The small guanosine triphosphate (GTP)–binding protein ADP-ribosylation factor (ARF) 6 regulates membrane recycling to regions of plasma membrane remodeling via the endocytic pathway. Here, we show that GTP–bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. We found that Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, we report that depletion of Sec5 exocyst subunit or dominant inhibition of Sec10 affects the function and the morphology of the recycling pathway. Sec10 is found to redistribute to ruffling areas of the plasma membrane in cells expressing GTP-ARF6, whereas dominant inhibition of Sec10 interferes with ARF6-induced cell spreading. Our paper suggests that ARF6 specifies delivery and insertion of recycling membranes to regions of dynamic reorganization of the plasma membrane through interaction with the vesicle-tethering exocyst complex.

The P2X7 receptor (P2X7R) is an ATP-gated cationic channel expressed by hematopoietic, epithelial, and neuronal cells. Prolonged ATP exposure leads to the formation of a nonselective pore, which can result in cell death. We show that P2X7R is associated with detergent-resistant membranes (DRMs) in both transfected human embryonic kidney (HEK) cells and primary macrophages independently from ATP binding. The DRM association requires the posttranslational modification of P2X7R by palmitic acid. Treatment of cells with the palmitic acid analog 2-bromopalmitate as well as mutations of cysteine to alanine residues abolished P2X7R palmitoylation. Substitution of the 17 intracellular cysteines of P2X7R revealed that 4 regions of the carboxyl terminus domain are involved in palmitoylation. Palmitoylation-defective P2X7R mutants showed a dramatic decrease in cell surface expression because of their retention in the endoplasmic reticulum and proteolytic degradation. Taken together, our data demonstrate that P2X7R palmitoylation plays a critical role in its association with the lipid microdomains of the plasma membrane and in the regulation of its half-life.

IB␣ inhibits the transcriptional activity of NF-B both in the cytoplasm by preventing the nuclear translocation of NF-B and in the nucleus where it dissociates NF-B from DNA and transports it back to the cytoplasm. Cytoplasmic localization of inactive NF-B/IB␣ complexes is controlled by mutual masking of nuclear import sequences of NF-B p65 and IB␣ and active CRM1-mediated nuclear export. Here, we describe an additional mechanism accounting for the cytoplasmic anchoring of IB␣ or NF-B/IB␣ complexes. The N-terminal domain of IB␣ contains a sequence responsible for the cytoplasmic retention of IB␣ that is specifically recognized by G3BP2, a cytoplasmic protein that interacts with both IB␣ and IB␣/NF-B complexes. G3BP2 is composed of an N-terminal domain homologous to the NTF2 protein, followed by an acidic domain sufficient for the interaction with the IB␣ cytoplasmic retention sequence, a region containing five PXXP motifs and a C-terminal domain containing RNA-binding motifs. Overexpression of G3BP2 directly promotes retention of IB␣ in the cytoplasm, indicating that subcellular distribution of IB␣ and NF-B/IB␣ complexes likely results from a equilibrium between nuclear import, nuclear export, and cytoplasmic retention. The molecular organization of G3BP2 suggests that this putative scaffold protein might connect the NF-B signal transduction cascade with cellular functions such as nuclear transport or RNA metabolism.Rel/NF-B transcription factors play a major role in inducible expression of a number of cellular genes involved in immune, inflammatory, and anti-apoptotic responses (1-3). Human NF-B is composed of a homo-or heterodimer of proteins that belong to the multigene family of transcription factors comprising p50, p52, p65/RelA, c-Rel, and RelB (4 -12). The prototypical NF-B is a heterodimeric p50/p65 molecule. Each member of NF-B/Rel family of proteins contains a Rel homology domain that is responsible for nuclear translocation, dimerization, and sequence-specific DNA binding. In most unstimulated cells, NF-B is retained in an inactive form in the cytoplasm through its association with the IB inhibitor proteins (13-16). IBs also belong to a multigene family of proteins including IB␣, IB␤, IB⑀, Bcl-3, and also the C-terminal domains of p50 and p52 precursors (p105 and p100, respectively) that in isolation are known as IB␥ and IB␦, respectively (17-25). Members of the IB family contain multiple conserved ankyrin repeat domains that interact with NF-B factors such that their nuclear localization sequences (NLS) 1 are masked, leading to cytoplasmic retention of the complex. IB proteins are also characterized by their ability to inhibit NF-B DNA binding activity.IB␣ is composed of a surface-exposed N-terminal domain, a central region containing six ankyrin repeat domains, and a highly acidic C-terminal domain. Upon stimulation of cells with appropriate signals such as tumor necrosis factor or interleukin 1, a signaling cascade is initiated leading to activation of two IB␣ kinases, IKK-1 and IKK-2, which phosph...