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ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

D'Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD, Peters PJ - J. Cell Biol. (1998)

Bottom Line: We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

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ARF6 colocalizes with cellubrevin but not with MPR or Lamp. CHO cells expressing wild-type ARF6 were doubled labeled  with an anticellubrevin antibody (15 nm gold), followed by anti–Tfn-R antibody (A, 10 nm), or with an anti-ARF6 antibody (5 nm gold)  followed by an anticellubrevin antibody (B, 10 nm gold). Arrows show clear examples of labeling for cellubrevin on ARF6-containing  vesicles. HEK-293 cells expressing ARF6 (T27N) were double labeled with antibodies against ARF6 (5 nm gold) followed by labeling  with antisera against MPR (C, 10 nm gold), or with anti-ARF6 antibody (10 nm gold) followed by anti–Lamp-3 antibody (D, 5 nm gold).  MPR and Lamp-3 labeling was done in 293 cells due to low cross-reactivity in CHO cells. c, centriole. Bar, 200 nm.
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Figure 5: ARF6 colocalizes with cellubrevin but not with MPR or Lamp. CHO cells expressing wild-type ARF6 were doubled labeled with an anticellubrevin antibody (15 nm gold), followed by anti–Tfn-R antibody (A, 10 nm), or with an anti-ARF6 antibody (5 nm gold) followed by an anticellubrevin antibody (B, 10 nm gold). Arrows show clear examples of labeling for cellubrevin on ARF6-containing vesicles. HEK-293 cells expressing ARF6 (T27N) were double labeled with antibodies against ARF6 (5 nm gold) followed by labeling with antisera against MPR (C, 10 nm gold), or with anti-ARF6 antibody (10 nm gold) followed by anti–Lamp-3 antibody (D, 5 nm gold). MPR and Lamp-3 labeling was done in 293 cells due to low cross-reactivity in CHO cells. c, centriole. Bar, 200 nm.

Mentions: Since we had previously shown that expression of ARF6(T27N) decreases the efficiency of Tfn recycling, we were prompted to examine whether proteins implicated in Tfn recycling colocalized with the ARF6 compartment. Cellubrevin, a member of the synaptobrevin/VAMP family of SNARES, is present on Tfn-R–containing vesicles in fibroblasts (McMohan et al., 1993) and tetanus toxin- mediated cleavage of cellubrevin impairs the recycling of Tfn-R–containing vesicles in CHO cells (Galli et al., 1994). Fig. 5 A shows the distribution of Tfn-Rs and cellubrevin in the pericentriolar endosomal compartment. We compared the distribution of ARF6 with that of cellubrevin. For these studies, cells expressing either wild-type ARF6 or ARF6(T27N) were double labeled with antibodies directed against ARF6 and cellubrevin. We found that cellubrevin-labeled, ARF6-positive vesicles in cells expressing wild-type ARF6 (Fig. 5 B) or ARF6(T27N; data not shown) and most of the colocalization was concentrated at the pericentriolar region of the cell. Double labeling for cation-dependent mannose-6-phosphate receptor (MPR; Fig 5 B) a late endosome marker, and LAMP-1 (not shown) and LAMP-3 (Fig. 5 D), markers for lysosomes, revealed little or no labeling of any of these markers in ARF6-positive vesicles. Furthermore, the ARF6-positive vesicles did not colocalize with γ-adaptin and other Golgi and endoplasmic reticulum markers (data not shown). Taken together, these findings indicate that intracellular ARF6 localizes to the perinuclear endosomal recycling compartment in CHO cells.


ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

D'Souza-Schorey C, van Donselaar E, Hsu VW, Yang C, Stahl PD, Peters PJ - J. Cell Biol. (1998)

ARF6 colocalizes with cellubrevin but not with MPR or Lamp. CHO cells expressing wild-type ARF6 were doubled labeled  with an anticellubrevin antibody (15 nm gold), followed by anti–Tfn-R antibody (A, 10 nm), or with an anti-ARF6 antibody (5 nm gold)  followed by an anticellubrevin antibody (B, 10 nm gold). Arrows show clear examples of labeling for cellubrevin on ARF6-containing  vesicles. HEK-293 cells expressing ARF6 (T27N) were double labeled with antibodies against ARF6 (5 nm gold) followed by labeling  with antisera against MPR (C, 10 nm gold), or with anti-ARF6 antibody (10 nm gold) followed by anti–Lamp-3 antibody (D, 5 nm gold).  MPR and Lamp-3 labeling was done in 293 cells due to low cross-reactivity in CHO cells. c, centriole. Bar, 200 nm.
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Figure 5: ARF6 colocalizes with cellubrevin but not with MPR or Lamp. CHO cells expressing wild-type ARF6 were doubled labeled with an anticellubrevin antibody (15 nm gold), followed by anti–Tfn-R antibody (A, 10 nm), or with an anti-ARF6 antibody (5 nm gold) followed by an anticellubrevin antibody (B, 10 nm gold). Arrows show clear examples of labeling for cellubrevin on ARF6-containing vesicles. HEK-293 cells expressing ARF6 (T27N) were double labeled with antibodies against ARF6 (5 nm gold) followed by labeling with antisera against MPR (C, 10 nm gold), or with anti-ARF6 antibody (10 nm gold) followed by anti–Lamp-3 antibody (D, 5 nm gold). MPR and Lamp-3 labeling was done in 293 cells due to low cross-reactivity in CHO cells. c, centriole. Bar, 200 nm.
Mentions: Since we had previously shown that expression of ARF6(T27N) decreases the efficiency of Tfn recycling, we were prompted to examine whether proteins implicated in Tfn recycling colocalized with the ARF6 compartment. Cellubrevin, a member of the synaptobrevin/VAMP family of SNARES, is present on Tfn-R–containing vesicles in fibroblasts (McMohan et al., 1993) and tetanus toxin- mediated cleavage of cellubrevin impairs the recycling of Tfn-R–containing vesicles in CHO cells (Galli et al., 1994). Fig. 5 A shows the distribution of Tfn-Rs and cellubrevin in the pericentriolar endosomal compartment. We compared the distribution of ARF6 with that of cellubrevin. For these studies, cells expressing either wild-type ARF6 or ARF6(T27N) were double labeled with antibodies directed against ARF6 and cellubrevin. We found that cellubrevin-labeled, ARF6-positive vesicles in cells expressing wild-type ARF6 (Fig. 5 B) or ARF6(T27N; data not shown) and most of the colocalization was concentrated at the pericentriolar region of the cell. Double labeling for cation-dependent mannose-6-phosphate receptor (MPR; Fig 5 B) a late endosome marker, and LAMP-1 (not shown) and LAMP-3 (Fig. 5 D), markers for lysosomes, revealed little or no labeling of any of these markers in ARF6-positive vesicles. Furthermore, the ARF6-positive vesicles did not colocalize with γ-adaptin and other Golgi and endoplasmic reticulum markers (data not shown). Taken together, these findings indicate that intracellular ARF6 localizes to the perinuclear endosomal recycling compartment in CHO cells.

Bottom Line: We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments.Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system.Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Washington University School of Medicine, St. Louis, Missouri 63110, USA.

ABSTRACT
We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

Show MeSH
Related in: MedlinePlus