Discrete determinants in ArfGAP2/3 conferring Golgi localization and regulation by the COPI coat

Abstract

From yeast to mammals, two types of GTPase-activating proteins, ArfGAP1 and ArfGAP2/3, control guanosine triphosphate (GTP) hydrolysis on the small G protein ADP-ribosylation factor (Arf) 1 at the Golgi apparatus. Although functionally interchangeable, they display little similarity outside the catalytic GTPase-activating protein (GAP) domain, suggesting differential regulation. ArfGAP1 is controlled by membrane curvature through its amphipathic lipid packing sensor motifs, whereas Golgi targeting of ArfGAP2 depends on coatomer, the building block of the COPI coat. Using a reporter fusion approach and in vitro assays, we identified several functional elements in ArfGAP2/3. We show that the Golgi localization of ArfGAP3 depends on both a central basic stretch and a carboxy-amphipathic motif. The basic stretch interacts directly with coatomer, which we found essential for the catalytic activity of ArfGAP3 on Arf1-GTP, whereas the carboxy-amphipathic motif interacts directly with lipid membranes but has minor role in the regulation of ArfGAP3 activity. Our findings indicate that the two types of ArfGAP proteins that reside at the Golgi use a different combination of protein-protein and protein-lipid interactions to promote GTP hydrolysis in Arf1-GTP.

Figure 1.

ER diversion of CD4 localization by its fusion to ArfGAPs. (A) This panel shows the switch of the localization of CD4 in HeLa cells from the plasma membrane to the ER upon fusion to ArfGAP1-3. Compartment markers used are cyan fluorescent protein (CFP)-SRβ (ER) and yellow fluorescent protein (YFP)-GPI anchor signal (plasma membrane). CD4-KKTN served as control for ER retrieval. (B) The cells were cotransfected with the CD4 fusions along with GFP used as transfection marker and were fixed and stained with anti-CD4 without permeabilization; note that only wild-type CD4 reacts under these conditions, which selectively detect the surface receptor. (C) The Golgi complex remains intact in CD4-ArfGAP–transfected cells. The Golgi was labeled by cotransfection with CFP-N-acetylgalactoseamine transferase.

Figure 2.

Truncation analysis of ArfGAP3. (A) Truncation analysis of CD4 fusions of ArfGAP3. Bars summarize the localizations observed, and regions containing determinants that contribute to ER localization of CD4 fusions are indicated as black boxes in the bottom bar. (B) Coatomer pull-down from liver cytosol by using nickel-nitrilotriacetic acid-agarose-immobilized ArfGAP3 fragments.

Figure 3.

Identification of ArfGAP3 residues involved in ER diversion of CD4-ArfGAP3(204-360). (A) The sequence of the middle part of ArfGAP3 is displayed, with mutations that abrogated ER localization in red, mutations that had no effect in blue, and those with partial effect in orange. The corresponding images are displayed in B. Note the abundance of abrogating mutations in the basic stretch including residues 215-242. Multispecies alignment (C) shows the high conservation of these residues (Hs, Homo sapiens; Mm, Mus musculus; Xl, Xenopus laevis; Tn, Tetraodon nigroviridis; Gg, Gallus gallus; Dm, Drosophila melanogaster; Sc, S. cerevisiae).

Figure 4.

The basic stretch is involved in coatomer binding. Effect of alanine replacement mutations on coatomer pull-down by ArfGAP3(204-360) (A) and by full-length ArfGAP3 (B).

Figure 5.

ArfGAP3 regions involved in Golgi targeting and effect of mutations that disrupt coatomer interaction. (A) Truncation analysis of GFP-ArfGAP3 fusions. (B) Mutations that abrogate coatomer interaction also diminish Golgi localization of GFP-full length ArfGAP3. Staining with antibodies against the cis-Golgi protein GM130 was used to mark the Golgi region.

Figure 6.

Golgi localization depends on amphipathic motif at the carboxy terminus of ArfGAP3. (A) Helical wheel presentation of carboxy-terminal parts of ArfGAP2/3 and Glo3; part of the ALPS1 motif of ArfGAP1 is displayed for comparison. (B) Circular dichroism spectra of a synthetic peptide representing residues 484-510 of ArfGAP3 (70 μM) in the absence or presence of sonicated liposomes (2 mM), demonstrating liposome-induced transition to helical conformation. (C) Mutations expected to abrogate helix amphipathicity diminish Golgi localization of the full-length ArfGAP3. (D) Appending the ALPS1 motif (ArfGAP1 residues 198-240) to ArfGAP3(1-483) restores Golgi localization. This effect is abolished in the W211D mutant of the ALPS1 motif.

Figure 7.

Effect of aluminum fluoride on the localization of ArfGAPs and their mutants. (A) Aluminum fluoride confers Golgi localization on ArfGAP3 fragments lacking the carboxy-amphipathic motif. Note the strong effect of fluoride on the 1-303 fragment, and its abrogation upon mutations in the coatomer binding motif. (B) Fluoride cannot confer Golgi localization on full-length ArfGAP1 containing mutations in the ALPS motifs. Cells that have been transfected with GFP-ArfGAP3 fragments and mutants thereof or with GFP-ArfGAP1 and its ALPS mutants (M204A-L207A-W211A for ALPS1; V279D for ALPS2) were incubated with 30 mM NaF plus 50 μM AlNH₄(SO₄)₂ for 30 min at 37°C or left untreated (control).

Figure 8.

The activity of ArfGAP2/3 but not ArfGAP1 is strictly coatomer-dependent. (A) Time course of GTP hydrolysis in liposome-bound myristoylated Arf1 and with the indicated concentration of coatomer. The liposomes were prepared by extrusion through 0.03-μm pore size filters (mean radius, 35 nm) and contained 2 mol% of lp23. GTP hydrolysis was initiated by the addition of 25 nM ArfGAP1 (top), ArfGAP2 (middle), or ArfGAP3 (bottom) and was followed by tryptophan fluorescence. Note the lack of GTP hydrolysis when ArfGAP2/3 were used in the absence of coatomer. (B) Dose–response curves. The rate of GTP hydrolysis in Arf1 was measured as in described in A, with various concentrations of ArfGAP3 and coatomer. Maximal rate of GTP hydrolysis was observed with roughly equal amounts of the two proteins (coatomer was ∼80% pure), suggesting a functional one-to-one complex. The bottom curves show the corresponding fluorescence recordings.

Figure 9.

Disassembly from liposomes of the COPI coat by ArfGAP1 and ArfGAP3. Light-scattering at right angle was used to follow the assembly–disassembly cycle of the COPI coat on liposomes of defined radii. In a first stage myrArf1 (0.75 μM) was activated by the addition of GTP and by lowering the concentration of free Mg²⁺ with EDTA. Next, addition of coatomer (0.3 μM) induced an instantaneous jump reflecting assembly of the coat. COPI disassembly was then initiated by the addition of ArfGAP1 (5 nM; red curves) or ArfGAP3 (10 nM; black curves). Note that ArfGAP3 was more efficient than ArfGAP1 in promoting COPI disassembly on large (top row) or medium-size (middle row) liposomes. In striking contrast ArfGAP1 promoted much faster coat disassembly than ArfGAP3 on small liposomes (bottom row). Complete coatomer disassembly was achieved after further addition of 95 nM ArfGAP1 (at t = 24 min).

Figure 10.

Localization of ArfGAP3 determinants involved in coatomer stimulation. In all panels, GTP hydrolysis in liposome-bound Arf1 was measured as in Figure 8A in the presence of 30 nM ArfGAP3 or the indicated truncated/mutated forms. (A) Activity measurements of truncated ArfGAP3 forms in presence of 50 nM coatomer. A gradual decrease in activity is observed upon shortening the C-terminal end of ArfGAP3. The last truncation caused the most severe effect suggesting a key role of the central (196-303) region. (B) Activity measurements of full-length ArfGAP3 with 30 nM coatomer and with the indicated concentration of the (195-303) ArfGAP3 fragment. Specificity of the inhibitory effect was checked by the addition of boiled peptide. (C) Effect of point mutations in the central basic stretch of ArfGAP3. The activity of the mutants was determined in the presence of 50 nM coatomer. 4K(1) and 4K(2) correspond to full-length ArfGAP3 4K(215/16/22/23)4A and 4K(222-223-228-229)4A mutants, respectively.