FYVE Domain-containing Proteins Research Articles

The plant FYVE domain-containing protein FREE1 associates with microprocessor components to repress miRNA biogenesis.

FYVE domain protein required for endosomal sorting 1 (FREE1), originally identified as a plant-specific component of the endosomal sorting complex required for transport (ESCRT) machinery, plays diverse roles either in endosomal sorting in the cytoplasm or in transcriptional regulation of abscisic acid signaling in the nucleus. However, to date, a role for FREE1 or other ESCRT components in the regulation of plant miRNA biology has not been discovered. Here, we demonstrate a nuclear function of FREE1 as a cofactor in miRNA biogenesis in plants. FREE1 directly interacts with the plant core microprocessor component CPL1 in nuclear bodies and disturbs the association between HYL1, SE and CPL1. Inactivation of FREE1 in the nucleus increases the binding affinity between HYL1, SE, and CPL1 and causes a transition of HYL1 from the inactive hyperphosphorylated version to the active hypophosphorylated form, thereby promoting miRNA biogenesis. Our results suggest that FREE1 has evolved as a negative regulator of miRNA biogenesis and provides evidence for a link between FYVE domain-containing proteins and miRNA biogenesis in plants.

Expression of Concern: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway.

Expression of Concern: The lysosome-associated apoptosis-inducing protein containing the pleckstrin homology (PH) and FYVE domains (LAPF), representative of a novel family of PH and FYVE domain-containing proteins, induces caspase-independent apoptosis via the lysosomal-mitochondrial pathway.

A Cross-Linking-Aided Immunoprecipitation/Mass Spectrometry Workflow Reveals Extensive Intracellular Trafficking in Time-Resolved, Signal-Dependent Epidermal Growth Factor Receptor Proteome.

Ligand binding to the cell surface receptors initiates signaling cascades that are commonly transduced through a protein-protein interaction (PPI) network to activate a plethora of response pathways. However, tools to capture the membrane PPI network are lacking. Here, we describe a cross-linking-aided mass spectrometry workflow for isolation and identification of signal-dependent epidermal growth factor receptor (EGFR) proteome. We performed protein cross-linking in cell culture at various time points following EGF treatment, followed by immunoprecipitation of endogenous EGFR and analysis of the associated proteins by quantitative mass spectrometry. We identified 140 proteins with high confidence during a 2 h time course by data-dependent acquisition and further validated the results by parallel reaction monitoring. A large proportion of proteins in the EGFR proteome function in endocytosis and intracellular protein transport. The EGFR proteome was highly dynamic with distinct temporal behavior; 10 proteins that appeared in all time points constitute the core proteome. Functional characterization showed that loss of the FYVE domain-containing proteins altered the EGFR intracellular distribution but had a minor effect on EGFR proteome or signaling. Thus, our results suggest that the EGFR proteome include functional regulators that influence EGFR signaling and bystanders that are captured as the components of endocytic vesicles. The high-resolution spatiotemporal information of these molecules facilitates the delineation of many pathways that could determine the strength and duration of the signaling, as well as the location and destination of the receptor.

SIMPLE binds specifically to PI4P through SIMPLE-like domain and participates in protein trafficking in the trans-Golgi network and/or recycling endosomes.

Small integral membrane protein of the lysosome/late endosome (SIMPLE) is a 161-amino acid cellular protein that contains a characteristic C-terminal domain known as the SIMPLE-like domain (SLD), which is well conserved among species. Several studies have demonstrated that SIMPLE localizes to the trans-Golgi network (TGN), early endosomes, lysosomes, multivesicular bodies, aggresomes and the plasma membrane. However, the amino acid regions responsible for its subcellular localization have not yet been identified. The SLD resembles the FYVE domain, which binds phosphatidylinositol (3)-phosphate (PI3P) and determines the subcellular localization of FYVE domain-containing proteins. In the present study, we have found that SIMPLE binds specifically to PI4P through its SLD. SIMPLE co-localized with PI4P and Rab11, a marker for recycling endosomes (REs, organelles enriched in PI4P) in both the IMS32 mouse Schwann cell line and Hela cells. Sucrose density-gradient centrifugation revealed that SIMPLE co-fractionated with syntaxin-6 (a TGN marker) and Rab11. We have also found that SIMPLE knockdown impeded recycling of transferrin and of transferrin receptor. Our overall results indicate that SIMPLE may regulate protein trafficking physiologically by localizing to the TGN and/or REs by binding PI4P.

Fluorescent FYVE Chimeras to Quantify PtdIns3P Synthesis During Autophagy.

Autophagy is the major cellular process of degradation and is modulated by several signaling pathways. Phosphatidylinositol 3-kinase (PtdIns3K) class III (Vps34) and PtdIns3K class I regulate the autophagy pathway positively and negatively, respectively. Both classes of PtdIns3K participate in the synthesis of phosphatidylinositol 3-phosphate (PtdIns3P), which plays a crucial role in autophagosome biogenesis and membrane traffic. PtdIns3P is a membrane phospholipid that is associated with endogenous FYVE domain-containing proteins. Indeed, such interactions facilitate autophagosome fusion with lysosomes and subsequent cargo degradation. During starvation-induced autophagy, the expression of FYVE domain-containing proteins increases, and their binding to PtdIns3P is strengthened. Nonetheless, not all FYVE domain proteins are related to the induction of autophagy. This method report presents the quantification of PtdIns3P synthesis by using cells either transiently transfected with or stably expressing FYVE-dsRed.

Identification and Evolution of FYVE Domain-Containing Proteins and Their Expression Patterns in Response to Abiotic Stresses in Rice

The FYVE domain is a typical zinc finger motif containing four conserved CxxC pairs and has been shown to specifically binds to PtdIns(3)P on the surface of cell membrane. FYVE domain-containing proteins are commonly distributed in eukaryotic cells and have been implicated in diverse functions like signal transduction, membrane trafficking, exocytosis and endocytosis, phosphoinositides (PIs) metabolism, and cytoskeletal regulation. Analysis of the rice genome using comprehensive online databases and research tools resulted in the identification of 19 putative rice FYVE (OsFVYE) proteins. Based on domain architectural and phylogenetic analyses, these OsFYVEs were further classified into six groups. Groups I–V were conserved in Arabidopsis (dicots) and rice (monocot) at both the genetic and protein levels, while group VI was widely present in plants but truncated in Arabidopsis and species of Brassicaceae. Comprehensive and comparative investigation of their expression profiles showed that FYVE genes in plant exhibited a variety of expression patterns during different developmental stages and in response to phytohormones and abiotic stresses. These findings indicated that both OsFYVE and AtFYVE genes may played potential roles in normal plant growth, hormone signal transduction, and abiotic stress tolerance. Results from our study shed light on the potential roles of FYVE proteins in plant growth, development, and stress responses.

Lipid kinases are essential for apicoplast homeostasis in Toxoplasma gondii.

Phosphoinositides regulate numerous cellular processes by recruiting cytosolic effector proteins and acting as membrane signalling entities. The cellular metabolism and localization of phosphoinositides are tightly regulated by distinct lipid kinases and phosphatases. Here, we identify and characterize a unique phosphatidylinositol 3 kinase (PI3K) in Toxoplasma gondii, a protozoan parasite belonging to the phylum Apicomplexa. Conditional depletion of this enzyme and subsequently of its product, PI(3)P, drastically alters the morphology and inheritance of the apicoplast, an endosymbiotic organelle of algal origin that is a unique feature of many Apicomplexa. We searched the T. gondii genome for PI(3)P-binding proteins and identified in total six PX and FYVE domain-containing proteins including a PIKfyve lipid kinase, which phosphorylates PI(3)P into PI(3,5)P2 . Although depletion of putative PI(3)P-binding proteins shows that they are not essential for parasite growth and apicoplast biology, conditional disruption of PIKfyve induces enlarged apicoplasts, as observed upon loss of PI(3)P. A similar defect of apicoplast homeostasis was also observed by knocking down the PIKfyve regulatory protein ArPIKfyve, suggesting that in T. gondii, PI(3)P-related function for the apicoplast might mainly be to serve as a precursor for the synthesis of PI(3,5)P2 . Accordingly, PI3K is conserved in all apicomplexan parasites whereas PIKfyve and ArPIKfyve are absent in Cryptosporidium species that lack an apicoplast, supporting a direct role of PI(3,5)P2 in apicoplast homeostasis. This study enriches the already diverse functions attributed to PI(3,5)P2 in eukaryotic cells and highlights these parasite lipid kinases as potential drug targets.

RUFY, Rab and Rap Family Proteins Involved in a Regulation of Cell Polarity and Membrane Trafficking

Cell survival, homeostasis and cell polarity rely on the control of membrane trafficking pathways. The RUN domain (comprised of the RPIP8, UNC-14, and NESCA proteins) has been suggested to be implicated in small GTPase-mediated membrane trafficking and cell polarity. Accumulating evidence supports the hypothesis that the RUN domain-containing proteins might be responsible for an interaction with a filamentous network linked to actin cytoskeleton and/or microtubules. In addition, several downstream molecules of PI3K are involved in regulation of the membrane trafficking by interacting with vesicle-associated RUN proteins such as RUFY family proteins. In this review, we summarize the background of RUN domain research with an emphasis on the interaction between RUN domain proteins including RUFY proteins (designated as RUN and FYVE domain-containing proteins) and several small GTPases with respect to the regulation of cell polarity and membrane trafficking on filamentous network.

Formins and membranes: anchoring cortical actin to the cell wall and beyond

Formins are evolutionarily conserved eukaryotic proteins participating in actin and microtubule organization. Land plants have three formin clades, with only two – Class I and II – present in angiosperms. Class I formins are often transmembrane proteins, residing at the plasmalemma and anchoring the cortical cytoskeleton across the membrane to the cell wall, while Class II formins possess a PTEN-related membrane-binding domain. Lower plant Class III and non-plant formins usually contain domains predicted to bind RHO GTPases that are membrane-associated. Thus, some kind of membrane anchorage appears to be a common formin feature. Direct interactions between various non-plant formins and integral or peripheral membrane proteins have indeed been reported, with varying mechanisms and biological implications. Besides of summarizing new data on Class I and Class II formin-membrane relationships, this review surveys such “non-classical” formin-membrane interactions and examines which, if any, of them may be evolutionarily conserved and operating also in plants. FYVE, SH3 and BAR domain-containing proteins emerge as possible candidates for such conserved membrane-associated formin partners.

The PtdIns3P‐Binding Protein Phafin 2 Mediates Epidermal Growth Factor Receptor Degradation by Promoting Endosome Fusion

Phosphatidylinositol 3-phosphate (PtdIns3P) orchestrates endosomal cargo transport, fusion and motility by recruiting FYVE or PX domain-containing effector proteins to endosomal membranes. In an attempt to discover novel PtdIns3P effectors involved in the termination of growth factor receptor signalling, we performed an siRNA screen for epidermal growth factor (EGF) degradation, targeting FYVE and PX domain proteins in the human proteome. This screen identified several potential regulators of EGF degradation, including HRS (used as positive control), PX kinase, MTMR4 and Phafin2/PLEKHF2. As Phafin2 has not previously been shown to be required for EGF receptor (EGFR) degradation, we performed further functional studies on this protein. Loss of Phafin2 was found to decrease early endosome size, whereas overexpression of Phafin2 resulted in enlarged endosomes. Moreover, both the EGFR and the fluid-phase marker dextran were retained in abnormally small endosomes in Phafin2-depleted cells. In yeast two-hybrid analysis we identified Phafin2 as a novel interactor of the endosomal-tethering protein EEA1, and Phafin2 colocalized strongly with EEA1 in microdomains of the endosome membrane. Our results suggest that Phafin2 controls receptor trafficking and fluid-phase transport through early endosomes by facilitating endosome fusion in concert with EEA1.

Identification and structural characterization of FYVE domain-containing proteins of Arabidopsis thaliana

BackgroundFYVE domains have emerged as membrane-targeting domains highly specific for phosphatidylinositol 3-phosphate (PtdIns(3)P). They are predominantly found in proteins involved in various trafficking pathways. Although FYVE domains may function as individual modules, dimers or in partnership with other proteins, structurally, all FYVE domains share a fold comprising two small characteristic double-stranded β-sheets, and a C-terminal α-helix, which houses eight conserved Zn2+ ion-binding cysteines. To date, the structural, biochemical, and biophysical mechanisms for subcellular targeting of FYVE domains for proteins from various model organisms have been worked out but plant FYVE domains remain noticeably under-investigated.ResultsWe carried out an extensive examination of all Arabidopsis FYVE domains, including their identification, classification, molecular modeling and biophysical characterization using computational approaches. Our classification of fifteen Arabidopsis FYVE proteins at the outset reveals unique domain architectures for FYVE containing proteins, which are not paralleled in other organisms. Detailed sequence analysis and biophysical characterization of the structural models are used to predict membrane interaction mechanisms previously described for other FYVE domains and their subtle variations as well as novel mechanisms that seem to be specific to plants.ConclusionsOur study contributes to the understanding of the molecular basis of FYVE-based membrane targeting in plants on a genomic scale. The results show that FYVE domain containing proteins in plants have evolved to incorporate significant differences from those in other organisms implying that they play a unique role in plant signaling pathways and/or play similar/parallel roles in signaling to other organisms but use different protein players/signaling mechanisms.

Phosphatidylinositol-phosphates mediate cytoskeletal reorganization during phagocytosis via a unique modular protein consisting of RhoGEF/DH and FYVE domains in the parasitic protozoonEntamoeba histolytica

To understand the roles of phosphoinositides [PtdIns] in phagocytosis of parasitic eukaryotes, we examined the interaction of phosphatidylinositol-3-phosphate [PtdIns(3)P] and putative PtdIns-P-binding proteins during phagocytosis in the enteric protozoan parasite Entamoeba histolytica. It was previously shown that phagocytosis in E. histolytica is indispensable for virulence and is inhibited by PtdIns 3-kinase inhibitors. We demonstrated by time-lapse live imaging that during the initiation of phagocytosis, the PtdIns(3)P biomarker GFP-Hrs-FYVE, was translocated to the phagocytic cup, phagosome, and to tunnel-like structures connecting the plasma membrane and phagosomes. E. histolytica possesses 12 FYVE domain-containing proteins (EhFP1-12), 11 of which also contain the RhoGEF/DH domain. Among them EhFP4 was shown to be recruited to the tunnel-like structures and to the proximal region of the phagosome. We further demonstrated that EhFP4 physically interacted with 4 of 10 predominant Rho/Rac small GTPases. Phosphoinositide binding assay showed that EhFP4 unexpectedly bound to PtdIns(4)P via the carboxyl-terminal domain and that the FYVE domain modulates the binding specificity of EhFP4 to PtdIns-P. Expression of the FYVE domain from EhFP4 inhibited phagocytosis while enhancement was observed when mammalian Hrs-FYVE domain was expressed. Altogether, we demonstrated that PtdIns(3)P, PtdIns(4)P and EhFP4 coordinately regulate phagocytosis and phagosome maturation in this parasitic eukaryote.

Evolutionarily conserved structural and functional roles of the FYVE domain

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12-13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4'. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

Evolutionarily conserved structural and functional roles of the FYVE domain

The FYVE domain is an approx. 80 amino acid motif that binds to the phosphoinositide PtdIns3P with high specificity and affinity. It is present in 38 predicted gene products within the human genome, but only in 12–13 in Caenorhabditis elegans and Drosophila melanogaster. Eight of these are highly conserved in all three organisms, and they include proteins that have not been characterized in any species. One of these, WDFY2, appears to play an important role in early endocytosis and was revealed in a RNAi (RNA interference) screen in C. elegans. Interestingly, some proteins contain FYVE-like domains in C. elegans and D. melanogaster, but have lost this domain during evolution. One of these is the homologue of Rabatin-5, a protein that, in mammalian cells, binds both Rab5 and Rabex-5, a guanine-nucleotide exchange factor for Rab5. Thus the Rabatin-5 homologue suggests that mechanisms to link PtdIns3P and Rab5 activation developed in evolution. In mammalian cells, these mechanisms are apparent in the existence of proteins that bind PtdIns3P and Rab GTPases, such as EEA1, Rabenosyn-5 and Rabip4′. Despite the comparable ability to bind to PtdIns3P in vitro, FYVE domains display widely variable abilities to interact with endosomes in intact cells. This variation is due to three distinct properties of FYVE domains conferred by residues that are not involved in PtdIns3P head group recognition: These properties are: (i) the propensity to oligomerize, (ii) the ability to insert into the membrane bilayer, and (iii) differing electrostatic interactions with the bilayer surface. The different binding properties are likely to regulate the extent and duration of the interaction of specific FYVE domain-containing proteins with early endosomes, and thereby their biological function.

The WD40 and FYVE domain containing protein 2 defines a class of early endosomes necessary for endocytosis

The FYVE domain binds with high specificity and avidity to phosphatidylinositol 3-phosphate. It is present in approximately 30 proteins in humans, some of which have been implicated in functions ranging from early endosome fusion to signal transduction through the TGF-beta receptor. To develop a further understanding of the biological roles of this protein family, we turned to the nematode Caenorhabditis elegans, which contains only 12 genes predicted to encode for phosphatidylinositol 3-phosphate binding, FYVE domain-containing proteins, all of which have homologs in the human genome. Each of these proteins was targeted individually by RNA interference. One protein, WDFY2, produced a strong inhibition of endocytosis when silenced. WDFY2 contains WD40 motifs and a FYVE domain, is highly conserved between species, and localizes to a set of small endosomes that reside within 100 nm from the plasma membrane. These endosomes are involved in transferrin uptake but lack the classical endosomal markers Rab5 and EEA1. Silencing of WDFY2 by siRNA in mammalian cells impaired transferrin endocytosis. These studies reveal the important, conserved role of WDFY2 in endocytosis, and the existence of a subset of early endosomes, closely associated with the plasma membrane, that may constitute the first stage of endocytic processing of internalized cargo.

The Lysosome-associated Apoptosis-inducing Protein Containing the Pleckstrin Homology (PH) and FYVE Domains (LAPF), Representative of a Novel Family of PH and FYVE Domain-containing Proteins, Induces Caspase-independent Apoptosis via the Lysosomal-Mitochondrial Pathway

Lysosomes have recently been identified as important apoptotic signal integrators in response to various stimuli. Here we report the functional characterization of LAPF, a novel lysosome-associated apoptosis-inducing protein containing PH and FYVE domains. LAPF is a representative of a new protein family, the Phafins (protein containing both PH and FYVE domains), which consists of 14 unidentified proteins from various species. Overexpression of LAPF in L929 cells induces apoptosis and also increases cell sensitivity to TNFalpha-induced apoptosis, concomitant with its translocation to lysosomes. Two mutants of LAPF, either lacking the PH or FYVE domain, failed to induce cell death and translocate to lysosomes, suggesting that both domains are required for its apoptosis-inducing activity and relocation. We demonstrate that LAPF may induce apoptosis via the following steps: LAPF translocation to lysosomes, lysosomal membrane permeabilization (LMP), release of cathepsin (cath) D and L, mitochondrial membrane permeabilization (MMP), release of apoptosis-inducing factor (AIF), and caspase-independent apoptosis. The cath D-specific inhibitor attenuates LAPF-induced apoptosis, indicating a pivotal role of lysosomes in LAPF-initiated apoptosis. We also demonstrate that the lysosomal pathway was employed in the typical apoptotic model in which high dose TNFalpha was used to stimulate L929 cells. Silencing of LAPF expression by small RNA interference protected L929 cells from hTNFalpha-induced apoptosis by impairing hTNFalpha-triggered LMP and MMP. Therefore, LAPF may launch caspase-independent apoptosis through the lysosomal-mitochondrial pathway.

Emerging roles of phosphatidylinositol monophosphates in cellular signaling and trafficking

The phosphoinositide metabolism that is highly controlled by a set of kinases, phosphatases and phospholipases leads to the production of several second messengers playing critical roles in intracellular signal transduction mechanisms. Recent discoveries have unraveled unexpected roles for the three phosphatidylinositol monophosphates, PtdIns(3)P, PtdIns(4)P and PtdIns(5)P, that appear now as important lipid messengers able to specifically interact with proteins. The formation of functionally distinct and independently regulated pools of phosphatidylinositol monophosphates probably contributes to the specificity of the interactions with their targets. The relative enrichment of organelles in a particular species of phosphoinositides (i.e. PtdIns(3)P in endosomes, PtdIns(4)P in Golgi and PtdIns(4,5)P2 in plasma membrane) suggests the notion of lipid-defined organelle identity. PtdIns(3)P is now clearly involved in vesicular trafficking by interaction with a set of FYVE domain-containing proteins both in yeast and in mammals. PtdIns(4)P, which until now was only considered as a precursor for PtdIns(4,5)P2, appears as a regulator on its own, by recruiting a set of proteins to the trans-Golgi network. PtdIns(5)P, the most recently discovered inositol lipid, is also emerging as a potentially important signaling molecule.

Novel PtdIns(3)P-binding protein Etf1 functions as an effector of the Vps34 PtdIns 3-kinase in autophagy.

Autophagy is the process whereby cytoplasmic cargo (e.g., protein and organelles) are sequestered within a double membrane–enclosed transport vesicle and degraded after vesicle fusion with the vacuole/lysosome. Current evidence suggests that the Vps34 phosphatidylinositol 3-kinase is essential for macroautophagy, a starvation-induced autophagy pathway (Kihara et al., 2001). Here, we characterize a requirement for Vps34 in constitutive autophagy by the cytoplasm-to-vacuole targeting (Cvt) pathway. First, we show that transient disruption of phosphatidylinositol (PtdIns) 3-phosphate (PtdIns[3]P) synthesis through inactivation of temperature-sensitive Vps34 or its upstream activator, Vps15, blocks the Cvt and macroautophagy pathways. Yet, PtdIns(3)P-binding FYVE domain-containing proteins, which mediate carboxypeptidase Y (CPY) transport to the vacuole by the CPY pathway, do not account for the requirement of Vps34 in autophagy. Using a genetic selection designed to isolate PtdIns(3)P-binding effectors of Vps34, we identify Etf1, an uncharacterized type II transmembrane protein. Although Etf1 does not contain a known 3-phosphoinositide–binding domain (i.e., FYVE or Phox), we find that Etf1 interacts with PtdIns(3)P and that this interaction requires a basic amino acid motif (KKPAKK) within the cytosolic region of the protein. Moreover, deletion of ETF1 or mutation of the KKPAKK motif results in strong sorting defects in the Cvt pathway but not in macroautophagy or in CPY sorting. We propose that Vps34 regulates the CPY, Cvt, and macroautophagy pathways through distinct sets of PtdIns(3)P-binding effectors and that Vps34 promotes protein trafficking in the Cvt pathway through activation/localization of the effector protein Etf1.

FENS-1 and DFCP1 are FYVE domain-containing proteins with distinct functions in the endosomal and Golgi compartments.

FENS-1 and DFCP1 are recently discovered proteins containing one or two FYVE-domains respectively. We show that the FYVE domains in these proteins can bind PtdIns3P in vitro with high specificity over other phosphoinositides. Exogenously expressed FENS-1 localises to early endosomes: this localisation requires an intact FYVE domain and is sensitive to wortmannin inhibition. The isolated FYVE domain of FENS-1 also localises to endosomes. These results are consistent with current models of FYVE-domain function in this cellular compartment. By contrast, exogenously expressed DFCP1 displays a predominantly Golgi, endoplasmic reticulum (ER) and vesicular distribution with little or no overlap with FENS-1 or other endosomal markers. Overexpression of DFCP1 was found to cause dispersal of the Golgi compartment defined by giantin and gpp130-staining. Disruption of the FYVE domains of DFCP1 causes a shift to more condensed and compact Golgi structures and overexpression of this mutant was found to confer significant protection to the Golgi against brefeldin-induced dispersal. These properties of DFCP1 are surprising, and suggest FYVE domain-localisation and function may not be exclusively endosomal. Movies available on-line

Hgs and SARA Recruit Smads

Smads 1, 2, 3, 5, and 8 are transcription factors that transmit the signal generated by activation of members of the transforming growth factor-β (TGF-β) receptor family. When activated, these receptor-regulated Smads form complexes with Smad4 and then translocate to the nucleus. Miura et al. investigated how the receptor-regulated Smads are anchored to the receptors. Their data suggest that two FYVE domain-containing proteins, Hgs and SARA, cooperate to recruit Smads to inactive receptors and that this interaction is essential for viability in mice. FYVE domains interact with phosphatidylinositol 3-phosphate and function in various signaling pathways (see Corvera). In a yeast two-hybrid screen, Hgs was identified as a binding partner for Smad 2. These results were confirmed by coimmunoprecipitation experiments in which Hgs interacted with Smads 1, 2, and 3 in the absence of ligand stimulation, but not with Smad 4 or 6. Cotransfection experiments with wild-type or mutated Hgs and Smad 2 or 3 showed that these interactions depended on the COOH-terminal domain of Hgs, but not the FYVE domain. Overexpression of Hgs increased recruitment of Smads to the activin receptor, increased phosphorylation of Smads, and was essential for activin-simulated signaling to reporter genes. The effect of Hgs was enhanced if a second FYVE-domain protein, SARA, was also cotransfected into the cells. Expression of a COOH-terminally truncated version of Hgs in "knockin" mice was lethal by embryonic day 10 (E10). Mutant cells isolated before E10 demonstrated a decreased responsiveness to TGF-β and activin A, which could be restored if SARA or Hgs or both proteins were overexpressed in the cells.Miura, S., Takeshita, T., Asao, H., Kimura, Y., Murata, K., Sasaki, Y., Hania, J.-I., Beppu, H., Tsukazaki, T., Wrana, J.L., Miyazono, K., and Sugamura, K. (2000) Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA. Mol. Cell. Biol. 20: 9346-9355. [Abstract] [Full Text]Corvera, S. (2000) Signal transduction: Stuck with FYVE domains.Science's STKE: http://www.stke.org/cgi/content/full/OC_sigtrans;2000/37/pe1. [Full Text]