with secondary antibodies in the absence of detergent to detect Tac antibody remaining at the surface (CD Surface only); ARF6 was subsequently localized in these cells after detergent permeabilization. Note that Tac antibodies were internalized into the tubular compartment in the absence of CD treatment (No Drug). During CD treatment, both ARF6 and the internalized Tac antibodies accumulated in the tubular compartment. In contrast, AlF treatment resulted in the sequestration of anti-Tac antibodies and ARF6 in surface protrusions and inhibited movement into the tubular compartment.
Figure 2
The ARF6-labeled tubular structures are distinct from transferrin-positive endosomes. Transfected HeLa cells expressing ARF6-HA were incubated with 30 μg/ ml iron-loaded human transferrin (Tfn) for 30 min at 37°C either in the absence (Untreated) or presence of 1 μM CD (CD), 1 μM BFA (BFA), or CD plus BFA (CD+BFA). Cells were washed, fixed, and processed for indirect immunofluorescence. ARF6-HA was labeled with a mouse anti-HA antibody, and transferrin was labeled with rabbit anti-Tfn antiserum; primary antibodies were visualized with appropriately labeled donkey secondary antibodies. Note that in the presence of both CD and BFA, the ARF6 tubules are distinct from the transferrin tubules.
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.
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.
Figure 7
CD treatment blocks the recycling of Tac from the tubular compartment back out to the PM. Anti-Tac antibodies bound to the surface of cells expressing ARF6 and Tac were loaded into the tubular compartment by incubation with 1 μM CD at 37°C for 30 min. The cells were chilled to 4°C, rinsed with low pH buffer to remove the remaining surface anti-Tac antibodies, and then either fixed immediately (Load) and assessed for the Tac antibody loaded, or warmed to 37°C for 30 min in the absence or presence of CD before fixation. Tac antibody that reappeared on the cell surface was detected by incubation with fluorescently labeled secondary antibodies in the absence of detergent permeabilization (Surface Reappearance); ARF6 was subsequently localized in these cells after permeabilization.
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.
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.
Figure 10
Model for the ARF6-regulated PM–endosomal recycling pathway. We propose that activation of ARF6, through nucleotide exchange, occurs at the tubular endosome and triggers the recycling of membrane to discrete sites at the PM where ARF6-GTP stimulates the formation of protrusive structures, rich in actin. Inactivation, through GTP hydrolysis, signals the return of ARF6 to the tubular endosome via membrane intermediates that are yet to be defined. CD treatment or expression of the GTP-binding–defective mutant of ARF6, T27N, blocks recycling out to the PM. In contrast, AlF treatment or expression of the GTPase-defective mutant, Q67L, blocks membrane internalization back into the tubular endosomal compartment.
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