A BAR Domain in the N Terminus of the Arf GAP ASAP1 Affects Membrane Structure and Trafficking of Epidermal Growth Factor Receptor

Abstract

The N-terminal BAR domain of ASAP1 mediates membrane bending and is necessary for ASAP1 function. The Arf dependence of the bending activity is consistent with ASAP1 functioning as an Arf effector.

Arf GTPase-activating Protein ASAP1 Interacts with Rab11 Effector FIP3 and Regulates Pericentrosomal Localization of Transferrin Receptor–positive Recycling Endosome

“…Homology modeling of the BAR domain of ASAP1 predicts positive electrostatic clusters on the concave surface. We found that, like other BAR domains, the BAR domain of ASAP1 mediates homodimerization and contributes to the formation of tubules from synthetic large unilamellar vesicles in vitro (Nie et al, 2006). Arf contributed to the tubulation induced by ASAP1, supporting the idea that the membrane curvature-inducing function is coupled with Arf signaling.…”

“…Arf GAP assay using ASAP1 was carried out as described previously (Che et al, 2005b;Nie et al, 2006). Briefly, purified myristoylated Arf1 or Arf6 were loaded with [␣-32 P]GTP in GTP loading buffer (25 mM HEPES, pH 7.4, 100 mM NaCl, 0.5 mM MgCl 2 , 1 mM EDTA, 1 mM ATP, and 1 mM DTT) with large unilamellar vesicles (LUVs) comprised of 500 M total phospholipids at 30°C.…”

“…IMPDH forms a homotetramer through its core domain (Sintchak et al, 1996). Because both ASAP1 and FIP3 form homodimers (Eathiraj et al, 2006;Nie et al, 2006;Shiba et al, 2006), they might function as heterotetramer. FIP3 is comprised of an N-terminal Pro-rich domain, two EF-hand motifs and, in the C-terminal half of the molecule, a coiled-coil domain.…”

ArfGAP1 promotes COPI vesicle formation by facilitating coatomer polymerization

“…Imaging of LUVs by negative staining was performed as previously described. 75 LUVs were prepared as described 10,13,75 and incubated at a total phospholipid concentration of 1 mM with the indicated combinations of 0.1 mM myrArf1•GTPgS or myrArf1•GTP, 0.1 mM ArfGAP1 or ArfGAP2, 124 nM coatomer and 5 mM palmitoylated p25 peptide in a total volume of 25 ml for 10 min at 30°C.…”

“…effect of arfGap1 and arfGap2 on LUVs. LUVs were formed by extrusion through membrane with 1.0 mm pore size as described 75 and consisted of 40% phosphatidylcholine (pC), 25% phosphatidylethanolamine (pe), 15% phosphatidylserine (ps), 9% phosphatidylinositol (pI), and 10% cholesterol and 1% phosphatidylinositol 4-phosphate (pI4p). LUVs were incubated with the proteins and peptide indicated in the figure at the following concentrations: bovine serum albumin (Bsa), 5 mM; myrArf1•GTPγS, 0.1 mM; Arf1•GTP, 0.1 mM; arfGap1, 0.1 mM; arfGap2, 0.1 mM; coatomer, 0.124 mM; palmitoylated p25 peptide, 5 mM.…”