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ARF-GEP100, A Guanine Nucleotide-Exchange Protein for ADP-Ribosylation Factor 6
Akimasa Someya, Makoto Sata, Kazuyo Takeda, Gustavo Pacheco-Rodriguez, Victor J. Ferrans, Joel Moss and Martha Vaughan
Proceedings of the National Academy of Sciences of the United States of America
Vol. 98, No. 5 (Feb. 27, 2001), pp. 2413-2418
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3055070
Page Count: 6
You can always find the topics here!Topics: Acute kidney failure, Proteins, Amino acids, Complementary DNA, Yeasts, Phospholipids, pH, Cell membranes, Antibodies, Phosphatidylinositols
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A human cDNA encoding an 841-aa guanine nucleotide-exchange protein (GEP) for ADP-ribosylation factors (ARFs), named ARF-GEP100, 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 ≈8-kb mRNA that hybridized with an ARF-GEP100 cDNA was abundant in peripheral blood leukocytes, brain, and spleen. ARF-GEP100 accelerated [35S]GTPγS binding to ARF1 (class I) and ARF5 (class II) 2- to 3-fold, and to ARF6 (class III) ca. 12-fold. The ARF-GEP100 Sec7 domain contains Asp543 and Met555, corresponding to residues associated with sensitivity to the inhibitory effect of the fungal metabolite brefeldin A (BFA) in yeast Sec7, but also Phe535 and Ala536, 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-GEP100 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-GEP100, 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-GEP100 in those areas. No similar coincidence of ARF-GEP100 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.
Proceedings of the National Academy of Sciences of the United States of America © 2001 National Academy of Sciences