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J G Donaldson

Publications and source records attributed to J G Donaldson.

At least 19 recordsLinked to original sources
PubMed2005

Localization and function of Arf family GTPases.

Arfs are a family of Ras-related GTP-binding proteins that function in the regulation of membrane trafficking and structure. The six mammalian Arf proteins are expressed ubiquitously and so it is anticipated that each will have a distinct localization and function within the cell. It has been assumed that much of this specificity will be defined by determining which regulators of Arfs, the GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins) function with which Arf proteins. Although in vitro assays may indicate Arf preferences for the numerous Arf GEFs and GAPs that have been identified, in the cell the different Arfs, GEFs and GAPs are targeted to specific compartments where they carry out their functions. We have embarked on studies to define regions of the Arf1 and Arf6 proteins that determine their sites of action and specific activities at the Golgi and plasma membrane respectively. Chimaeras were made between Arf1 and Arf6 in order to identify regions of the protein that contributed to targeting and function. Whereas Arf6 is targeted to the plasma membrane through multiple regions along the protein, we have found a Golgi-targeting region in Arf1 that is sufficient to target Arf6 to the Golgi complex.

ADP-Ribosylation Factor 1↗
PubMed2005

Arfs, phosphoinositides and membrane traffic.

Arf (ADP-ribosylation factor) GTP-binding proteins function in cells to regulate membrane traffic and structure. Arfs accomplish this task through modification of membrane lipids and the recruitment of proteins, including coat proteins and actin, to membrane surfaces. Arf1 and Arf6 are the most divergent and most studied human Arf proteins that localize predominantly to the Golgi complex and plasma membrane respectively. We have been studying the targeting of Arf1 and Arf6 to these specific compartments and the common and divergent activities that they exert on these membranes. We have found that Arf6 acts through activation of type I phosphatidylinositol 4-phosphate 5-kinases to generate phosphatidylinositol 4,5-bisphosphate and that this activity is instrumental in facilitating the actin cytoskeletal rearrangements and alterations in endosomal membrane trafficking observed with increased Arf6 activation. Arf1 can also stimulate the activity of phosphatidylinositol kinases and recruit coat proteins and actin cytoskeletal elements to the Golgi complex.

ADP-Ribosylation Factor 1↗
PubMed2001

Phosphatidylinositol 4,5-bisphosphate and Arf6-regulated membrane traffic.

ADP-ribosylation factor (Arf) 6 regulates the movement of membrane between the plasma membrane (PM) and a nonclathrin-derived endosomal compartment and activates phosphatidylinositol 4-phosphate 5-kinase (PIP 5-kinase), an enzyme that generates phosphatidylinositol 4,5-bisphosphate (PIP2). Here, we show that PIP2 visualized by expressing a fusion protein of the pleckstrin homology domain from PLCdelta and green fluorescent protein (PH-GFP), colocalized with Arf6 at the PM and on tubular endosomal structures. Activation of Arf6 by expression of its exchange factor EFA6 stimulated protrusion formation, the uptake of PM into macropinosomes enriched in PIP2, and recycling of this membrane back to the PM. By contrast, expression of Arf6 Q67L, a GTP hydrolysis-resistant mutant, induced the formation of PIP2-positive actin-coated vacuoles that were unable to recycle membrane back to the PM. PM proteins, such as beta1-integrin, plakoglobin, and major histocompatibility complex class I, that normally traffic through the Arf6 endosomal compartment became trapped in this vacuolar compartment. Overexpression of human PIP 5-kinase alpha mimicked the effects seen with Arf6 Q67L. These results demonstrate that PIP 5-kinase activity and PIP2 turnover controlled by activation and inactivation of Arf6 is critical for trafficking through the Arf6 PM-endosomal recycling pathway.

ADP-Ribosylation Factor 6↗
PubMed2001

Membrane-cytoskeletal dynamics in a new dimension.

The recent Airlie House meeting on 'Cytoplasmic Organization and Membrane Traffic' (22-25 March 2001), sponsored by the Keith Porter Endowment, proved not to be the typical exchange of advances among specialists familiar with each other's work, but rather a series of interesting and diverse presentations that together illuminated the pace and pattern of membrane and cytoskeletal interactions in living cells.

Animals↗
PubMed2000

ACAPs are arf6 GTPase-activating proteins that function in the cell periphery.

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.

3T3 Cells↗
PubMed2000

Regulators and effectors of the ARF GTPases.

The small G proteins of the ARF family are key regulators of membrane dynamics. Many functions of ARF proteins in cells are being revealed by studies of their regulators and effectors. Significant progress has been made over the past year, with the identification of a surprisingly large family of novel ARF GTPase-activating proteins. In addition, two new classes of effectors, the PIP kinases and a novel family of monomeric coat-like proteins have been discovered.

ADP-Ribosylation Factors↗
PubMed2000

Separation of membrane trafficking and actin remodeling functions of ARF6 with an effector domain mutant.

The ADP-ribosylation factor 6 (ARF6) GTPase has a dual function in cells, regulating membrane traffic and organizing cortical actin. ARF6 activation is required for recycling of the endosomal membrane back to the plasma membrane (PM) and also for ruffling at the PM induced by Rac. Additionally, ARF6 at the PM induces the formation of actin-containing protrusions. To identify sequences in ARF6 that are necessary for these distinct functions, we examined the behavior of a chimeric protein of ARF1 and ARF6. The 1-6 chimera (with the amino half of ARF1 and the carboxyl half of ARF6) localized like ARF6 in HeLa cells and moved between the endosome and PM, but it did not form protrusions, an ARF6 effector function. Two residues in the amino-terminal half of ARF6, Q37 and S38, when substituted into the 1-6 chimera allowed protrusion formation, whereas removal of these residues from ARF6 resulted in an inability to form protrusions. Interestingly, expression of 1-6 in cells selectively inhibited protrusions induced by wild-type ARF6 but had no effect on ARF6-regulated membrane movement or Rac-induced ruffling. Thus, we have uncoupled two functions of ARF6, one involved in membrane trafficking, which is necessary for Rac ruffling, and another involved in protrusion formation.

ADP-Ribosylation Factor 6↗
PubMed1999

ARF6 requirement for Rac ruffling suggests a role for membrane trafficking in cortical actin rearrangements.

The ARF6 GTPase regulates a novel endosomal-plasma membrane recycling pathway and influences cortical actin remodeling. Here we examined the relationship between ARF6 and Rac1, a Rho family GTPase, implicated in cortical actin rearrangements. Endogenous Rac1 colocalized with ARF6 at the plasma membrane and on the ARF6 recycling endosome in untransfected HeLa and primary human fibroblast cells. In transfected HeLa cells Rac1 and ARF6 also colocalized. Cells expressing wild-type ARF6 or Rac1 formed actin-containing surface protrusions and membrane ruffles, respectively, upon treatment with the G protein activator aluminum fluoride. Aluminum fluoride-treatment of cells transfected with equivalent amounts of plasmid resulted in enhanced membrane ruffling, with protrusions appearing as Rac expression was lowered. Co-expression of the dominant negative, GTP binding-defective ARF6 T27N mutant inhibited the aluminum fluoride-induced ruffling observed in cells expressing Rac1, and the constitutive ruffling observed in cells expressing the activated Rac1 Q61L mutant. In contrast, co-expression of the GTP-binding-defective, T17N mutant of either Rac1 or Cdc42 with ARF6 did not inhibit the aluminum fluoride-induced surface protrusions, nor did inactivation of Rho with C3-transferase. These observations suggest that ARF6, a non-Rho family GTPase, can, by itself, alter cortical actin and can influence the ability of Rac1 to form lamellipodia, in part, by regulating its trafficking to the plasma membrane.

ADP-Ribosylation Factors↗
PubMed1998

A requirement for ARF6 in Fcgamma receptor-mediated phagocytosis in macrophages.

Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcgamma receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 microM BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of beta-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcgamma receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcgamma receptor activation.

ADP-Ribosylation Factors↗
PubMed1998

Brefeldin A-inhibited guanine nucleotide-exchange activity of Sec7 domain from yeast Sec7 with yeast and mammalian ADP ribosylation factors.

The Saccharomyces cerevisiae Sec7 protein (ySec7p), which is an important component of the yeast secretory pathway, contains a sequence of approximately 200 amino acids referred to as a Sec7 domain. Similar Sec7 domain sequences have been recognized in several guanine nucleotide-exchange proteins (GEPs) for ADP ribosylation factors (ARFs). ARFs are approximately 20-kDa GTPases that regulate intracellular vesicular membrane trafficking and activate phospholipase D. GEPs activate ARFs by catalyzing the replacement of bound GDP with GTP. We, therefore, undertook to determine whether a Sec7 domain itself could catalyze nucleotide exchange on ARF and found that it exhibited brefeldin A (BFA)-inhibitable ARF GEP activity. BFA is known to inhibit ARF GEP activity in Golgi membranes, thereby causing reversible apparent dissolution of the Golgi complex in many cells. The His6-tagged Sec7 domain from ySec7p (rySec7d) synthesized in Escherichia coli enhanced binding of guanosine 5'-[gamma-[35S]thio]triphosphate by recombinant yeast ARF1 (ryARF1) and ryARF2 but not by ryARF3. The effects of rySec7d on ryARF2 were inhibited by BFA in a concentration-dependent manner but not by inactive analogues of BFA (B-17, B-27, and B-36). rySec7d also promoted BFA-sensitive guanosine 5'-[gamma-thio]triphosphate binding by nonmyristoylated recombinant human ARF1 (rhARF1), rhARF5, and rhARF6, although the effect on rhARF6 was very small. These results are consistent with the conclusion that the yeast Sec7 domain itself contains the elements necessary for ARF GEP activity and its inhibition by BFA.

ADP-Ribosylation Factor 1↗
PubMed1998

ASAP1, a phospholipid-dependent arf GTPase-activating protein that associates with and is phosphorylated by Src.

Membrane trafficking is regulated in part by small GTP-binding proteins of the ADP-ribosylation factor (Arf) family. Arf function depends on the controlled exchange and hydrolysis of GTP. We have purified and cloned two variants of a 130-kDa phosphatidylinositol 4, 5-biphosphate (PIP2)-dependent Arf1 GTPase-activating protein (GAP), which we call ASAP1a and ASAP1b. Both contain a pleckstrin homology (PH) domain, a zinc finger similar to that found in another Arf GAP, three ankyrin (ANK) repeats, a proline-rich region with alternative splicing and SH3 binding motifs, eight repeats of the sequence E/DLPPKP, and an SH3 domain. Together, the PH, zinc finger, and ANK repeat regions possess PIP2-dependent GAP activity on Arf1 and Arf5, less activity on Arf6, and no detectable activity on Arl2 in vitro. The cDNA for ASAP1 was independently identified in a screen for proteins that interact with the SH3 domain of the tyrosine kinase Src. ASAP1 associates in vitro with the SH3 domains of Src family members and with the Crk adapter protein. ASAP1 coprecipitates with Src from cell lysates and is phosphorylated on tyrosine residues in cells expressing activated Src. Both coimmunoprecipitation and tyrosine phosphorylation depend on the same proline-rich class II Src SH3 binding site required for in vitro association. By directly interacting with both Arfs and tyrosine kinases involved in regulating cell growth and cytoskeletal organization, ASAP1 could coordinate membrane remodeling events with these processes.

ADP-Ribosylation Factor 1↗
PubMed1998

Localization of endogenous ARF6 to sites of cortical actin rearrangement and involvement of ARF6 in cell spreading.

To study the function of the endogenous ARF6 GTP binding protein in cells, we generated an antibody which specifically recognizes ARF6, and not the other ARF proteins. Using this antibody, ARF6 was detected in all mouse organs tested and in a variety of cultured cell lines including RBL, MDCK, NRK, BHK, COS, and HeLa cells. In NRK cells, by immunofluorescence, ARF6 localized to the plasma membrane, especially at regions exhibiting membrane ruffling, and was also concentrated in a fine punctate distribution in the juxtanuclear region. This pattern of localization of the endogenous protein was similar to the localization of ARF6 when overexpressed in NRK, or HeLa, cells. Treatments which perturb cortical actin in NRK cells, such as replating of cells after trypsinization or treatment with phorbol ester, resulted in the recruitment of endogenous ARF6 to the regions of cortical actin rearrangement. ARF6 activation and subsequent membrane recycling was required for cell spreading activity since expression of the dominant-negative, GTP-binding defective mutant of ARF6, T27N, previously shown to inhibit ARF6-regulated membrane recycling, inhibited cell attachment and spreading in HeLa cells. Furthermore, phorbol ester treatment enhanced the cell spreading activities in NRK cells, and in HeLa cells, but was not observed in cells expressing T27N. Taken together, these observations support a role for endogenous ARF6 in modeling the plasma membrane and cortical actin cytoskeleton.

ADP-Ribosylation Factors↗
PubMed1997

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

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.

ADP-Ribosylation Factor 6↗
PubMed1997

Characterization of chemical inhibitors of brefeldin A-activated mono-ADP-ribosylation.

Brefeldin A, a toxin inhibitor of vesicular traffic, induces the selective mono-ADP-ribosylation of two cytosolic proteins, glyceraldehyde-3-phosphate dehydrogenase and the novel GTP-binding protein BARS-50. Here, we have used a new quantitative assay for the characterization of this reaction and the development of specific pharmacological inhibitors. Mono-ADP-ribosylation is activated by brefeldin A with an EC50 of 17.0 +/- 3.1 microg/ml, but not by biologically inactive analogs including a brefeldin A stereoisomer. Brefeldin A acts by increasing the Vmax of the reaction, whereas it does not influence the Km of the enzyme for NAD+ (154 +/- 13 microM). The enzyme is an integral membrane protein present in most tissues and is modulated by Zn2+, Cu2+, ATP (but not by other nucleotides), pH, temperature, and ionic strength. To identify inhibitors of the reaction, a large number of drugs previously tested as blockers of bacterial ADP-ribosyltransferases were screened. Two classes of molecules, one belonging to the coumarin group (dicumarol, coumermycin A1, and novobiocin) and the other to the quinone group (ilimaquinone, benzoquinone, and naphthoquinone), rather potently and specifically inhibited brefeldin A-dependent mono-ADP-ribosylation. When tested in living cells, these molecules antagonized the tubular reticular redistribution of the Golgi complex caused by brefeldin A at concentrations similar to those active in the mono-ADP-ribosylation assay in vitro, suggesting a role for mono-ADP-ribosylation in the cellular actions of brefeldin A.

Adenosine Diphosphate↗
PubMed1996

Aluminum fluoride stimulates surface protrusions in cells overexpressing the ARF6 GTPase.

To study the effector function of the ADP- ribosylation factor (ARF) 6 GTP-binding protein, we transfected HeLa cells with wild-type, epitope-tagged ARF6. Previously shown to indirectly activate the ARF1 GTPase, aluminum fluoride (AIF) treatment of ARF6-transfected cells resulted in a redistribution of both ARF6 and actin to discrete sites on the plasma membrane, which became increasingly protrusive over time. The effects of AIF were reversible, specific to cells transfected with wild-type ARF6, and resembled the cellular protrusions observed in cells expressing the GTPase defective mutant of ARF6. Importantly, the protrusions observed in cells transfected with ARF6 were distinct from the enhanced stress fibers and membrane ruffles observed in cells transfected with RhoA and Rac1, respectively. In cells forming protrusions, there was an apparent stimulation of macropinocytosis and membrane recycling within the protrusive structures. In contrast, no block in transferrin uptake or alteration of the distribution of clathrin AP-2 complexes was detected in these cells. The AIF-induced, ARF6- dependent formation of protrusive structures was blocked by cytochalasin D and inhibitors of the lipoxygenase pathway. These observations support a novel role for the ARF6 GTPase in modeling the plasma membrane and underlying cytoskeleton.

ADP-Ribosylation Factor 1↗