The sec6/8 complex or exocyst is an octameric protein complex that functions during cell polarization by regulating the site of exocytic vesicle docking to the plasma membrane, in concert with small GTP-binding proteins. The Sec5 subunit of the mammalian sec6/8 complex binds Ral in a GTP-dependent manner. This study reports the crystal structure of the complex between the Ral-binding domain of Sec5 and RalA bound to a non-hydrolyzable GTP analog (GppNHp) at 2.1 Å resolution, providing the first structural insights into the mechanism and specificity of sec6/8 regulation. The Sec5 Ral-binding domain folds into an immunoglobulin-like beta-sandwich structure, which represents a novel fold for an effector of a GTP-binding protein. The interface between the two proteins involves a continuous antiparallel beta-sheet, similar to that found in other effector/G-protein complexes, such as Ras and Rap1A. Specific interactions unique to the RalA.Sec5 complex include Sec5 Thr11 and Arg27, and RalA Glu38, which are required for complex formation by isothermal titration calorimetry. Comparison of the structures of GppNHp- and GDP-bound RalA suggests a nucleotide-dependent switch mechanism for Sec5 binding (Fukai, 2003).
The exocyst complex is involved in the final stages of exocytosis, when vesicles are targeted to the plasma membrane and dock. The regulation of exocytosis is vital for a number of processes, for example, cell polarity, embryogenesis, and neuronal growth formation. Regulation of the exocyst complex in mammals was recently shown to be dependent upon binding of the small G protein, Ral, to Sec5, a central component of the exocyst. This interaction is thought to be necessary for anchoring the exocyst to secretory vesicles. The structure of the Ral-binding domain of Sec5 has been determined; it adopts a fold that has not been observed in a G protein effector before. This fold belongs to the immunoglobulin superfamily in a subclass known as IPT domains. The Ral binding site on this domain has been mapped; it overlaps with protein-protein interaction sites on other IPT domains but it is completely different from the G protein-geranyl-geranyl interaction face of the Ig-like domain of the Rho guanine nucleotide dissociation inhibitor. This mapping, along with available site-directed mutagenesis data, allows predictions of how Ral and Sec5 may interact (Mott, 2003).
The exocyst is a multiprotein complex that plays an important role in secretory vesicle targeting and docking at the plasma membrane. A new component of the exocyst, Exo84p, has been identified and characterized in the yeast Saccharomyces cerevisiae. Yeast cells depleted of Exo84p cannot survive. These cells are defective in invertase secretion and accumulate vesicles similar to those in the late sec mutants. Exo84p co-immunoprecipitates with the exocyst components, and a portion of the Exo84p co-sediments with the exocyst complex in velocity gradients. The assembly of Exo84p into the exocyst complex requires two other subunits, Sec5p and Sec10p. Exo84p interacts with both Sec5p and Sec10p in a two-hybrid assay. Overexpression of Exo84p selectively suppresses the temperature sensitivity of a sec5 mutant. Exo84p specifically localizes to the bud tip or mother/daughter connection, sites of polarized secretion in the yeast S. cerevisiae. Exo84p is mislocalized in a sec5 mutant. These studies suggest that Exo84p is an essential protein that plays an important role in polarized secretion (Guo, 1999a).
The exocyst is a protein complex required for the late stages of secretion in yeast. Unlike the SNAREs (SNAP receptors), important secretory proteins that are broadly distributed on the target membrane, the exocyst is specifically located at sites of vesicle fusion. cDNAs encoding the rexo70, rsec5, and rsec15 subunits of the mammalian complex have been isolated. The amino acid sequences encoded by these genes are between 21% and 24% identical to their yeast homologs. All three genes are broadly expressed and multiple transcripts are observed for rexo70 and rsec15. Characterization of cDNAs encoding the 84-kDa subunit of the mammalian complex revealed a novel protein. mAbs were generated to the mammalian rsec6 subunit of the exocyst complex. rsec6 immunoreactivity is found in a punctate distribution at terminals of PC12 cell processes at or near sites of granule exocytosis (Kee, 1997).
Delivery of cytoplasmic vesicles to discrete plasma-membrane domains is critical for establishing and maintaining cell polarity, neurite differentiation and regulated exocytosis. The exocyst is a multisubunit complex required for vectorial targeting of a subset of secretory vesicles. Mechanisms that regulate the activity of this complex in mammals are unknown. Sec5, an integral component of the exocyst, has been shown to be a direct target for activated Ral GTPases. Ral GTPases regulate targeting of basolateral proteins in epithelial cells, secretagogue-dependent exocytosis in neuroendocrine cells and assembly of exocyst complexes. These observations define Ral GTPases as critical regulators of vesicle trafficking (Moskalenko, 2002).
The Ras-related small GTPase RalA is involved in controlling actin cytoskeletal remodelling and vesicle transport in mammalian cells. The mammalian homolog of Sec5, a subunit of the exocyst complex determining yeast cell polarity, has been identified as a specific binding partner for GTP-ligated RalA. Inhibition of RalA binding to Sec5 prevents filopod production by tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 (IL-1) and by activated forms of RalA and Cdc42, signalling intermediates downstream of these inflammatory cytokines. It is proposed that the RalA-exocyst complex interaction integrates the secretory and cytoskeletal pathways (Sugihara, 2002).
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. GTP-bound ARF6 interacts with Sec10, a subunit of the exocyst complex involved in docking of vesicles with the plasma membrane. Sec10 localization in the perinuclear region is not restricted to the trans-Golgi network, but extends to recycling endosomes. In addition, 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. This 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 (Prigent, 2003).
Many secretory cells utilize a GTP-dependent pathway, in addition to the well characterized Ca(2+)-dependent pathway, to trigger exocytotic secretion. However, little is currently known about the mechanism by which this may occur. This study shows the key signaling pathway that mediates GTP-dependent exocytosis. Incubation of permeabilized PC12 cells with soluble RalA GTPase, but not RhoA or Rab3A GTPases, strongly inhibited GTP-dependent exocytosis. A Ral-binding fragment from Sec5, a component of the exocyst complex, showed a similar inhibition. Point mutations in both RalA [RalA(E38R)] and the Sec5 [Sec5(T11A)] fragment that abolish the RalA-Sec5 interaction also abolish the inhibition of GTP-dependent exocytosis. Moreover, transfection with wild-type RalA, but not RalA(E38R), enhances GTP-dependent exocytosis. In contrast RalA and the Sec5 fragment shows no inhibition of Ca(2+)-dependent exocytosis, but cleavage of a SNARE (soluble-N-ethylmaleimide-sensitive factor attachment protein receptor) protein by Botulinum neurotoxin blocked both GTP- and Ca(2+)-dependent exocytosis. These results indicate that the interaction between RalA and the exocyst complex (containing Sec5) is essential for GTP-dependent exocytosis. Furthermore, GTP- and Ca(2+)-dependent exocytosis use different sensors and effectors for triggering exocytosis while their final fusion steps are both SNARE-dependent (Wang, 2004).
RalA, a member of the Ras-family GTPases, regulates various cellular functions such as filopodia formation, endocytosis, and exocytosis. On epidermal growth factor (EGF) stimulation, activated Ras recruits guanine nucleotide exchange factors (GEFs) for RalA, followed by RalA activation. By using FRET-based probes for RalA activity, it was found that the EGF-induced RalA activation in Cos7 cells is restricted at the EGF-induced nascent lamellipodia, whereas under a similar condition both Ras activation and Ras-dependent translocation of Ral GEFs occurs more diffusely at the plasma membrane. This EGF-induced RalA activation is not observed when lamellipodial protrusion is suppressed by a dominant negative mutant of Rac1, a GAP for Cdc42, inhibitors of PI 3-kinase, or inhibitors of actin polymerization. In contrast, EGF-induced lamellipodial protrusion is inhibited by microinjection of the RalA-binding domains (RBD) of RalBP1 and Sec5. Furthermore, RalA activity is high at the lamellipodia of migrating MDCK cells and the migration of MDCK cells is perturbed by the microinjection of RalBP1-RBD. Thus, RalA activation is required for the induction of lamellipodia and, conversely, lamellipodial protrusion seems to be required for the RalA activation, suggesting the presence of a positive feedback loop between RalA activation and lamellipodial protrusion. These observations also demonstrate that the spatial regulation of RalA is conducted by a mechanism distinct from the temporal regulation conducted by Ras-dependent plasma membrane recruitment of Ral GEFs (Takaya, 2004).
Ral GTPases have been implicated in the regulation of a variety of dynamic cellular processes including proliferation, oncogenic transformation, actin-cytoskeletal dynamics, endocytosis, and exocytosis. Recently the Sec6/8 complex, or exocyst, a multisubunit complex facilitating post-Golgi targeting of distinct subclasses of secretory vesicles, has been identified as a bona fide Ral effector complex. Ral GTPases regulate exocyst-dependent vesicle trafficking and are required for exocyst complex assembly. Sec5, a membrane-associated exocyst subunit, has been identified as a direct target of activated Ral; however, the mechanism by which Ral can modulate exocyst assembly is unknown. An additional component of the exocyst, Exo84, has been shown to be a direct target of activated Ral. Evidence is provided that mammalian exocyst components are present as distinct subcomplexes on vesicles and the plasma membrane, and Ral GTPases regulate the assembly interface of a full octameric exocyst complex through interaction with Sec5 and Exo84 (Moskalenko, 2003).
In order to identify the function of deafness locus putative guanine nucleotide exchange factor (DelGEF), a protein homologous to the nucleotide exchange factor for the small GTPase Ran, a cDNA library was screened for interacting proteins using a yeast two-hybrid system. The human homolog of Sec5, a protein involved in vesicle transport and secretion, was identified as a binding partner. The interaction between DelGEF and Sec5 was found to be dependent on Mg2+ and stimulated by guanosine triphosphate (GTP) or deoxycytidine triphosphate (dCTP). Downregulation of endogenous DelGEF in HeLa cells induces increased extracellular secretion of proteoglycans indicating a possible role for DelGEF in the secretion process (Sjolinder, 2002).
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