exocytosis; Golgi; ARF-1

Exo1 modifies the GTPase activity of Golgi ARF1. Exo1 offers a separate method of interfering with Golgi activity because it blocks a portion of the membrane events that BFA blocks, although it appears to target a different protein target and likely has different side effects. Exo1 Disrupts Golgi structures to disrupt vesicular traffic from the ER to the Golgi apparatus. Exo1 has an IC50 of about 20 μM, which inhibits exocytosis. Exo1 inhibits newly synthesized proteins like VSVGts-GFP, transferrin, and MHC class I from acquiring endoglycosidase H resistance[1].

Tryptophan Fluorescence Assay for GDP/GTP Exchange.[1]
The fluorescence assay for measuring GTP binding to ARF1D17 stimulated by sec7 domain of ARF nucleotide binding site opener (ARNO) mutated in positions F190Y, A191S, S198M, and P208D was performed as described. ARF1D17 (5 mM) with 100 nM recombinant ARNO sec7 domain was assayed with 100 mM of Exo1 or 10 mM of BFA.

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Guanine Nucleotide Exchange Assays[1]
Whole membrane. Golgi membranes containing 5 mg of protein and different amounts of compound were incubated with 4 mM myristoylated recombinant ARF1 and 100 mM GTP[g-35S] at 30°C for 15 min. Radioactivity that bound to filters was measured by scintillation counting, and the result was corrected by the amount that bound in the absence of recombinant ARF (background was <20% of total). Recombinant GFB1, BFA-Inhibited ARF-GEF 1, and ARFs[1]
Reactions were performed at 30°C in the presence of 50 mM Hepes (pH 7.5), 100 mM KCl, 1 mM MgCl2, 1 mM DTT, 1.5 mg/ml of azolectin vesicles, and 4 mM 5'-[g-thio]-triphosphate (GTP[gS]) spiked with [35S]GTP[gS] (1250 Ci/mmol) to a final specific activity of 3 ´ 105 cpm/pmol, as described. Reactions contained either DMSO (final concentration 2.5%) or the indicated concentrations of BFA or Exo1 dissolved in DMSO. Reactions were initiated by addition of either 2 mM bovine ARF3/1 or 2 mM recombinant myristoylated human ARF5. Reactions (15 ml) were terminated by dilution into 2 ml of ice-cold wash buffer (50 mM Hepes, pH 7.5/100 mM KCl/10 mM MgCl2). Bound ARF-GTP[gS] was quantified by filtering samples through 0.45 mM nitrocellulose membranes, followed by four rinses with 2 ml of wash buffer. Dried filters in 3 ml of liquid scintillant were analyzed in an LS 6500. The amount of GEF-dependent GTP loaded on ARFs was calculated after subtracting background values measured in assays containing only azolectin vesicles, only M1, and only ARF-GEF.[1]
Bovine ARFs (mixture of ARF3 and ARF1) and recombinant myristoylated human ARF5 were purified as described. A hexahistidine-tagged fragment containing the Sec7 domain of the BFA-inhibited ARF-GEF 1 was purified as described. A GST-tagged form of GBF1 was constructed by subcloning a fragment encoding full-length GBF1 into a modified version of pCEP4 expressing GST. Monolayers of HEK-293 expressing GST-GBF1 were extracted in lysis buffer (PBS/0.1% Triton X-100/1 mM PMSF) containing a mixture of protease inhibitors. GST-GFB1 was partially purified by using Glutathione-Sepharose as per the manufacturer’s instructions. Beads recovered by centrifugation at 1,500 ´ g were washed twice with 2 vol of lysis buffer and once with buffer lacking detergent. Bound material was eluted with wash buffer containing 10 mM glutathione and assayed as described previously.

Endoglycosidase H (Endo H) Sensitivity[1]
. Cells (106) grown in a 6-cm dish were transduced with VSVGts-GFP adenovirus overnight at 40°C. Afterward, the cells were pulse-labeled with 140 mCi 35S-trans (ICN) for 30 min at 40°C followed by incubation with or without 100 mM Exo1 or 5 mM BFA for various times at 32°C and processed for endo H sensitivity of newly synthesized proteins.

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Time-Lapse Fluorescence Microscopy[1]
Cells were plated on 25-mm glass coverslips and transfected with the appropriate GFP fusion proteins. After 16-24 h at 37°C, fluorescence images were acquired at 37°C in living cells with a Nikon E300 inverted microscope (20 ´ 0.45 numerical aperture objective lens) by using a Hamamatsu ER charge-coupled device camera under control of METAMORPH. For dissociation experiments, images were collected 10 min before and 30 min after adding Exo1. To measure the amount of GFP signal associated with membranes, we first applied a threshold to each image such that only signals above the cytosolic background were considered to determine integrated intensity. The signal in the first image (100%) was used to normalize the values of the remaining time points. No corrections were imposed to correct for possible photobleaching, because no significant changes were detected during this interval. In most cases we present the averaged values corresponding to 4-6 cells from three independent experiments.

[1]. Exo1: a new chemical inhibitor of the exocytic pathway. Proc Natl Acad Sci U S A. 2003 May 27;100(11):6469-74.

2-[[(4-fluorophenyl)-oxomethyl]amino]benzoic acid methyl ester is a member of benzamides.

C15H12FNO3

273.26

273.08

C, 65.93; H, 4.43; F, 6.95; N, 5.13; O, 17.56

75541-83-2

310557

White to off-white solid powder

1.298 g/cm3

333.9ºC at 760 mmHg

155.8ºC

2.937

1

4

4

20

353

0

O=C(OC)C1=CC=CC=C1NC(C2=CC=C(F)C=C2)=O

KIAPWMKFHIKQOZ-UHFFFAOYSA-N

InChI=1S/C15H12FNO3/c1-20-15(19)12-4-2-3-5-13(12)17-14(18)10-6-8-11(16)9-7-10/h2-9H,1H3,(H,17,18)

methyl 2-[(4-fluorobenzoyl)amino]benzoate

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.5 mg/mL (9.15 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)