Distribution of ARF6 between Membrane and Cytosol Is Regulated by Its GTPase Cycle
1999; Elsevier BV; Volume: 274; Issue: 28 Linguagem: Inglês
10.1074/jbc.274.28.20040
ISSN1083-351X
AutoresJoëlle Gaschet, Victor W. Hsu,
Tópico(s)Erythrocyte Function and Pathophysiology
ResumoThe ADP-ribosylation factor (ARF) subfamily of small GTPases regulates intracellular transport. Although much is known about how ARF1 regulates transport in the secretory pathways, regulation of the endocytic pathways by ARF6 remains less understood. In particular, whereas cycling of ARF1 between membrane and cytosol represents a major mechanism of regulating its function, this regulation has been questioned for ARF6. In this study, we found that ARF6 is distributed both on membranes and in the cytosol. Cytosolic ARF6 is recruited to membranes in a GTP-dependent manner that is fundamentally similar to ARF1. However, unlike ARF1, release of membrane-bound ARF6 to the cytosol requires hydrolysis of GTP that is sensitive to the level of magnesium. These findings suggest that the GTPase cycle of ARF6 also regulates its distribution between membrane and cytosol and that this form of regulation will also likely be important for the function of ARF6. Moreover, as ARF6 has little intrinsic ability to hydrolyze GTP, magnesium concentration most likely affects the release of membrane-bound ARF6 by altering the activity of its GTPase-activating protein. The ADP-ribosylation factor (ARF) subfamily of small GTPases regulates intracellular transport. Although much is known about how ARF1 regulates transport in the secretory pathways, regulation of the endocytic pathways by ARF6 remains less understood. In particular, whereas cycling of ARF1 between membrane and cytosol represents a major mechanism of regulating its function, this regulation has been questioned for ARF6. In this study, we found that ARF6 is distributed both on membranes and in the cytosol. Cytosolic ARF6 is recruited to membranes in a GTP-dependent manner that is fundamentally similar to ARF1. However, unlike ARF1, release of membrane-bound ARF6 to the cytosol requires hydrolysis of GTP that is sensitive to the level of magnesium. These findings suggest that the GTPase cycle of ARF6 also regulates its distribution between membrane and cytosol and that this form of regulation will also likely be important for the function of ARF6. Moreover, as ARF6 has little intrinsic ability to hydrolyze GTP, magnesium concentration most likely affects the release of membrane-bound ARF6 by altering the activity of its GTPase-activating protein. ADP-ribosylation factor guanine nucleotide exchange factor GTPase-activating protein Chinese hamster ovary guanosine 5′-O-(3-thiotriphosphate) guanosine 5′-O-(2-thiodiphosphate) adaptor protein Intracellular transport by vesicular carriers is initiated from a membrane compartment by the recruitment of cytosolic coat proteins to their target membranes. These local membrane sites become transformed into coated buds that then mature into coated transport vesicles. Subsequently, the membrane-bound coat proteins must be released to the cytosol before a vesicle can fuse with its target compartment. Thus, regulating movement of coat proteins between membranes and cytosol represents a major mechanism of regulating intracellular transport pathways (1Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1023) Google Scholar, 2Schekman R. Orci L. Science. 1996; 271: 1526-1532Crossref PubMed Scopus (818) Google Scholar). The recruitment of cytosolic coat proteins to membranes is regulated by the ADP-ribosylation factor (ARF)1 subfamily of small GTPases. Among members of this family, ARF1 serves as the prototype and has been shown to regulate coat proteins of the coat promoter I (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar) and clathrin AP-1 (5Stamnes M.A. Rothman J.E. Cell. 1993; 73: 999-1005Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 6Traub L.M. Ostrom J.A. Kornfeld S. J. Cell Biol. 1993; 123: 561-573Crossref PubMed Scopus (252) Google Scholar), AP-2 (7West M.A. Bright N.A. Robinson M.S. J. Cell Biol. 1997; 138: 1239-1254Crossref PubMed Scopus (105) Google Scholar), and AP-3 (8Ooi C.E. Dell'Angelica E.C. Bonifacino J.S. J. Cell Biol. 1998; 142: 391-402Crossref PubMed Scopus (166) Google Scholar) complexes. Binding of GTP activates ARF1 and stabilizes its interaction with target membranes. As a result, coat proteins regulated by ARF1 are also recruited from the cytosol to target membranes (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar, 9Randazzo P.A. Yang Y.C. Rulka C. Kahn R.A. J. Biol. Chem. 1993; 268: 9555-9563Abstract Full Text PDF PubMed Google Scholar, 10Tsai S.-C. Adamik R. Haun R.S. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9272-9276Crossref PubMed Scopus (40) Google Scholar). Subsequently, hydrolysis of its bound GTP deactivates ARF1 and releases ARF1 and its coat proteins from their target membranes to the cytosol (11Tanigawa G. Orci L. Amherdt M. Ravazzola M. Helms J.B. Rothman J.E. J. Cell Biol. 1993; 123: 1365-1371Crossref PubMed Scopus (200) Google Scholar, 12Teal S.B. Hsu V.W. Peters P.J. Klausner R.D. Donaldson J.G. J. Biol. Chem. 1994; 269: 3135-3138Abstract Full Text PDF PubMed Google Scholar, 13Randazzo P.A. Kahn R.A. J. Biol. Chem. 1994; 269: 10758-10763Abstract Full Text PDF PubMed Google Scholar). Thus, as with coat proteins, translocation of ARF1 between membranes and cytosol also represents a major mechanism of regulating its function. Moreover, like all members of the Ras-like small GTPase family, interconversion of ARF1 between its active and inactive states requires catalysis whereby a guanine nucleotide exchange factor (GEF) catalyzes the exchange of GDP for GTP (14Peyroche A. Paris S. Jackson C.L. Nature. 1996; 384: 479-481Crossref PubMed Scopus (236) Google Scholar, 15Chardin P. Paris S. Antonny B. Robineau S. Beraud-Dufour S. Jackson C.L. Chabre M. Nature. 1996; 384: 481-484Crossref PubMed Scopus (409) Google Scholar, 16Klarlund J.K. Guilherme A. Holik J.J. Virbasius J.V. Chawla A. Czech M.P. Science. 1997; 275: 1927-1930Crossref PubMed Scopus (371) Google Scholar, 17Meacci E. Tsai S.-C. Adamik R. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 1745-1748Crossref PubMed Scopus (136) Google Scholar, 18Morinaga N. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12926-12931Crossref PubMed Scopus (83) Google Scholar), and a GTPase-activating protein (GAP) catalyzes the hydrolysis of GTP to GDP (19Cukierman E. Huber I. Rotman M. Cassel D. Science. 1995; 270: 1999-2002Crossref PubMed Scopus (270) Google Scholar, 20Ding M. Vitale N. Tsai S.-C. Adamik R. Moss J. Vaughan M. J. Biol. Chem. 1996; 271: 24005-24009Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar, 21Randazzo P.A. Biochem. J. 1997; 324: 413-419Crossref PubMed Scopus (35) Google Scholar, 22Premont R.T. Claing A. Vitale N. Freeman J.L.R. Pitcher J.A. Patton W.A. Moss J. Vaughan M. Lefkowitz R.J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14082-14087Crossref PubMed Scopus (255) Google Scholar). ARF6 is the most distant relative of ARF1 and is the first member of the ARF subfamily characterized to regulate both membrane transport (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar, 24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar) and cytoskeletal organization (25Radhakrishna H. Klausner R.D. Donaldson J.G. J. Cell Biol. 1996; 134: 935-947Crossref PubMed Scopus (214) Google Scholar). ARF6 induces actin rearrangement through POR-1 (26D'Souza-Schorey C. Boshans R.L. McDonough M. Stahl P.D. Van Aelst L. EMBO J. 1997; 16: 5445-5454Crossref PubMed Scopus (205) Google Scholar), a protein that has been characterized previously to mediate actin rearrangement induced by the small GTPase Rac (27Van Aelst L. Joneson T. Bar-Sagi D. EMBO J. 1996; 15: 3778-3786Crossref PubMed Scopus (144) Google Scholar). However, the mechanistic details of how ARF6 regulates membrane transport of the endocytic pathways remain somewhat enigmatic. Overexpression of an activating mutant of ARF6 (Q67L) that is predicted to poorly hydrolyze GTP blocks internalization of the transferrin receptor from the cell surface to the early endosome (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). Overexpression of a mutant ARF6 (T27N) that is predicted to prevent exchange of GDP for GTP blocks the internalized transferrin receptor from recycling to the cell surface (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). A recent ultrastructural study suggests that membrane-bound ARF6 localizes mainly to the recycling early endosome, suggesting that activation of ARF6 occurs at this compartment (28D'Souza-Schorey C. van Donselaar E. Hsu V.W. Yang C. Stahl P.D. Peters P.J. J. Cell Biol. 1998; 140: 603-616Crossref PubMed Scopus (196) Google Scholar). Thus, inhibition of internalization by the activating mutant of ARF6 (Q67L) may be an indirect result of the redistribution of the early endosome to the plasma membrane such that material from the cell surface can no longer be internalized (24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). Moreover, an initial fractionation study on ARF6 suggested that it is all membrane-bound, whereas all other ARFs have a significant cytosolic pool that can be recruited to membranes in a GTP-dependent manner (29Cavenagh C.M. Whitney J.A. Carroll K. Zhang C. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), suggesting that ARF6 does not move between membrane and cytosol like all other ARF members. A subsequent study revealed a cytosolic form of ARF6, and its abundance varied by tissue type and developmental stage (30Yang C.Z. Heimberg H. D'Souza-Schorey C. Mueckler M.M. Stahl P.D. J. Biol. Chem. 1998; 273: 4006-4011Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). It remains unclear how this finding can be reconciled with the earlier finding that ARF6 is all membrane-bound and whether the GTPase cycle of ARF6 regulates its distribution between membrane and cytosol. In this study, we found that the distribution of ARF6 between membrane and cytosol is regulated by its GTPase cycle. Recruitment of cytosolic ARF6 to membranes requires its binding of GTP. However, unlike ARF1, release of membrane-bound ARF6 to the cytosol that requires hydrolysis of GTP is sensitive to changes in magnesium concentration, and this effect is attributable to alterations in the GAP activity on ARF6. Thus, as the GTPase cycle also regulates the distribution of ARF6 between membrane and cytosol, our finding also suggests that this form of regulation is a general characteristic of all members of the ARF subfamily. All cell lines were grown in media that were supplemented with 10% fetal calf serum, 100 μm glutamine, and 20 μg/ml gentamycin. Dulbecco's modified Eagle's medium was used for HeLa, RBL, and RD4 cells. α-Minimal essential medium was used for HEK 293 cells, whereas RPMI 1640 medium was used for Jurkat cells. All media were obtained from Life Technologies, Inc. All nucleotides were obtained from Sigma. Anti-ARF6 antibody was generated as described previously (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). Anti-hemagglutinin antibody (12CA5) was purchased from BabCo (Richmond, CA). Anti-human transferrin receptor antibody (H68.4) was purchased from Zymed Laboratories Inc. (South San Francisco, CA). Different GTPase mutant constructs of ARF1 and ARF6 have been described (24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar) and were transiently transfected into HeLa cells using the calcium phosphate precipitation method, as described previously (24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). Cells (1 × 107) were collected from culture flasks by scraping and washed once with phosphate-buffered saline, followed by another wash with a buffer that contained either magnesium or EDTA. The magnesium-containing buffer consisted of 25 mm Hepes (pH 7.4), 2.5 mmMgCl2, and 250 mm sucrose, as described previously (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar). The EDTA-containing buffer consisted of 10 mm triethanolamine (pH 7.4), 1 mm EDTA, and 250 mm sucrose and was referred to as TEAS buffer previously (29Cavenagh C.M. Whitney J.A. Carroll K. Zhang C. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Cells that were washed in either of these two buffers were then resuspended in 4 volumes of either buffer and subjected to shearing using a ball-bearing homogenizer (EMBL, Heidelberg, Germany) with four passes at 36-μm clearance. The resulting homogenate was centrifuged at 1000 × g for 10 min to obtain a post-nuclear supernatant. This supernatant was further centrifuged at 200,000 × g for 1 h (TFT 80.4 rotor, Sorvall, Newtown, CT) to obtain the cytosol (supernatant) and membranes (pellet). Equal fractions of both were analyzed by SDS-polyacrylamide gel electrophoresis followed by immunoblotting using an anti-ARF6 antibody and an anti-transferrin receptor antibody that assessed for quantitative recovery of total membranes. Blots were then visualized using enhanced chemiluminescence (ECL, NEN Life Science Products). CHO cells (5 × 108) were washed as described above and then resuspended in 4 volumes of either the magnesium- or EDTA-containing buffer and homogenized, followed by centrifugation as described above. The resulting post-nuclear supernatant was layered onto a sucrose step gradient in an SW 41 tube (Beckman Instruments). Sucrose solutions were prepared in either the magnesium- or EDTA-containing buffer and layered as follows: 1 ml of 60% sucrose at the bottom, followed by 1.5 ml of 30% sucrose, 1.5 ml of 20% sucrose, and then 7 ml of homogenate. The step gradient was centrifuged (SW 41 rotor, Beckman Instruments) at 200,000 ×g for 2.5 h. The interface between 30 and 60% sucrose of the step gradient was collected as the membrane fraction, whereas those above the 20% sucrose interface was collected as cytosol. Both fractions were stored at −80 °C. After thawing, the cytosol fraction was centrifuged at 200,000 × g for 40 min, and the supernatant was used for assays. 100 μl of cytosol (total protein concentration of 6–7 mg/ml) and 6 μl of total membranes (total protein concentration of 2–3 mg/ml), prepared as described above, were thawed and incubated together with various concentrations of nucleotides (as indicated in figure legends) for 45 min at 37 °C. The incubation mixture was then centrifuged at 200,000 × g for 30 min at 4 °C. The supernatant was collected, and the remaining membrane pellet was washed once. Equal fractions of both membranes and supernatant were then analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotted using an anti-ARF6 antibody. Blots were visualized using ECL and then scanned for densitometry using ScionImage. Total membranes (6 μl) prepared in the EDTA-containing buffer, as described above, were incubated with 100 μl of either cytosol prepared in the magnesium-containing buffer or simply the magnesium-containing buffer alone at 4 °C for 1 h. After incubation, the incubation mixture was centrifuged at 200,000 × g for 30 min at 4 °C and analyzed as membrane and cytosol fractions as described above. In experiments in which cytosol was used, calculation of membrane-bound ARF released into the cytosol was performed by quantifying the amount of ARF6 already in the cytosol and then subtracting this value from the total amount of soluble ARF6 obtained after the incubation. A subcellular fractionation study that had revealed ARF6 as all membrane-bound was performed in the presence of EDTA, where cells were homogenized in a 4-fold volume of buffer that contained 1 mm EDTA (29Cavenagh C.M. Whitney J.A. Carroll K. Zhang C. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). As physiologic magnesium is in the range of 1 mm (31Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), its dilution into the EDTA-containing buffer would likely result in a free magnesium concentration substantially <1 mm (probably in the submicromolar range). However, studies on how the GTPase cycle of ARF1 regulates its distribution between membrane and cytosol were performed using magnesium concentrations in the millimolar range (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar, 9Randazzo P.A. Yang Y.C. Rulka C. Kahn R.A. J. Biol. Chem. 1993; 268: 9555-9563Abstract Full Text PDF PubMed Google Scholar, 10Tsai S.-C. Adamik R. Haun R.S. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9272-9276Crossref PubMed Scopus (40) Google Scholar,31Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Thus, we first examined whether this difference in the concentration of magnesium affected the distribution of ARF6 between membrane and cytosol. Subcellular fractionation that separated total membranes from the cytosol was performed initially on a CHO cell line. ARF6 was detected by Western blotting using an anti-ARF6 antibody that was generated as described previously (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar). Consistent with previous observation using CHO cells (29Cavenagh C.M. Whitney J.A. Carroll K. Zhang C. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), when fractionation was performed in the EDTA-containing buffer, ARF6 remained virtually all membrane-bound. However, using a buffer that contained physiologic levels of magnesium (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar), we found a significant pool of cytosolic ARF6 (Fig.1). In contrast, the distribution of ARF1 remained unaffected (Fig. 1), suggesting that the effect of increased magnesium was selective for ARF6. This ability of increased magnesium to alter the ARF6 distribution between membrane and cytosol was not unique to CHO cells. Subcellular fractionation using the magnesium-containing buffer revealed a significant level of cytosolic ARF6 in human embryonal kidney cells (293), human cervical epithelial cells (HeLa), human fibrosarcoma cells (RD4), human T cells (Jurkat), and rat basophilic granulocytic cells (RBL) (Fig.2).Figure 2Magnesium-induced redistribution of ARF6 from membranes to the cytosol is independent of cell type. The cell lines as shown were homogenized with the magnesium-containing buffer and then fractionated into total membranes (M) and the cytosol (C), followed by immunoblotting with an anti-ARF6 antibody (lower panel) and an anti-transferrin receptor (TfR) antibody to assess quantitative recovery of total membranes (upper panel). The relative fractions of ARF6 on membranes and in the cytosol were then quantified by densitometry.View Large Image Figure ViewerDownload (PPT) As the GTPase cycle regulates the distribution of ARF1 between membrane and cytosol (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar, 9Randazzo P.A. Yang Y.C. Rulka C. Kahn R.A. J. Biol. Chem. 1993; 268: 9555-9563Abstract Full Text PDF PubMed Google Scholar, 10Tsai S.-C. Adamik R. Haun R.S. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9272-9276Crossref PubMed Scopus (40) Google Scholar, 31Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar), we examined whether the cytosolic form of ARF6 induced by increased magnesium could be recruited to membranes in a GTP-dependent manner. In a recruitment assay, we found that more cytosolic ARF6 was recruited to membranes with increasing concentrations of GTPγS (Fig. 3). Significantly, this recruitment assay was performed using the magnesium-containing buffer. Thus, the finding that the effect of GTPγS in promoting more membrane-bound ARF6 dominated over the effect of magnesium that induced more cytosolic ARF6 suggested that the ARF6 distribution was regulated more directly by its GTPase cycle than by the effect of magnesium. In support of this finding, we sought to inhibit hydrolysis of GTP bound to ARF6 more selectively by examining a GTPase mutant of ARF6 (Q67L) that is predicted to poorly hydrolyze GTP (24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). Transfecting ARF6 Q67L into cells followed by subcellular fractionation using the magnesium-containing buffer, we found that the Q67L mutant localized on membranes more efficiently than wild-type ARF6 (Fig.4). This difference was similar to that observed between an activating mutant of ARF1 (Q71I) that poorly hydrolyzed GTP (12Teal S.B. Hsu V.W. Peters P.J. Klausner R.D. Donaldson J.G. J. Biol. Chem. 1994; 269: 3135-3138Abstract Full Text PDF PubMed Google Scholar) and wild-type ARF1 (Fig. 4). For both ARF1 and ARF6, the activating mutation caused a relative, but not complete, shift in the distribution of the mutants to membranes. This partial shift can be attributed to the previous finding that the activating point mutation only partially inhibits hydrolysis of GTP on ARF1, as compared with a more complete block of hydrolysis induced by GTPγS (11Tanigawa G. Orci L. Amherdt M. Ravazzola M. Helms J.B. Rothman J.E. J. Cell Biol. 1993; 123: 1365-1371Crossref PubMed Scopus (200) Google Scholar, 12Teal S.B. Hsu V.W. Peters P.J. Klausner R.D. Donaldson J.G. J. Biol. Chem. 1994; 269: 3135-3138Abstract Full Text PDF PubMed Google Scholar). Thus, the similar effects of the activating point mutation on the distribution of ARF1 and ARF6 further suggested that the GTPase cycle of ARF6, like that of ARF1, directly regulated its movement between membrane and cytosol. In contrast to GTPγS, GDPβS did not stabilize ARF6 on membranes (Fig. 5). Thus, this result corroborated with the above findings suggesting that the GTP-bound form stabilized ARF6 on membranes. Significantly, GTP was also unable to induce the membrane-bound form of ARF6 (Fig. 5). This result was reminiscent of the recruitment of cytosolic ARF1 to membranes. GTPγS, but not GTP, stabilized ARF1 on membranes because its GAP was too active to allow accumulation of membrane-bound ARF1 in the presence of GTP (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar). These findings suggested why subcellular fractionation revealed mainly cytosolic ARF1, as the ARF1 GAP activity appeared highly active even at 4 °C during subcellular fractionation (32Ostermann J. Orci L. Tani K. Amherdt M. Ravazzola M. Elazar Z. Rothman J.E. Cell. 1993; 75: 1015-1025Abstract Full Text PDF PubMed Scopus (232) Google Scholar). To begin investigating whether increasing magnesium used a similar mechanism to redistribute ARF6 from membranes to the cytosol, we first isolated the membrane-bound fraction of ARF6 in the EDTA-containing buffer that allowed significant accumulation of ARF6 on membranes. This membrane was then incubated at 4 °C with cytosol that was prepared in the magnesium-containing buffer. When incubated together, a significant fraction of membrane-bound ARF6 became soluble (Fig. 6). To ensure that this redistribution of ARF6 is not simply due to a re-mixing of membranes with cytosol, total membranes and cytosol were both prepared in the EDTA-containing buffer and then incubated together. This incubation revealed no significant redistribution of ARF6 to the cytosol (Fig. 6), suggesting that increasing magnesium also induced an activity at 4 °C to redistribute ARF6 from membranes to the cytosol. As cytosolic ARF1 could be induced by either inhibiting its GEF activity that prevents its recruitment from the cytosol to membranes or enhancing its GAP activity that facilitates its release from membranes to the cytosol, we also examined which regulatory activity on ARF6 was affected by magnesium to induce cytosolic ARF6. Thus, to examine whether increasing magnesium induced cytosolic ARF6 by inhibiting its GEF activity, we performed a recruitment assay in the presence of GTPγS that blocked the possibility of magnesium acting through a GAP activity to induce more cytosolic ARF6. Under this condition, more membrane-bound ARF6 was observed using the magnesium-containing buffer compared with the EDTA-containing buffer (Fig.7). Thus, cytosolic ARF6 induced by increasing magnesium could not be attributed to inhibition of the GEF activity on ARF6, suggesting that magnesium most likely induced its effect by enhancing the GAP activity on ARF6. To test more directly whether a GAP activity on ARF6 would enhance its release from membranes, we prepared total membranes in the EDTA-containing buffer to accumulate a significant fraction of membrane-bound ARF6. This membrane fraction was then incubated with the magnesium-containing buffer that lacked cytosol. In this type of simplified incubation, a significant fraction of membrane-bound ARF6 was released (Fig. 8). Thus, as this incubation started with ARF6 only in the membrane-bound fraction, its result suggested that a membrane-localized activity was activated by magnesium to release ARF6 from membranes. Significantly, the degree of this release was less than that seen when incubation was performed using cytosol prepared from the magnesium-containing buffer (compare Figs. 6 and 8), suggesting that the cytosol had additional factor(s) that potentiated a membrane-localized activity to release membrane-bound ARF6. To show that the magnesium-induced release of membrane-bound ARF6 was attributable to hydrolysis of GTP, we next examined whether ARF6 that had been loaded onto membranes with GTPγS could be released when subsequently incubated in the magnesium-containing buffer. For this purpose, membranes and cytosol that were prepared in the magnesium-containing buffer were incubated with GTPγS, and membrane-bound ARF6 was isolated by centrifugation. When isolated in this manner, membrane-bound ARF6 became resistant to release by the magnesium-containing buffer (Fig. 8), suggesting that increasing magnesium enhanced the release of membrane-bound ARF6 by facilitating the hydrolysis of GTP. In this study, we found that a difference in the level of magnesium alters the distribution of ARF6 between membrane and cytosol. Elucidating the underlying mechanism, we found that ARF6 is recruited from the cytosol to membranes in a GTP-dependent manner and is released back to the cytosol by increasing magnesium, which facilitates hydrolysis of GTP. Thus, these findings suggest that translocation of ARF6 between membrane and cytosol is regulated by its GTPase cycle. As ARF6 has little intrinsic ability to hydrolyze GTP (33Welsh C.F. Moss J. Vaughan M. J. Biol. Chem. 1994; 269: 15583-15587Abstract Full Text PDF PubMed Google Scholar), our findings also suggest that increasing magnesium induces cytosolic ARF6 by enhancing its GAP activity. Our study used indirect approaches to show that the ARF6 distribution is regulated by its GTPase cycle because approaches that might be more direct are also technically unfeasible. For example, one can envisage immunoprecipitating ARF6 from membrane and cytosol fractions and then examining the guanine nucleotide bound to ARF6 derived from these two fractions. However, this direct approach requires an immunoprecipitating antibody that is capable of stabilizing binding of a small GTPase to its guanine nucleotide (34Downward J. Graves J.D. Warne P.H. Rayter S. Cantrell D.A. Nature. 1990; 346: 719-723Crossref PubMed Scopus (687) Google Scholar), and such an antibody has not been identified for small GTPases other than Ras (35Ren X.D. Kiosses W.B. Schwartz M.A. EMBO J. 1999; 18: 578-585Crossref PubMed Scopus (1366) Google Scholar). Thus, as done for ARF1 (3Donaldson J.G. Cassel D. Kahn R.A. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6408-6412Crossref PubMed Scopus (380) Google Scholar, 4Palmer D.J. Helms J.B. Beckers C.J. Orci L. Rothman J.E. J. Biol. Chem. 1993; 268: 12083-12089Abstract Full Text PDF PubMed Google Scholar, 9Randazzo P.A. Yang Y.C. Rulka C. Kahn R.A. J. Biol. Chem. 1993; 268: 9555-9563Abstract Full Text PDF PubMed Google Scholar, 10Tsai S.-C. Adamik R. Haun R.S. Moss J. Vaughan M. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9272-9276Crossref PubMed Scopus (40) Google Scholar), we also used indirect approaches to show that binding of GTP localizes ARF6 to membranes. We found that GTPγS, but not GDPβS, supports recruitment of cytosolic ARF6 to membranes. Moreover, a point mutant of ARF6 (Q67L) that is predicted to poorly hydrolyze its bound GTP associates with membranes better than its wild-type counterpart. Thus, these results suggest that the GTPase cycle of ARF6 regulates its distribution between membrane and cytosol in a manner that is fundamentally similar to that seen for ARF1. Significantly, these results also suggest that potential mechanisms independent of the GTPase cycle as an explanation for how magnesium redistributes ARF6 would be unlikely. For example, one can envisage a mechanism of proteolysis whereby a magnesium-sensitive protease is activated to directly cleave a proteinaceous docking site used by ARF6 to bind membranes. However, such a mechanism cannot readily explain our finding that the magnesium-induced redistribution of membrane-bound ARF6 to the cytosol is blocked when GTP hydrolysis is inhibited. As magnesium appears to affect the ARF6 distribution through its GTPase cycle, this finding has mechanistic parallels to how brefeldin A affects the ARF1 distribution. Brefeldin A induces the cytosolic form of ARF1 by inhibiting its GEF activity, which then allows the GDP-bound form of ARF1 to predominate (36Donaldson J.G. Finazzi D. Klausner R.D. Nature. 1992; 360: 350-352Crossref PubMed Scopus (595) Google Scholar, 37Helms J.B. Rothman J.E. Nature. 1992; 360: 352-354Crossref PubMed Scopus (581) Google Scholar). In this regard, increasing magnesium can potentially induce cytosolic ARF6 by either inhibiting a GEF activity to prevent the recruitment of cytosolic ARF6 to membranes or enhancing a GAP activity to facilitate the release of membrane-bound ARF6 to the cytosol. To address this issue, we assessed whether increasing magnesium inhibits the recruitment of cytosolic ARF6 to membranes by examining its recruitment in the presence of GTPγS, which blocks the contribution of the ARF6 GAP activity. As this recruitment is enhanced upon increasing magnesium (see Fig. 7), this result suggests that the redistribution of ARF6 from membrane to cytosol cannot be explained by inhibition of the ARF6 GEF activity by magnesium. Thus, this result suggests that magnesium affects the ARF6 distribution mainly by enhancing its GAP activity. A seeming contradiction to our findings is the previous observation that both the activating (Q67L) and deactivating (T27N) mutants of ARF6 are localized on membranes (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar, 24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). This observation has the appearance that both the GTP- and GDP-bound forms of ARF6 are localized on membranes and thus reinforces the previous notion that the GTPase cycle of ARF6 does not regulate its distribution between membrane and cytosol (29Cavenagh C.M. Whitney J.A. Carroll K. Zhang C. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). An explanation for this apparent contradiction is suggested, however, by the elucidation of how the equivalent point mutations in Ras affect its GTPase cycle (38Boguski M.S. McCormick F. Nature. 1993; 366: 643-654Crossref PubMed Scopus (1761) Google Scholar, 39Dascher C. Balch W.E. J. Biol. Chem. 1994; 269: 1437-1448Abstract Full Text PDF PubMed Google Scholar). Significantly, whereas the equivalent mutation of ARF6 Q67L in Ras represents a GTP-bound form of Ras, the equivalent mutation of ARF6 T27N in Ras does not appear to represent a GDP-bound form of Ras. The exchange of GDP for GTP on Ras represents a two-step mechanism whereby a GEF activity displaces GDP bound to Ras and results in a transition state of Ras that has an empty GTP-binding pocket. Subsequently, GTP fills the pocket to generate the GTP-bound form of Ras. Thus, ARF6 T27N is likely to represent a transition state of ARF6 that is devoid of a guanine nucleotide, rather than its GDP-bound form. An important implication for the transition state of Ras is that it interacts more tightly with its GEF than the wild-type counterpart. Thus, overexpression of the transition state mutant results in a dominant-negative phenotype because the GEF can no longer act on endogenous wild-type Ras. In this regard, the phenotypic manifestations of ARF6 T27N could be attributed to its ability to block the function of endogenous wild-type ARF6 by sequestering GEFs that act on ARF6 (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar,24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). Thus, because this GEF activity is likely to be membrane-bound, as seen for ARF1 GEF (36, 37, and 39), ARF6 T27N would be predicted to associate with membranes rather than to reside in the cytosol. Indeed, we have found recently, by both subcellular fractionation and immunogold electron microscopy, that ARF6 T27N and its equivalent mutant in ARF1 (T31N) are associated with membranes to similar degrees. 2P. J. Peters and V. W. Hsu, manuscript in preparation. Thus, the fundamental parallels that we have seen for how the GTPase cycle regulates the distribution of both ARF1 and ARF6 are further supported by the behavior of their point mutants that affect the GTPase cycle. As cytosolic magnesium is thought to exist in the millimolar range (31Franco M. Chardin P. Chabre M. Paris S. J. Biol. Chem. 1995; 270: 1337-1341Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar) rather than the submicromolar range, which was used initially to study the distribution of ARF6 (29Cavenagh C.M. Whitney J.A. Carroll K. Zhang C. Boman A.L. Rosenwald A.G. Mellman I. Kahn R.A. J. Biol. Chem. 1996; 271: 21767-21774Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), our study suggests that ARF6 is likely to have a significant cytosol fractionin vivo. In considering how our findings might relate to regulation of endocytic transport by ARF6, we are led by the recent suggestion that ARF6 is likely to be activated on endosomal membranes to regulate the recycling pathway of endocytosis (28D'Souza-Schorey C. van Donselaar E. Hsu V.W. Yang C. Stahl P.D. Peters P.J. J. Cell Biol. 1998; 140: 603-616Crossref PubMed Scopus (196) Google Scholar). Thus, we propose that a GEF activates ARF6 for its recruitment from the cytosol to endosomal membranes to initiate the formation of transport carriers from this compartment. However, given what is currently known, the role of a GAP that acts on ARF6 in this transport pathway is more difficult to pinpoint. Although studies on ARF1 suggest that GAP should act to release ARF6 from its transport vesicles prior to their fusion with the plasma membrane (11Tanigawa G. Orci L. Amherdt M. Ravazzola M. Helms J.B. Rothman J.E. J. Cell Biol. 1993; 123: 1365-1371Crossref PubMed Scopus (200) Google Scholar), the activating mutant of ARF6 (Q67L) has been shown to reside on the plasma membrane (23D'Souza-Schorey C. Li G. Colombo M.I. Stahl P.D. Science. 1995; 267: 1175-1178Crossref PubMed Scopus (371) Google Scholar, 24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). This observation suggests that GAP acts to release ARF6 only after its transport vesicles have fused with the plasma membrane. Because coated vesicles are thought to fuse only with their target compartment after uncoating (1Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1023) Google Scholar, 2Schekman R. Orci L. Science. 1996; 271: 1526-1532Crossref PubMed Scopus (818) Google Scholar), the effect of ARF6 Q67L suggests the possibility that deactivation of ARF6 does not regulate the uncoating of its transport vesicles. As current evidence does not support this possibility, an alternative explanation that seems more likely is that constitutive activation of ARF6 through the use of the Q67L mutant may lead to secondary effects that confound the true nature of how GAP acts. Significantly, ARF6 Q67L also induces the redistribution of the early endosome to the plasma membrane (24Peters P.J. Hsu V.W. Ooi C.E. Finazzi D. Teal S.B. Oorschot V. Donaldson J.G. Klausner R.D. J. Cell Biol. 1995; 128: 1003-1017Crossref PubMed Scopus (320) Google Scholar). Thus, although this activating mutant can localize on the resulting mixed membrane compartment, it might not be capable of recruiting the ARF6-regulated coat to this compartment because this coat recruitment might occur only in an endosome-specific context. This explanation suggests that the GTPase cycle of ARF6 can be uncoupled with respect to its ability to recruit coat, when a mixed compartment of plasma membrane and early endosome is induced. Insight into the actual explanation will likely be forthcoming once the putative coat regulated by ARF6 is identified and characterized. On the other hand, another aspect of our study may provide insight into potential regulatory mechanisms that govern the GAP activity on ARF6. Although more cytosolic ARF6 induced by increasing magnesium could be attributed to enhancing the ARF6 GAP activity, the ARF1 distribution is not affected similarly, suggesting that its GAP activity does not vary over the range of magnesium concentrations examined in our study. This disparity between the GAP activities of ARF1 and ARF6 suggests that the GAPs for ARF1 and ARF6 may be regulated in significantly different ways. Relevant to this possibility, ARF6 has been shown to regulate not only endocytic transport, but also actin rearrangement (25Radhakrishna H. Klausner R.D. Donaldson J.G. J. Cell Biol. 1996; 134: 935-947Crossref PubMed Scopus (214) Google Scholar, 26D'Souza-Schorey C. Boshans R.L. McDonough M. Stahl P.D. Van Aelst L. EMBO J. 1997; 16: 5445-5454Crossref PubMed Scopus (205) Google Scholar), and these two events are also regulated by the Rho family of small GTPases during the formation of surface membrane protrusions (40Nobes C.D. Hall A. Cell. 1995; 81: 53-62Abstract Full Text PDF PubMed Scopus (3735) Google Scholar, 41Lamaze C. Chuan T.-H. Terlecky L.J. Bokoch G.M. Schmid S.L. Nature. 1996; 382: 177-179Crossref PubMed Scopus (331) Google Scholar). Thus, an intriguing possibility is that upstream regulation of ARF6 through its GAP might be subject to intracellular signaling mechanisms that regulate membrane protrusions (42Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1660) Google Scholar, 43Sanchez-Madrid F. Angel del Pozo M. EMBO J. 1999; 18: 501-511Crossref PubMed Scopus (521) Google Scholar). Consistent with this possibility, ARF6 has been shown recently to regulate cell spreading (44Song J. Khachikian Z. Radhakrishna H. Donaldson J.G. J. Cell Sci. 1998; 111: 2257-2267Crossref PubMed Google Scholar), a process that occurs through the formation of membrane protrusions. Moreover, this possibility would explain why different cell types exhibit different degrees of membrane-bound ARF6 (see Fig.2), as different cell types are likely to exhibit different capacities to form membrane protrusions. Finally, as this process is regulated by calcium signaling (42Burridge K. Chrzanowska-Wodnicka M. Annu. Rev. Cell Dev. Biol. 1996; 12: 463-519Crossref PubMed Scopus (1660) Google Scholar, 43Sanchez-Madrid F. Angel del Pozo M. EMBO J. 1999; 18: 501-511Crossref PubMed Scopus (521) Google Scholar), it will be interesting to examine in the future whether calcium also affects the distribution of ARF6 between membrane and cytosol. We thank Michael Brenner and Frank Austen for support; Eddie Koo for generating the anti-ARF6 antibody; and Peter Peters, Dan Cassel, Andreas Ambach, Tomohiko Aoe, James Casanova, James Collawn, Timothy McGraw, and Kathleen Buckley for helpful discussions.
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