Rab family of small GTPases: an updated view on their regulation and functions.

The Rab family of small GTPases regulates intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. Like other small GTPases, Rabs cycle between two states, an active (GTP-loaded) state and an inactive (GDP-loaded) state, and their cycling is catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Because an active form of each Rab localizes on a specific organelle (or vesicle) and recruits various effector proteins to facilitate each step of membrane trafficking, knowing when and where Rabs are activated and what effectors Rabs recruit is crucial to understand their functions. Since the discovery of Rabs, they have been regarded as one of the central hubs for membrane trafficking, and numerous biochemical and genetic studies have revealed the mechanisms of Rab functions in recent years. The results of these studies have included the identification and characterization of novel GEFs, GAPs, and effectors, as well as post-translational modifications, for example, phosphorylation, of Rabs. Rab functions beyond the simple effector-recruiting model are also emerging. Furthermore, the recently developed CRISPR/Cas technology has enabled acceleration of knockout analyses in both animals and cultured cells and revealed previously unknown physiological roles of many Rabs. In this review article, we provide the most up-to-date and comprehensive lists of GEFs, GAPs, effectors, and knockout phenotypes of mammalian Rabs and discuss recent findings in regard to their regulation and functions.

A single class of ARF GTPase activated by several pathway-specific ARF-GEFs regulates essential membrane traffic in Arabidopsis.

In eukaryotes, GTP-bound ARF GTPases promote intracellular membrane traffic by mediating the recruitment of coat proteins, which in turn sort cargo proteins into the forming membrane vesicles. Mammals employ several classes of ARF GTPases which are activated by different ARF guanine-nucleotide exchange factors (ARF-GEFs). In contrast, flowering plants only encode evolutionarily conserved ARF1 GTPases (class I) but not the other classes II and III known from mammals, as suggested by phylogenetic analysis of ARF family members across the five major clades of eukaryotes. Instead, flowering plants express plant-specific putative ARF GTPases such as ARFA and ARFB, in addition to evolutionarily conserved ARF-LIKE (ARL) proteins. Here we show that all eight ARF-GEFs of Arabidopsis interact with the same ARF1 GTPase, whereas only a subset of post-Golgi ARF-GEFs also interacts with ARFA, as assayed by immunoprecipitation. Both ARF1 and ARFA were detected at the Golgi stacks and the trans-Golgi network (TGN) by both live-imaging with the confocal microscope and nano-gold labeling followed by EM analysis. ARFB representing another plant-specific putative ARF GTPase was detected at both the plasma membrane and the TGN. The activation-impaired form (T31N) of ARF1, but neither ARFA nor ARFB, interfered with development, although ARFA-T31N interfered, like ARF1-T31N, with the GDP-GTP exchange. Mutant plants lacking both ARFA and ARFB transcripts were viable, suggesting that ARF1 is sufficient for all essential trafficking pathways under laboratory conditions. Detailed imaging of molecular markers revealed that ARF1 mediated all known trafficking pathways whereas ARFA was not essential to any major pathway. In contrast, the hydrolysis-impaired form (Q71L) of both ARF1 and ARFA, but not ARFB, had deleterious effects on development and various trafficking pathways. However, the deleterious effects of ARFA-Q71L were abolished by ARFA-T31N inhibiting cognate ARF-GEFs, both in cis (ARFA-T31N,Q71L) and in trans (ARFA-T31N + ARFA-Q71L), suggesting indirect effects of ARFA-Q71L on ARF1-mediated trafficking. The deleterious effects of ARFA-Q71L were also suppressed by strong over-expression of ARF1, which was consistent with a subset of BIG1-4 ARF-GEFs interacting with both ARF1 and ARFA. Indeed, the SEC7 domain of BIG5 activated both ARF1 and ARFA whereas the SEC7 domain of BIG3 only activated ARF1. Furthermore, ARFA-T31N impaired root growth if ARF1-specific BIG3 was knocked out and only ARF1- and ARFA-activating BIG4 was functional. Activated ARF1 recruits different coat proteins to different endomembrane compartments, depending on its activation by different ARF-GEFs. Unlike ARF GTPases, ARF-GEFs not only localize at distinct compartments but also regulate specific trafficking pathways, suggesting that ARF-GEFs might play specific roles in traffic regulation beyond the activation of ARF1 by GDP-GTP exchange.

Multiplexed Real-Time NMR GTPase Assay for Simultaneous Monitoring of Multiple Guanine Nucleotide Exchange Factor Activities from Human Cancer Cells and Organoids.

Small GTPases (sGTPases) are critical switch-like regulators that mediate several important cellular functions and are often mutated in human cancers. They are activated by guanine nucleotide exchange factors (GEFs), which specifically catalyze the exchange of GTP for GDP. GEFs coordinate signaling networks in normal cells, and are frequently deregulated in cancers. sGTPase signaling pathways are complex and interconnected; however, most GEF assays do not reveal such complexity. In this Communication, we describe the development of a unique real-time NMR-based multiplexed GEF assay that employs distinct isotopic labeling schemes for each sGTPase protein to enable simultaneous observation of six proteins of interest. We monitor nucleotide exchange of KRas, Rheb, RalB, RhoA, Cdc42 and Rac1 in a single system, and assayed the activities of GEFs in lysates of cultured human cells and 3D organoids derived from pancreatic cancer patients. We observed potent activation of RhoA by lysates of HEK293a cells transfected with GEF-H1, along with weak stimulation of Rac1, which we showed is indirect. Our functional analyses of pancreatic cancer-derived organoids revealed higher GEF activity for RhoA than other sGTPases, in line with RNA-seq data indicating high expression of RhoA-specific GEFs.

FgMon1, a guanine nucleotide exchange factor of FgRab7, is important for vacuole fusion, autophagy and plant infection in Fusarium graminearum.

The Ccz1-Mon1 protein complex, the guanine nucleotide exchange factor (GEF) of the late endosomal Rab7 homolog Ypt7, is required for the late step of multiple vacuole delivery pathways, such as cytoplasm-to-vacuole targeting (Cvt) pathway and autophagy processes. Here, we identified and characterized the yeast Mon1 homolog in Fusarium graminearum, named FgMon1. FgMON1 encodes a trafficking protein and is well conserved in filamentous fungi. Targeted gene deletion showed that the ∆Fgmon1 mutant was defective in vegetative growth, asexual/sexual development, conidial germination and morphology, plant infection and deoxynivalenol production. Cytological examination revealed that the ∆Fgmon1 mutant was also defective in vacuole fusion and autophagy, and delayed in endocytosis. Yeast two hybrid and in vitro GST-pull down assays approved that FgMon1 physically interacts with a Rab GTPase FgRab7 which is also important for the development, infection, membrane fusion and autophagy in F. graminearum. FgMon1 likely acts as a GEF of FgRab7 and constitutively activated FgRab7 was able to rescue the defects of the ∆Fgmon1 mutant. In summary, our study provides evidences that FgMon1 and FgRab7 are critical components that modulate vesicle trafficking, endocytosis and autophagy, and thereby affect the development, plant infection and DON production of F. graminearum.

Regulating Rac in the nervous system: molecular function and disease implication of Rac GEFs and GAPs.

Rho family GTPases, including RhoA, Rac1, and Cdc42 as the most studied members, are master regulators of actin cytoskeletal organization. Rho GTPases control various aspects of the nervous system and are associated with a number of neuropsychiatric and neurodegenerative diseases. The activity of Rho GTPases is controlled by two families of regulators, guanine nucleotide exchange factors (GEFs) as the activators and GTPase-activating proteins (GAPs) as the inhibitors. Through coordinated regulation by GEFs and GAPs, Rho GTPases act as converging signaling molecules that convey different upstream signals in the nervous system. So far, more than 70 members of either GEFs or GAPs of Rho GTPases have been identified in mammals, but only a small subset of them have well-known functions. Thus, characterization of important GEFs and GAPs in the nervous system is crucial for the understanding of spatiotemporal dynamics of Rho GTPase activity in different neuronal functions. In this review, we summarize the current understanding of GEFs and GAPs for Rac1, with emphasis on the molecular function and disease implication of these regulators in the nervous system.

Rapgef2 connects GPCR-mediated cAMP signals to ERK activation in neuronal and endocrine cells.

G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptor (GPCR)-mediated increases in the second messenger cyclic adenosine monophosphate (cAMP) activate the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase (ERK), and in neuroendocrine cells, this pathway leads to cAMP-dependent neuritogenesis mediated through Rap1 and B-Raf. We found that the Rap guanine nucleotide exchange factor Rapgef2 was enriched from primary bovine neuroendocrine cells by cAMP-agarose affinity chromatography and that it was specifically eluted by cAMP. With loss-of-function experiments in the rat neuronal cell line Neuroscreen-1 (NS-1) and gain-of-function experiments in human embryonic kidney 293T cells, we demonstrated that Rapgef2 connected GPCR-dependent activation of adenylate cyclase and increased cAMP concentration with the activation of ERK in neurons and endocrine cells. Furthermore, knockdown of Rapgef2 blocked cAMP- and ERK-dependent neuritogenesis. Our data are consistent with a pathway involving the cAMP-mediated activation of Rapgef2, which then stimulates Rap1, leading to increases in B-Raf, MEK, and ERK activity.

The product of C9orf72, a gene strongly implicated in neurodegeneration, is structurally related to DENN Rab-GEFs.

MOTIVATION: Fronto-temporal dementia (FTD) and amyotrophic lateral sclerosis (ALS, also called motor neuron disease, MND) are severe neurodegenerative diseases that show considerable overlap at the clinical and cellular level. The most common single mutation in families with FTD or ALS has recently been mapped to a non-coding repeat expansion in the uncharacterized gene C9ORF72. Although a plausible mechanism for disease is that aberrant C9ORF72 mRNA poisons splicing, it is important to determine the cellular function of C9ORF72, about which nothing is known. RESULTS: Sensitive homology searches showed that C9ORF72 is a full-length distant homologue of proteins related to Differentially Expressed in Normal and Neoplasia (DENN), which is a GDP/GTP exchange factor (GEF) that activates Rab-GTPases. Our results suggest that C9ORF72 is likely to regulate membrane traffic in conjunction with Rab-GTPase switches, and we propose to name the gene and its product DENN-like 72 (DENNL72).

Deciphering the molecular and functional basis of Dbl family proteins: a novel systematic approach toward classification of selective activation of the Rho family proteins.

The diffuse B-cell lymphoma (Dbl) family of the guanine nucleotide exchange factors is a direct activator of the Rho family proteins. The Rho family proteins are involved in almost every cellular process that ranges from fundamental (e.g. the establishment of cell polarity) to highly specialized processes (e.g. the contraction of vascular smooth muscle cells). Abnormal activation of the Rho proteins is known to play a crucial role in cancer, infectious and cognitive disorders, and cardiovascular diseases. However, the existence of 74 Dbl proteins and 25 Rho-related proteins in humans, which are largely uncharacterized, has led to increasing complexity in identifying specific upstream pathways. Thus, we comprehensively investigated sequence-structure-function-property relationships of 21 representatives of the Dbl protein family regarding their specificities and activities toward 12 Rho family proteins. The meta-analysis approach provides an unprecedented opportunity to broadly profile functional properties of Dbl family proteins, including catalytic efficiency, substrate selectivity, and signaling specificity. Our analysis has provided novel insights into the following: (i) understanding of the relative differences of various Rho protein members in nucleotide exchange; (ii) comparing and defining individual and overall guanine nucleotide exchange factor activities of a large representative set of the Dbl proteins toward 12 Rho proteins; (iii) grouping the Dbl family into functionally distinct categories based on both their catalytic efficiencies and their sequence-structural relationships; (iv) identifying conserved amino acids as fingerprints of the Dbl and Rho protein interaction; and (v) defining amino acid sequences conserved within, but not between, Dbl subfamilies. Therefore, the characteristics of such specificity-determining residues identified the regions or clusters conserved within the Dbl subfamilies.

ß-Amyloid 42/40 ratio and kalirin expression in Alzheimer disease with psychosis.

Psychosis in Alzheimer disease differentiates a subgroup with more rapid decline, is heritable, and aggregates within families, suggesting a distinct neurobiology. Evidence indicates that greater impairments of cerebral cortical synapses, particularly in dorsolateral prefrontal cortex, may contribute to the pathogenesis of psychosis in Alzheimer disease (AD) phenotype. Soluble ß-amyloid induces loss of dendritic spine synapses through impairment of long-term potentiation. In contrast, the Rho guanine nucleotide exchange factor (GEF) kalirin is an essential mediator of spine maintenance and growth in cerebral cortex. We therefore hypothesized that psychosis in AD would be associated with increased soluble ß-amyloid and reduced expression of kalirin in the cortex. We tested this hypothesis in postmortem cortical gray matter extracts from 52 AD subjects with and without psychosis. In subjects with psychosis, the ß-amyloid(1-42)/ß-amyloid(1-40) ratio was increased, due primarily to reduced soluble ß-amyloid(1-40), and kalirin-7, -9, and -12 were reduced. These findings suggest that increased cortical ß-amyloid(1-42)/ß-amyloid(1-40) ratio and decreased kalirin expression may both contribute to the pathogenesis of psychosis in AD.

[Characterization and expression of DjPreb gene in the planarian Dugesia japonica].

In this study we report the expression and identification of a PREB-related gene from the planarian Dugesia japonica, DjPreb. The planarian DjPreb cDNA is comprised of 1101 bp and contains a 972 bp open reading frame corresponding to a deduced protein of 323 amino acids with a 69 bp 5'-UTR and a 60 bp 3'-UTR. Phylogenetic analysis shows that DjPreb belongs to PREB/PREB-like members. We examined its spatial and temporal expression and distribution in both intact and regenerating planarians by Relative quantitative real-time PCR and Whole-mount in situ hybridization. The analysis indicates that DjPreb shows a gradient of expression with peak levels present in the anterior and posterior regions and progressively lower levels in central regions in intact and regenerating planarians. During regeneration the expression of DjPreb is upregulated. Strong expression of DjPreb is observed in the anterior and posterior blastemas. These results suggest that DjPreb may participate in head and tail formation.

A cytohesin homolog in Dictyostelium amoebae.

BACKGROUND: Dictyostelium, an amoeboid motile cell, harbors several paralogous Sec7 genes that encode members of three distinct subfamilies of the Sec7 superfamily of Guanine nucleotide exchange factors. Among them are proteins of the GBF/BIG family present in all eukaryotes. The third subfamily represented with three members in D. discoideum is the cytohesin family that has been thought to be metazoan specific. Cytohesins are characterized by a Sec7 PH tandem domain and have roles in cell adhesion and migration. PRINCIPAL FINDINGS: Dictyostelium SecG exhibits highest homologies to the cytohesins. It harbors at its amino terminus several ankyrin repeats that are followed by the Sec7 PH tandem domain. Mutants lacking SecG show reduced cell-substratum adhesion whereas cell-cell adhesion that is important for development is not affected. Accordingly, multicellular development proceeds normally in the mutant. During chemotaxis secG(-) cells elongate and migrate in a directed fashion towards cAMP, however speed is moderately reduced. SIGNIFICANCE: The data indicate that SecG is a relevant factor for cell-substrate adhesion and reveal the basic function of a cytohesin in a lower eukaryote.

Role of the GNOM gene in Arabidopsis apical-basal patterning--From mutant phenotype to cellular mechanism of protein action.

How the apical-basal axis of polarity is established in embryogenesis is still a mystery in plant development. This axis appeared specifically compromised by mutations in the Arabidopsis GNOM gene. Surprisingly, GNOM encodes an ARF guanine-nucleotide exchange factor (ARF-GEF) that regulates the formation of vesicles in membrane trafficking. In-depth functional analysis of GNOM and its closest relative, GNOM-LIKE 1 (GNL1), has provided a mechanistic explanation for the development-specific role of a seemingly mundane trafficking regulator. The current model proposes that GNOM is specifically involved in the endosomal recycling of the auxin-efflux carrier PIN1 to the basal plasma membrane in provascular cells, which in turn is required for the accumulation of the plant hormone auxin at the future root pole through polar auxin transport. Thus, the analysis of GNOM highlights the importance of cell-biological processes for a mechanistic understanding of development.

[Research progress in ARF-GEF gene family].

ARF-GEFs are a family of guanine-nucleotide exchange factors catalyzing the exchange of GDP for GTP on ADP-ribosylation factor. Large ARF-GEFs are highly conserved in all eukaryotes. It is an important regulator in both membrane dynamics and protein trafficking. Significant progress has been made in elucidating the structure, subcellular localization, and physiological functions of ARF-GEFs as a hot topic of cell biology recently. In this review, the character-istics and distribution of ARF-GEFs in different species, and recent research progress of ARF-GEFs and their regulation system are summarized.

GNOM-LIKE 2, encoding an adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factor protein homologous to GNOM and GNL1, is essential for pollen germination in Arabidopsis.

In flowering plants, male gametes are delivered to female gametophytes by pollen tubes. Although it is important for sexual plant reproduction, little is known about the genetic mechanism that controls pollen germination and pollen tube growth. Here we report the identification and characterization of two novel mutants, gnom-like 2-1 (gnl2-1) and gnl2-2 in Arabidopsis thaliana, in which the pollen grains failed to germinate in vitro and in vivo. GNL2 encodes a protein homologous to the adenosine diphosphate-ribosylation factor-guanine nucleotide exchange factors, GNOM and GNL1 that are involved in endosomal recycling and endoplasmic reticulum-Golgi vesicular trafficking. It was prolifically expressed in pollen grains and pollen tubes. The results of the present study suggest that GNL2 plays an important role in pollen germination.

Large ARF guanine nucleotide exchange factors in membrane trafficking.

In eukaryotic cells membrane compartments are connected through cargo-selective vesicle trafficking mediating the exchange of components between different organelles. This exchange is essential to maintain their structural integrity and specific composition. A fundamental regulatory step in vesicle formation is the activation of small ARF GTPases by exchanging their bound GDP for GTP, which is a prerequisite for ARF-mediated effector recruitment. Activation of ARFs is catalyzed by the characteristic SEC7 domain of guanine nucleotide exchange factors (ARF-GEFs), which are classified according to their additional protein domains.The only group of ARF-GEFs conserved in mammals, yeast and plants are the large ARF-GEFs. This review summarizes recent findings on the function of large ARF-GEFs, and the use of the inhibitor Brefeldin A as a potent tool in understanding membrane trafficking. Furthermore we highlight common themes and apparent differences in large ARF-GEF function between eukaryotic kingdoms.

Role of the C-terminal SH3 domain and N-terminal tyrosine phosphorylation in regulation of Tim and related Dbl-family proteins.

Dbl-related oncoproteins are guanine nucleotide exchange factors (GEFs) specific for Rho-family GTPases and typically possess tandem Dbl (DH) and pleckstrin homology (PH) domains that act in concert to catalyze exchange. Although the exchange potential of many Dbl-family proteins is constitutively activated by truncation, the precise mechanisms of regulation for many Dbl-family proteins are unknown. Tim and Vav are distantly related Dbl-family proteins that are similarly regulated; their Dbl homology (DH) domains interact with N-terminal helices to exclude and prevent activation of Rho GTPases. Phosphorylation, substitution, or deletion of the blocking helices relieves this autoinhibition. Here we show that two other Dbl-family proteins, Ngef and Wgef, which like Tim contain a C-terminal SH3 domain, are also activated by tyrosine phosphorylation of a blocking helix. Consequently, basal autoinhibition of DH domains by direct steric exclusion using short N-terminal helices likely represents a conserved mechanism of regulation for the large family of Dbl-related proteins. N-Terminal truncation or phosphorylation of many other Dbl-family GEFs leads to their activation; similar autoinhibition mechanisms could explain some of these events. In addition, we show that the C-terminal SH3 domain binding to a polyproline region N-terminal to the DH domain of the Tim subgroup of Dbl-family proteins provides a unique mechanism of regulated autoinhibition of exchange activity that is functionally linked to the interactions between the autoinhibitory helix and the DH domain.

The RCC1 superfamily: from genes, to function, to disease.

The Regulator of Chromosome Condensation 1 (RCC1) was identified over 20 years ago as a critical cell cycle regulator. By analyzing its amino acid sequence, RCC1 was found to consist of seven homologous repeats of 51-68 amino acid residues, which were later shown to adopt a seven-bladed beta-propeller fold. Since the initial identification of RCC1, a number of proteins have been discovered that contain one or more RCC1-like domains (RLDs). As we show here, these RCC1 superfamily proteins can be subdivided in five subgroups based on structural criteria. In recent years, a number of studies have been published regarding the functions of RCC1 superfamily proteins. From these studies, the emerging picture is that the RLD is a versatile domain which may perform many different functions, including guanine nucleotide exchange on small GTP-binding proteins, enzyme inhibition or interaction with proteins and lipids. Here, we review the available structural and functional data on RCC1 superfamily members, paying special attention to the human proteins and their involvement in disease.

A distinct mechanism regulating a pollen-specific guanine nucleotide exchange factor for the small GTPase Rop in Arabidopsis thaliana.

Rop/Rac small GTPases are central to diverse developmental and cellular activities in plants, playing an especially important role in polar growth of pollen tubes. Although it is established that a class of plant-specific RopGEFs promotes the activity of Rop/Rac through the catalytic PRONE (Plant-specific Rop nucleotide exchanger) domain, not much is known about how RopGEF function is controlled to allow a spatiotemporally regulated Rop activity. To understand such a process in pollen, we performed functional analysis with a pollen-specific RopGEF, AtRopGEF12. Overexpression of AtRopGEF12 had minimal phenotypic effects, whereas overexpression of a C-terminally truncated version disturbed tube growth, suggesting that the C terminus was inhibitory to GEF function. In contrast to non-pollen-expressed RopGEFs, pollen-expressed RopGEFs have conserved C termini. A phospho-mimicking mutation at an invariant serine within the C terminus of AtRopGEF12 resulted in loss of the C-terminal inhibition, suggesting that phosphorylation regulates GEF activity in vivo. The PRONE domain of AtRopGEF12 (PRONE12) was not sufficient to induce isotropic tube growth. We used mbSUS to show that AtRopGEF12 interacts with an Arabidopsis pollen receptor kinase AtPRK2a through its C terminus, and BiFC to show that they interact in pollen tubes. Coexpression of AtRopGEF12 and AtPRK2a caused isotropic growth reminiscent of that seen upon overexpression of a constitutively active (CA) Rop. Coexpression of AtPRK2a with an N-terminally truncated AtRopGEF12 did not induce isotropic growth, indicating a positive role for the N-terminal domain. Our results suggest a mechanism by which the noncatalytic domains of pollen-specific/enriched RopGEFs regulate PRONE function, leading to polarized pollen tube growth.

Guanine nucleotide exchange factors of the cytohesin family and their roles in signal transduction.

Members of the cytohesin protein family, a group of guanine nucleotide exchange factors for adenosine diphosphate ribosylation factor (ARF) guanosine triphosphatases, have recently emerged as important regulators of signal transduction in vertebrate and invertebrate biology. These proteins share a modular domain structure, comprising carboxy-terminal membrane recruitment elements, a Sec7 homology effector domain, and an amino-terminal coiled-coil domain that serve as a platform for their integration into larger signaling complexes. Although these proteins have a highly similar overall build, their individual biological functions appear to be at least partly specific. Cytohesin-1 had been identified as a regulator of beta2 integrin inside-out regulation in immune cells and was subsequently shown to be involved in mitogen-associated protein kinase signaling in tumor cell proliferation as well as in T-helper cell activation and differentiation. Cytohesin-3, which had been discovered to be strongly associated with T-cell anergy, was very recently described as an essential component of insulin signal transduction in Drosophila and in human and murine liver cells. Future work will aim to dissect the mechanistic details of the modes of action of the cytohesins as well as to define the precise roles of these versatile proteins in vertebrates at the genetic level.

Trix, a novel Rac guanine-nucleotide exchange factor from Dictyostelium discoideum is an actin-binding protein and accumulates at endosomes.

Small Rho family GTPases are involved in regulation of actin cytoskeleton dynamics. These molecular switches are themselves mainly controlled by specific GTPase-activating proteins (GAPs) and guanine-nucleotide exchange factors (GEFs). We have cloned and initially characterized a novel putative RhoGEF from Dictyostelium discoideum. The predicted 135-kDa protein displays a unique domain organization in its N-terminus by harboring two type3 calponin homology (CH) domains followed by a single type1 CH domain. The C-terminal region encompasses a diffuse B-cell lymphoma homology/pleckstrin homology tandem domain that is typically found in RhoGEFs. We therefore refer to this protein as Trix (triple CH-domain array exchange factor). A recombinant N-terminal region of Trix carrying all three CH domains binds to F-actin and bundles actin filaments. Trix-null mutants are viable and display only subtle defects when compared to wild-type cells with the exception of a substantial decrease in exocytosis of a fluid-phase marker. GFP fusions with the full-length protein or the N-terminal part containing all three CH domains revealed that Trix localizes to the cortical region and strongly accumulates on late endosomes. Our results suggest that Trix is specifically involved in a Rho GTPase-signaling pathway that is required for regulation of the actin cytoskeleton during exocytosis.