Large Guanine Nucleotide Exchange Factors Research Articles

Structural basis for activation of Arf1 at the Golgi complex.

SUMMARYThe Golgi complex is the central sorting station of the eukaryotic secretory pathway. Traffic through the Golgi requires activation of Arf guanosine triphosphatases that orchestrate cargo sorting and vesicle formation by recruiting an array of effector proteins. Arf activation and Golgi membrane association is controlled by large guanine nucleotide exchange factors (GEFs) possessing multiple conserved regulatory domains. Here we present cryoelectron microscopy (cryoEM) structures of full-length Gea2, the yeast paralog of the human Arf-GEF GBF1, that reveal the organization of these regulatory domains and explain how Gea2 binds to the Golgi membrane surface. We find that the GEF domain adopts two different conformations compatible with different stages of the Arf activation reaction. The structure of a Gea2-Arf1 activation intermediate suggests that the movement of the GEF domain primes Arf1 for membrane insertion upon guanosine triphosphate binding. We propose that conformational switching of Gea2 during the nucleotide exchange reaction promotes membrane insertion of Arf1.

The Pleckstrin Homology Domain of the Arf6-specific Exchange Factor EFA6 Localizes to the Plasma Membrane by Interacting with Phosphatidylinositol 4,5-Bisphosphate and F-actin

The Arf6-specific exchange factor EFA6 coordinates membrane trafficking with actin cytoskeleton remodeling. It localizes to the plasma membrane where it catalyzes Arf6 activation and induces the formation of actin-based membrane ruffles. We have shown previously that the pleckstrin homology (PH) domain of EFA6 was responsible for its membrane localization. In this study we looked for the partners of the PH domain at the plasma membrane. Mutations of the conserved basic residues suspected to be involved in the binding to phosphoinositides redistribute EFA6-PH to the cytosol. In addition, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) breakdown also leads to the solubilization of EFA6-PH. Direct binding measured by surface plasmon resonance gives an apparent affinity of approximately 0.5 microm EFA6-PH for PI(4,5)P2. Moreover, we observed in vitro that the catalytic activity of EFA6 is strongly increased by PI(4,5)P2. These results indicate that the plasma membrane localization of EFA6-PH is based on its interaction with PI(4,5)P2, and this interaction is necessary for an optimal catalytic activity of EFA6. Furthermore, we demonstrated by fluorescence recovery after photobleaching and Triton X-100 detergent solubility experiments that in addition to the phophoinositides, EFA6-PH is linked to the actin cytoskeleton. We observed both in vivo and in vitro that EFA6-PH interacts directly with F-actin. Finally, we demonstrated that EFA6 could bind simultaneously filamentous actin and phospholipids vesicles. Our results explain how the exchange factor EFA6 via its PH domain could coordinate at the plasma membrane actin cytoskeleton organization with membrane trafficking.

The domain architecture of large guanine nucleotide exchange factors for the small GTP-binding protein Arf

BackgroundSmall G proteins, which are essential regulators of multiple cellular functions, are activated by guanine nucleotide exchange factors (GEFs) that stimulate the exchange of the tightly bound GDP nucleotide by GTP. The catalytic domain responsible for nucleotide exchange is in general associated with non-catalytic domains that define the spatio-temporal conditions of activation. In the case of small G proteins of the Arf subfamily, which are major regulators of membrane trafficking, GEFs form a heterogeneous family whose only common characteristic is the well-characterized Sec7 catalytic domain. In contrast, the function of non-catalytic domains and how they regulate/cooperate with the catalytic domain is essentially unknown.ResultsBased on Sec7-containing sequences from fully-annotated eukaryotic genomes, including our annotation of these sequences from Paramecium, we have investigated the domain architecture of large ArfGEFs of the BIG and GBF subfamilies, which are involved in Golgi traffic. Multiple sequence alignments combined with the analysis of predicted secondary structures, non-structured regions and splicing patterns, identifies five novel non-catalytic structural domains which are common to both subfamilies, revealing that they share a conserved modular organization. We also report a novel ArfGEF subfamily with a domain organization so far unique to alveolates, which we name TBS (TBC-Sec7).ConclusionOur analysis unifies the BIG and GBF subfamilies into a higher order subfamily, which, together with their being the only subfamilies common to all eukaryotes, suggests that they descend from a common ancestor from which species-specific ArfGEFs have subsequently evolved. Our identification of a conserved modular architecture provides a background for future functional investigation of non-catalytic domains.

Molecular characterization of radiation- and chemically induced mutations associated with neuromuscular tremors, runting, juvenile lethality, and sperm defects in jdf2 mice.

The juvenile development and fertility-2 (jdf2) locus, also called runty-jerky-sterile (rjs), was originally identified through complementation studies of radiation-induced p-locus mutations. Studies with a series of ethylnitrosourea (ENU)-induced jdf2 alleles later indicated that the pleiotropic effects of these mutations were probably caused by disruption of a single gene. Recent work has demonstrated that the jdf2 phenotype is associated with deletions and point mutations in Herc2, a gene encoding an exceptionally large guanine nucleotide exchange factor protein thought to play a role in vesicular trafficking. Here we describe the molecular characterization of a collection of radiation- and chemically induced jdf2/Herc2 alleles. Ten of the 13 radiation-induced jdf2 alleles we studied are deletions that remove specific portions of the Herc2 coding sequence; DNA rearrangements were also detected in two additional mutations. Our studies also revealed that Herc2 transcripts are rearranged, not expressed, or are present in significantly altered quantities in animals carrying most of the jdf2 mutations we analyzed, including six independent ENU-induced alleles. These data provide new molecular clues regarding the wide range of jdf2 and p phenotypes that are expressed by this collection of recently generated and classical p-region mutations.