ARF-GEP(100), a guanine nucleotide-exchange protein for ADP-ribosylation factor 6 - PubMed (original) (raw)

ARF-GEP(100), a guanine nucleotide-exchange protein for ADP-ribosylation factor 6

A Someya et al. Proc Natl Acad Sci U S A. 2001.

Abstract

A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP(100), which contains a Sec7 domain, a pleckstrin homology (PH)-like domain, and an incomplete IQ-motif, was identified. On Northern blot analysis of human tissues, a approximately 8-kb mRNA that hybridized with an ARF-GEP(100) cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP(100) accelerated [(35)S]GTPgammaS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP(100) Sec7 domain contains Asp(543) and Met(555), corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe(535) and Ala(536), associated with BFA-insensitivity. The PH-like domain differs greatly from those of other ARF GEPs in regions involved in phospholipid binding. Consistent with its structure, ARF-GEP(100) activity was not affected by BFA or phospholipids. After subcellular fractionation of cultured T98G human glioblastoma cells, ARF6 was almost entirely in the crude membrane fraction, whereas ARF-GEP(100), a 100-kDa protein detected with antipeptide antibodies, was cytosolic. On immunofluorescence microscopy, both proteins had a punctate pattern of distribution throughout the cells, with apparent colocalization only in peripheral areas. The coarse punctate distribution of EEA-1 in regions nearer the nucleus appeared to coincide with that of ARF-GEP(100) in those areas. No similar coincidence of ARF-GEP(100) with AP-1, AP-2, catenin, LAMP-1, or 58K was observed. The new human BFA-insensitive GEP may function with ARF6 in specific endocytic processes.

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Figures

Figure 1

Figure 1

Predicted structure of ARF-GEP100. (A) Schematic representation of putative functional domains and location of cDNA probe sequence in ARF-GEP100. (B) Amino acid sequence alignment of Sec7 domain of ARF-GEP100 (p100) with those of cytohesin 1 (C-1) and BIG1. (C) Alignment of Sec7 domain motif 2 and adjacent sequence from p100, EFA6, ARNO, C-1, yeast Sec7 domain (Sec7), Gea2, and BIG1.

Figure 2

Figure 2

Northern blot analysis of ARF-GEP100 mRNA in human tissues. A blot with poly(A)+ RNA from the indicated tissues was hybridized with the 586-bp ARF-GEP100 (p100) cDNA. After stripping, the blot was hybridized with GAPDH cDNA.

Figure 3

Figure 3

Binding of [35S]GTPγS to ARFs 1, 5, and 6. (A) Samples (25 pmol) of ARF1 (●), ARF5 (▴), or ARF6 (■) were incubated for 20 min at 30°C with 4 μM [35S]GTPγS and the indicated amount of ARF-GEP100. (B) Samples (25 pmol) of ARF1, ARF5, or ARF6 were incubated with 4 μM [35S]GTPγS for indicated time at 30°C without (○), or with 2.5 pmol of ARF-GEP100 (■) or cytohesin-1 Sec7 (▴). Data are means ± SEM of values from triplicate assays. Findings were replicated at least three times with different protein preparations.

Figure 4

Figure 4

Effect of BFA and phospholipids on [35S]GTPγS binding to ARF 6. (A) Samples (25 pmol) of ARF6 plus 2.5 pmol of ARF-GEP100 (■) or yeast ARF2 (25 pmol) plus 2.5 pmol of yeast Sec7 (●) were incubated for 4 h at 4°C with 4 μM [35S]GTPγS and the indicated amounts of BFA. (B) Samples (25 pmol) of ARF6 or ARF1 were incubated without or with 2.5 pmol of ARF-GEP100 (p100) for 20 min at 30°C with 4 μM [35S]GTPγS and the indicated amount of phospholipid (PS, PIP2, PIP3). Data are means ± SEM of values from triplicate assays. Experiments were repeated twice with similar results.

Figure 5

Figure 5

Effect of calcium and calmodulin on [35S]GTPγS. Samples (25 pmol) of ARF6 were incubated for 20 min at 30°C with 2.5 pmol of ARF-GEP100 and 4 μM [35S]GTPγS without (□) or with (■) 100 nM calmodulin and the indicated concentration of CaCl2(Ca2+) (Left), or without (○) or without (●) 100 μM CaCl2 and the indicated concentration of CaM (Right). Data are means ± SEM of values from triplicate assays. IQ motif sequence above is compared with sequence of ARF-GEP100 (p100).

Figure 6

Figure 6

Subcellular distribution of ARF-GEP100 and ARF6. T98G cells were homogenized in buffer with 1 mM MgCl2 (Mg2+) or 1 mM EDTA, and postnuclear supernatant, crude membrane (M), and cytosol (C) fractions were prepared. Samples were subjected to SDS/PAGE and immunoblotting with anti-ARF-GEP100 (p100) and anti-ARF6 antibodies. Experiment was replicated three times.

Figure 7

Figure 7

Intracellular distribution of ARF-GEP100 and ARF6 in T98G cells. Cells were reacted with rabbit anti-ARF-GEP100 (p100) antibody and mouse anti-ARF6, followed by Texas Red-labeled anti-rabbit IgG and FITC-labeled anti-mouse IgG. Lower right panel is a composite of the superimposed images. Lower left panel shows the corresponding Normarski image.

Figure 8

Figure 8

Confocal images of T98G cells stained for ARF-GEP100 and organelle marker proteins. Cells were reacted with rabbit anti-ARF-GEP100 (p100) antibody and mouse monoclonal antibody against β-catenin, LAMP-1, Golgi 58K protein, EEA-1, AP-1, or AP-2, followed by Texas Red-labeled anti-rabbit IgG and FITC-labeled anti-mouse IgG. In the first two columns are pairs of images of the same cells. Images in the third column are superimposed images of the preceding panels. In the fourth column are Nomarski images.

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