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. 2008 Nov 17;183(4):725-35.
doi: 10.1083/jcb.200806140.

Differential Roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI Trafficking

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Free PMC article

Differential Roles of ArfGAP1, ArfGAP2, and ArfGAP3 in COPI Trafficking

Carolin Weimer et al. J Cell Biol. .
Free PMC article

Abstract

The formation of coat protein complex I (COPI)-coated vesicles is regulated by the small guanosine triphosphatase (GTPase) adenosine diphosphate ribosylation factor 1 (Arf1), which in its GTP-bound form recruits coatomer to the Golgi membrane. Arf GTPase-activating protein (GAP) catalyzed GTP hydrolysis in Arf1 triggers uncoating and is required for uptake of cargo molecules into vesicles. Three mammalian ArfGAPs are involved in COPI vesicle trafficking; however, their individual functions remain obscure. ArfGAP1 binds to membranes depending on their curvature. In this study, we show that ArfGAP2 and ArfGAP3 do not bind directly to membranes but are recruited via interactions with coatomer. In the presence of coatomer, ArfGAP2 and ArfGAP3 activities are comparable with or even higher than ArfGAP1 activity. Although previously speculated, our results now demonstrate a function for coatomer in ArfGAP-catalyzed GTP hydrolysis by Arf1. We suggest that ArfGAP2 and ArfGAP3 are coat protein-dependent ArfGAPs, whereas ArfGAP1 has a more general function.

Figures

Figure 1.
Figure 1.
Expression and purification of recombinant ArfGAP1, ArfGAP2, and ArfGAP3 in Sf9 insect cells. (A) Schematic view of the recombinant fusion proteins of ArfGAP1, ArfGAP2, and ArfGAP3 used in this study. H, His6 tag. (B) Expression and purification of the recombinant ArfGAPs. Suspension cultures of Sf9 insect cells were infected with recombinant baculovirus containing the coding sequences for rat ArfGAP1, ArfGAP2, or ArfGAP3 fused to an N-terminal His6 tag. Cells were harvested after 72 h of incubation, and lysates (L) of cells infected with recombinant baculovirus and of noninfected control cells (C) were analyzed for expression of the recombinant proteins by SDS-PAGE and Coomassie staining. Proteins were purified by Ni affinity chromatography, and 2 μg of the eluates (E) were loaded for SDS-PAGE.
Figure 2.
Figure 2.
Quantification and ultrastructural localization of ArfGAPs in mammalian cells. (A and B) Ultrastructural localization of ArfGAPs. Ultrathin cryosections of NRK cells were stained with antibodies specific for ArfGAP1, ArfGAP2, or ArfGAP3 followed by incubation with protein A–coupled gold particles of 15-nm size. GM130 antibody was used as a cis-Golgi marker and visualized with protein A–coupled gold particles of 10-nm size. (A) Micrographs of representative evaluated Golgis. (B) Statistical evaluation of the normalized relative labeling densities of the three ArfGAPs at cis- and trans-halves of the Golgi. n, number of Golgis evaluated. (C) Quantification of ArfGAPs in mammalian cells. Lysates of NRK cells were separated by SDS-PAGE and stained with antibodies against ArfGAP1, ArfGAP2, ArfGAP3, Arf1, and ε-COP in Western blots. Signals were quantified on the basis of standards of the respective recombinant proteins. The diagram shows the amounts in picomoles per milligram of total protein in the lysates of seven independent experiments. (B and C) Error bars represent standard deviations of the mean.
Figure 3.
Figure 3.
Activity of ArfGAP1, ArfGAP2, and ArfGAP3 on liposomes of different sizes. GAP activity was measured by following the change of tryptophan fluorescence in Arf1 (Bigay et al., 2005). Buffer, liposomes extruded through 200- or 30-nm polycarbonate filters, myristoylated full-length Arf1 (1-μM final concentration), and GTP were mixed in a cuvette. After the addition of EDTA, the fluorescence increases because of the exchange of GDP for GTP in Arf1. The GTP state was stabilized by increasing the Mg2+ concentration at the indicated time points. ArfGAPs were added to a final concentration of 50 nM, 500 nM, or 1 μM, resulting in a decrease of tryptophan fluorescence caused by GTP hydrolysis. For comparative analysis, fluorescence at time point 0 was set to 0 arbitrary units (AU), fluorescence of the GTP state was normalized to 100 AU, and the shift in fluorescence resulting from tryptophan residues in the ArfGAP proteins was subtracted. For each ArfGAP concentration and liposomal diameter, at least three curves were averaged.
Figure 4.
Figure 4.
Binding of ArfGAPs to membranes. (A) Binding of ArfGAPs to liposomes of different sizes. ArfGAPs were mixed with liposomes (extruded through 200-, 80-, or 30-nm filters, or no liposomes as a control) to a final concentration of 0.5 μM of protein and 0.5 mM of lipids in a total volume of 200 μl. After 5 min of incubation at RT, the samples were floated to an interphase between 25 and 0% (wt/vol) sucrose. 10% of the collected fractions were analyzed for the presence of ArfGAPs by SDS-PAGE and Western blotting with specific antibodies against ArfGAP1, ArfGAP2, and ArfGAP3 and were compared with 1 and 5% of the input. (B) Binding of ArfGAPs to Golgi membranes. Rat liver Golgi membranes were incubated with ArfGAP proteins, myristoylated Arf1, coatomer, and the slowly hydrolyzable GTP analogue GTPγS in the combinations indicated. Golgi membranes were pelleted through a 20% (vol/vol) sucrose cushion, and 30% of the sample was analyzed for bound proteins by SDS-PAGE and Western blotting with antibodies directed against the indicated proteins. MW, molecular weight.
Figure 5.
Figure 5.
Quantification of uncoating activities of ArfGAPs in COPI vesicle formation. (A) COPI vesicles were formed using salt-washed Golgi membranes and recombinant proteins in the presence of varying amounts of ArfGAP1, ArfGAP2, or ArfGAP3. COPI-coated vesicles were purified via sucrose density centrifugation. 50% of the vesicle fractions (V) and 0.5% of input (I) were analyzed by Western blotting with antibodies directed against δ-COP, Arf1, and p23. MW, molecular weight. (B) Western blot signals for p23, Arf1, and δ-COP were quantified and normalized to the signals in control reactions without the addition of ArfGAP. Results of three independent experiments were averaged. Error bars represent standard deviations of the mean.
Figure 6.
Figure 6.
Uncoating activities of ArfGAPs measured by light scattering. (A–D) The coat assembly–disassembly cycle was measured by light scattering at 350 nm in a spectrofluorometer. Liposomes supplemented with 2 mol% of p23 lipopeptide and extruded through 200-, 80-, or 30-nm polycarbonate filters (dynamic light-scattering assays showed a mean diameter of 160 nm, 60 nm, and 40 nm, respectively; Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200806140/DC1) or salt-washed Golgi membranes were incubated with Arf1, coatomer, and EDTA. At the time points indicated, GTP or its slowly hydrolyzable analogue GTPγS was added to start the coating reaction. After 10 min, the GTP state was stabilized with MgCl2. ArfGAPs (ArfGAP1, blue lines; ArfGAP2, gray lines; ArfGAP3, black lines) were added at the indicated time points to induce coat disassembly. Scattering at time point 0 was set to 0 AU, and scattering of the coated state was normalized to 100 AU. (A) 160 nm liposomes and 2.5 nM ArfGAPs; (B) 60 nm liposomes and 2.5 nM ArfGAPs; (C) 40 nm liposomes and 2.5 nM ArfGAPs; (D) Golgi membranes and 1 and 5 nM ArfGAPs.

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