Rab Binding Research Articles

Raf kinases in lung tumor development.

The specific response of a cell to an extracellular signal is mediated by signaling cascades that relay the excitation of one or more receptor(s) to the co-ordinated activity of a set of effector molecules which regulate cell division and growth and prevent an intrinsic cell death program. The small G protein Ras is an essential component in the transduction of extracellular signals. Raf is a serine/threonine protein kinase and a direct downstream effector of Ras, which is activated to its GTP-bound state in response to various ligands binding to their cognate receptors. Raf kinases are located at the head of one of the best characterized signaling cascades, the Raf-MEK-ERK kinase cascade which is involved in many signaling processes (Fig. 1). In multicellular organisms, the family of Raf kinases consists of three members, ARaf, BRaf and CRaf. Although all of these Raf kinases phosphorylate MEK, they differ in the way they are activated (Hagemann and Rapp, 1999). For example, cAMP potentiates BRaf kinase activity but inhibits CRaf (Erhardt et al., 1995; Cook and McCormick, 1993; Dhillon et al., 2002). Genetic and biochemical data also indicate that isoform specific functions exist for Raf kinases which correlate with specific protein interactions (Hagemann and Rapp, 1999). Recent findings regarding interaction of Raf with lipid membrane microdomains (Hekman et al., 2002; Raabe and Rapp, 2002) and the discovery of Rab binding scaffold proteins that may be regulated by MAP kinases in their ability to activate actin comet formation for vesicle transport (Kerkhoff et al., 2001; Eden et al., 2002) have suggested to us a revised model for membrane translocation of Raf (Fig. 2). The model incorporates suggestions on the role of rafts in protein sorting (Simons and Ikonen, 1997) and anticipates a self organizing element in the maturation of signaling complexes. As pictured in Fig. 2, joint sorting of Raf, Ras and upstream signal regulators, e.g., growth factor receptors together with elements of the Ras cycle as well as effectors of Ras/Raf signaling such as MEK and ERK (perhaps

A conserved ER targeting motif in three families of lipid binding proteins and in Opi1p binds VAP.

Intracellular lipid traffic is mediated both by membrane vesicles and by a number of non-vesicular pathways facilitated by cytoplasmic lipid binding proteins. For these proteins to act effectively they must be targeted accurately to specific membranes. Here we identify a novel short conserved determinant called the FFAT motif that is shared by several seemingly unrelated lipid binding proteins and is also found in Opi1p, a transcriptional regulator of phospholipid synthesis in yeast. FFAT motifs act as membrane- targeting determinants by their direct interaction with homologues of VAMP-associated protein (VAP), a conserved endoplasmic reticulum (ER) protein. In budding yeast, all four proteins with FFAT motifs interact with Scs2p, a homologue of VAP, to target the ER to some extent. The precise intracellular distribution of each of these proteins depends on the integration of the FFAT-Scs2p interaction with other targeting determinants, and the interaction is functionally significant. We conclude that binding to a VAP homologue is a common mechanism by which proteins with FFAT motifs, most of which are involved in lipid metabolism, target ER membranes.

Arfophilins are dual Arf/Rab 11 binding proteins that regulate recycling endosome distribution and are related to Drosophila nuclear fallout.

Arfophilin is an ADP ribosylation factor (Arf) binding protein of unknown function. It is identical to the Rab11 binding protein eferin/Rab11-FIP3, and we show it binds both Arf5 and Rab11. We describe a related protein, arfophilin-2, that interacts with Arf5 in a nucleotide-dependent manner, but not Arf1, 4, or 6 and also binds Rab11. Arfophilin-2 localized to a perinuclear compartment, the centrosomal area, and focal adhesions. The localization of arfophilin-2 to the perinuclear compartment was selectively blocked by overexpression of Arf5-T31N. In contrast, a green fluorescent protein-arfophilin-2 chimera or arfophilin-2 deletions were localized around the centrosome in a region that was also enriched for transferrin receptors and Rab11 but not early endosome markers, suggesting that the distribution of the endosomal recycling compartment was altered. The arfophilins belong to a conserved family that includes Drosophila melanogaster nuclear fallout, a centrosomal protein required for cellularization. Expression of green fluorescent protein-nuclear fallout in HeLa cells resulted in a similar phenotype, indicative of functional homology and thus implicating the arfophilins in mitosis/cytokinesis. We suggest that the novel dual GTPase-binding capacity of the arfophilins could serve as an interface of signals from Rab and Arf GTPases to regulate membrane traffic and integrate distinct signals in the late endosomal recycling compartment.

Distinct Rab Binding Specificity of Rim1, Rim2, Rabphilin, and Noc2: IDENTIFICATION OF A CRITICAL DETERMINANT OF Rab3A/Rab27A RECOGNITION BY Rim2

Rabphilin, Rim, and Noc2 have generally been believed to be the Rab3 isoform (Rab3A/B/C/D)-specific effectors that regulate secretory vesicle exocytosis in neurons and in some endocrine cells. The results of recent genetic analysis of rabphilin knock-out animals, however, strongly refute this notion, because there are no obvious genetic interactions between Rab3 and rabphilin in nematoda (Staunton, J., Ganetzky, B., and Nonet, M. L. (2001) J. Neurosci. 21, 9255-9264), suggesting that Rab3 is not a major ligand of rabphilin in vivo. In this study, I tested the interaction of rabphilin, Rim1, Rim2, and Noc2 with 42 different Rab proteins by cotransfection assay and found differences in rabphilin, Rim1, Rim2, and Noc2 binding to several Rab proteins that belong to the Rab functional group III (Rab3A/B/C/D, Rab26, Rab27A/B, and Rab37) and/or VIII (Rab8A and Rab10). Rim1 interacts with Rab3A/B/C/D, Rab10, Rab26, and Rab37; Rim2 interacts with Rab3A/B/C/D and Rab8A; and rabphilin and Noc2 interact with Rab3A/B/C/D, Rab8A, and Rab27A/B. By contrast, the synaptotagmin-like protein homology domain of Slp homologue lacking C2 domains-a (Slac2-a)/melanophilin specifically recognizes Rab27A/B but not other Rabs. I also found that alternative splicing events in the first alpha-helical region (alpha(1)) of the Rab binding domain of Rim1 alter the Rab binding specificity of Rim1. Site-directed mutagenesis and chimeric analyses of Rim2 and Slac2-a indicate that the acidic cluster (Glu-50, Glu-51, and Glu-52) in the alpha(1) region of the Rab binding domain of Rim2, which is not conserved in the synaptotagmin-like pro tein homology domain of Slac2-a, is a critical determinant of Rab3A recognition. Based on these results, I propose that Rim, rabphilin, and Noc2 function differently in concert with functional group III and/or VIII Rab proteins, including Rab3 isoforms.

Geranylgeranyl Switching Regulates

Rab GTPases, key regulators of membrane targeting and fusion, require the covalent attachment of geranylgeranyl lipids to their C terminus for function. To elucidate the role of lipid in Rab recycling, we have determined the crystal structure of Rab guanine nucleotide dissociation inhibitor (αGDI) in complex with a geranylgeranyl (GG) ligand (H 2N-Cys-(S-GG)-OMe). The lipid is bound beneath the Rab binding platform in a shallow hydrophobic groove. Mutation of the binding pocket in the brain-specific αGDI leads to mental retardation. Strikingly, lipid binding acts through a conserved allosteric switching mechanism to promote release of the GDI-Rab[GDP] complex from the membrane.

Structural determinants of Rab and Rab Escort Protein interaction: Rab family motifs define a conserved binding surface

Rab proteins are a large family of monomeric GTPases with 60 members identified in the human genome. Rab GTPases require an isoprenyl modification to their C-terminus for membrane association and function in the regulation of vesicular trafficking pathways. This reaction is catalysed by Rab geranylgeranyl transferase, which recognises as protein substrate any given Rab in a 1:1 complex with Rab Escort Protein (REP). REP is therefore able to bind many distinct Rab proteins but the molecular basis for this activity is still unclear. We recently identified conserved motifs in Rabs termed RabF motifs, which we proposed to mediate a conserved mode of interaction between Rabs and REPs. Here, we tested this hypothesis. We first used REP1 as a bait in the yeast two-hybrid system and isolated strictly full-length Rabs, suggesting that REP recognises multiple regions within and properly folded Rabs. We introduced point mutations in Rab3a as a model Rab and assessed the ability of the mutants to interact with REP using the yeast two-hybrid system and an in vitro prenylation assay. We identified several residues that affect REP:Rab binding in the RabF1, RabF3, and RabF4 regions (which include parts of the switch I and II regions), but not other RabF regions. These results support the hypothesis that Rabs bind REP via conserved RabF motifs and provide a molecular explanation for the preferential recognition of the GDP-bound conformation of Rab by REP.

Expression of mammalian Rab Escort Protein-1 and -2 in yeast Saccharomyces cerevisiae

Rab GTPases are post-translationally geranylgeranylated on their C-terminal cysteines by Rab geranylgeranyl transferase (RabGGTase) and this modification is essential for their biological activity. Rab Escort Protein (REP) is a molecular chaperone that assists in the prenylation reaction carried out by RabGGTase. Mutations in the REP-1 gene lead to progressive retinal degradation and blindness in humans. Despite the significant interest in REP proteins, their preparative production remains challenging. We report here an inducible expression system for the production of REP-1 and REP-2 in yeast Saccharomyces cerevisiae at levels 3.5 and 2 mg/L, respectively. The REP-1 was found to be toxic for yeast cells and its toxicity is proposed to be associated with the formation of unproductive Ypt:REP-1 complexes. To minimize the toxic effect of REP-1, the recombinant protein expression was induced at the end of the exponential phase. Under these conditions, the GAL1 promoter is no longer repressed due to exhaustion of glucose and utilization of ethanol as a carbon source. This expression procedure was successfully scaled up to 30 L for both proteins. The REP-1 and REP-2 were purified using a combination of affinity and anion-exchange chromatography. Purified proteins were functionally active, as determined by a fluorescent Rab binding assay and in vitro prenylation. The reported procedure provides a reliable source of REP proteins for biochemical and structural studies.

Disruption of Golgi Morphology and Trafficking in Cells Expressing Mutant Prenylated Rab Acceptor-1

Prenylated Rab acceptor (PRA1) is a protein that binds Rab GTPases and the v-SNARE VAMP2. The protein is localized to the Golgi complex and post-Golgi vesicles. To determine its functional role, we generated a number of point mutations and divided them into three classes based on cellular localization. Class A mutants were retained in the endoplasmic reticulum (ER) and exerted an inhibitory effect on transport of vesicular stomatitis virus envelope glycoprotein (VSVG) from the ER to Golgi as well as to the plasma membrane. Class B mutants exhibited a highly condensed Golgi complex and inhibited exit of anterograde cargo from this organelle. Class C mutants exhibited an intermediate phenotype with Golgi and ER localization along with extensive tubular structures emanating from the Golgi complex. There was a direct correlation between the cellular phenotype and binding to Rab and VAMP2. Class A and C mutants showed a significant decrease in Rab and VAMP2 binding, whereas an increase in binding was observed in the class B mutants. Thus, PRA1 is required for vesicle formation from the Golgi complex and might be involved in recruitment of Rab effectors and SNARE proteins during cargo sequestration.

Rab11-FIP2 Functions in Transferrin Recycling and Associates with Endosomal Membranes via Its COOH-terminal Domain

Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells (Hales, C. M., Griner, R., Hobdy-Henderson, K. C., Dorn, M. C., Hardy, D., Kumar, R., Navarre, J., Chan, E. K., Lapierre, L. A., and Goldenring, J. R. (2001) J. Biol. Chem. 276, 39067-390751). Here, we characterized the specificity of the interaction between Rab11-FIP2 and Rab11 and report that it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. We demonstrate that the COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself was insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, caused tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 was also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking.

Rab Coupling Protein (RCP), a Novel Rab4 and Rab11 Effector Protein

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4- and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.

The Novel Rab11-FIP/Rip/RCP Family of Proteins Displays Extensive Homo- and Hetero-Interacting Abilities

The Rab11-FIP/Rip/RCP proteins are a recently described novel protein family, whose members interact with Rab GTPases that function in endosomal recycling. To date, five such proteins have been described in humans, all of which interact with Rab11, and one (RCP) also interacts with Rab4. Here, we characterise several of these proteins with respect to their ability to interact with Rab4, as well as their ability to self-interact, and to interact with each other. We now demonstrate that two of the family members—pp75/Rip11 and Rab11-FIP3 do not bind Rab4 and show that several members of the family can self-interact and interact with each other. These interactions primarily involve their C-terminal end which includes the Rab binding domain (RBD) that is contained within a predicted coiled-coil, or ERM motif. We identify a new (sixth) member of the protein family, which we propose to name Rab11-FIP4, and report the family evolutionary complexity and chromosomal distribution. Furthermore, we propose that the ability of these proteins to bind each other will be important in effecting membrane trafficking events by forming protein ‘platforms,’ regulated by Rab11 and/or Rab4 activity.

Identification of a Novel Rab11/25 Binding Domain Present in Eferin and Rip Proteins

Rab11, a low molecular weight GTP-binding protein, has been shown to play a key role in a variety of cellular processes, including endosomal recycling, phagocytosis, and transport of secretory proteins from the trans-Golgi network. In this study we have described a novel Rab11 effector, EF-hands-containing Rab11-interacting protein (Eferin). In addition, we have identified a 20-amino acid domain that is present at the C terminus of Eferin and other Rab11/25-interacting proteins, such as Rip11 and nRip11. Using biochemical techniques we have demonstrated that this domain is necessary and sufficient for Rab11 binding in vitro and that it is required for localization of Rab11 effector proteins in vivo. The data suggest that various Rab effectors compete with each other for binding to Rab11/25 possibly accounting for the diversity of Rab11 functions.

Mutations in Mlph, encoding a member of the Rab effector family, cause the melanosome transport defects observed in leaden mice.

The d, ash, and ln coat color mutations provide a unique model system for the study of vesicle transport in mammals. All three mutant loci encode genes that are required for the polarized transport of melanosomes, the specialized, pigment-containing organelles of melanocytes, to the neighboring keratinocytes and eventually into coat hairs. Genetic studies suggest that these genes function in the same or overlapping pathways and are supported by biochemical studies showing that d encodes an actin-based melanosome transport motor, MyoVa, whereas ash encodes Rab27a, a protein that localizes to the melanosome and is postulated to serve as the MyoVa receptor. Here we show that ln encodes melanophilin (Mlph), a previously undescribed protein with homology to Rab effectors such as granuphilin, Slp3-a, and rabphilin-3A. Like all of these effectors, Mlph possesses two Zn(2+)-binding CX(2)CX(13,14)CX(2)C motifs and a short aromatic-rich amino acid region that is critical for Rab binding. However, Mlph does not contain the two Ca(2+)-binding C(2) domains found in these and other proteins involved in vesicle transport, suggesting that it represents a previously unrecognized class of Rab effectors. Collectively, our data show that Mlph is a critical component of the melanosome transport machinery and suggest that Mlph might function as part of a transport complex with Rab27a and MyoVa.

GDP Dissociation Inhibitor Domain II Required for Rab GTPase Recycling

Rab GTPases are localized to distinct subsets of organelles within the cell, where they regulate SNARE-mediated membrane trafficking between organelles. One factor required for Rab localization and function is Rab GDP dissociation inhibitor (GDI), which is proposed to recycle Rab after vesicle fusion by extracting Rab from the membrane and loading Rab onto newly formed transport intermediates. GDI is composed of two domains; Rab binding is mediated by Domain I, and the function of Domain II is not known. In this study, Domain II of yeast GDI, encoded by the essential GDI1/SEC19 gene, was targeted in a genetic screen to obtain mutants that might lend insight into the function of this domain. In one gdi1 mutant, the cytosolic pools of all Rabs tested were depleted, and Rab accumulated on membranes, suggesting that this mutant Gdi1 protein has a general defect in extraction of Rab from membranes. In a second gdi1 mutant, the endosomal/vacuolar Rabs Vps21/Ypt51p and Ypt7p accumulated in the cytosol bound to Gdi1p, but localization of Ypt1p and Sec4p were not significantly affected. Using an in vitro assay which reconstitutes Gdi1p-mediated membrane loading of Rab, this mutant Gdi1p was found to be defective in loading of Vps21p but not Ypt1p. Loading of Vps21p by loading-defective Gdi1p was restored when acceptor membranes prepared from a deletion strain lacking Vps21p were used. These results suggest that membrane-associated Rab may regulate recruitment of GDI-Rab from the cytosol, possibly by regulating a GDI-Rab receptor. We conclude that Domain II of Gdi1p is essential for Rab loading and Rab extraction, and confirm that each of these activities is required for Gdi1p function in vivo.

Molecular basis for Rab prenylation.

Rab escort proteins (REP) 1 and 2 are closely related mammalian proteins required for prenylation of newly synthesized Rab GTPases by the cytosolic heterodimeric Rab geranylgeranyl transferase II complex (RabGG transferase). REP1 in mammalian cells is the product of the choroideremia gene (CHM). CHM/REP1 deficiency in inherited disease leads to degeneration of retinal pigmented epithelium and loss of vision. We now show that amino acid residues required for Rab recognition are critical for function of the yeast REP homologue Mrs6p, an essential protein that shows 50% homology to mammalian REPs. Mutant Mrs6p unable to bind Rabs failed to complement growth of a mrs6Delta null strain and were found to be dominant inhibitors of growth in a wild-type MRS6 strain. Mutants were identified that did not affect Rab binding, yet prevented prenylation in vitro and failed to support growth of the mrs6Delta null strain. These results suggest that in the absence of Rab binding, REP interaction with RabGG transferase is maintained through Rab-independent binding sites, providing a molecular explanation for the kinetic properties of Rab prenylation in vitro. Analysis of the effects of thermoreversible temperature-sensitive (mrs6(ts)) mutants on vesicular traffic in vivo showed prenylation activity is only transiently required to maintain normal growth, a result promising for therapeutic approaches to disease.

Molecular Role for the Rab Binding Platform of Guanine Nucleotide Dissociation Inhibitor in Endoplasmic Reticulum to Golgi Transport

Guanine nucleotide dissociation inhibitor (GDI) regulates the recycling of Rab GTPases involved in vesicle targeting and fusion. We have analyzed the requirement for conserved amino acid residues in the binding of Rab1A and the function of GDI in transport of cargo between the endoplasmic reticulum (ER) and the Golgi apparatus. Using a new approach to monitor GDI-Rab interactions based on the change in fluorescence associated with the release of methylanthraniloyl guanosine di(tri)phosphate-GDP (mGDP) from Rab, we show that residues previously implicated in the binding of the synapse-specific Rab3A, including Gln-236, Arg-240, and Thr-248, are essential for the binding of Rab1A. Mutation of each of these residues has potent effects on the ability of GDI to remove Rab1A from membranes and inhibit ER to Golgi transport in vitro. Given the sequence divergence between Rab1A and 3A (35% identity), these residues are proposed to play a general role in GDI function in the cell. In contrast, several other residues found within or flanking the Rab-binding region were found to have differential effects in the recognition and recycling of Rab1A and 3A, and therefore direct selective interaction of GDI with individual Rab proteins. Intriguingly, mutation of one residue, Arg-70, led to a reduction of Rab1A binding, failed to extract Rab1A from membranes in vitro, yet bound membranes tightly and potently inhibited ER to Golgi transport. These results provide evidence that novel membrane-associated factor(s) mediate Rab-independent GDI interaction with membranes.

Chimeras Containing Influenza NS1 and HIV-1 Rev Protein Sequences: Mechanism of Their Inhibition of Nuclear Export of Rev Protein–RNA Complexes

Nuclear RNA export mediated by the HIV-1 Rev protein is inhibited by chimeric proteins in which the wild-type Rev protein is covalently linked to amino acid sequences of the NS1 protein of influenza A virus (NS1A protein), a protein that inhibits nuclear RNA export. These chimeric molecules function not only incisbut also intrans:they inhibit nuclear RNA export mediated by Rev protein molecules that are not covalently linked to the NS1A protein sequence. Here we show that inhibition occurs with a NS1-Rev chimera in which the 78 amino-terminal amino acids of the NS1A protein comprising its entire RNA-binding domain is deleted, thereby establishing that this carboxyl portion of the NS1A protein can function as an independent effector domain. The mechanism by which this NS1-Rev chimera inhibits Rev function intranswas determined. The Rev sequence in this chimera oligomerizes with Rev molecules intrans,and the resulting mixed oligomers are retained in the nucleus because the nuclear retention activity of the NS1 effector domain is dominant over the nuclear transport activity of the Rev effector domain. Binding of the FG-containing nucleoporin-like Rab protein to this NS1-Rev chimera, as measured in yeast two-hybrid assays, is much stronger than that to the Rev protein itself, yet nuclear export does not occur in the presence of the chimera. Unexpectedly, the introduction of specific mutations into the NS1A portion of this NS1-Rev chimera not only restores Rev-mediated nuclear export of RNA but also eliminates detectable Rab binding, indicating that this nuclear export can occur without detectable Rab binding. A different NS1-Rev chimera, one in which the NS1A protein is full-length but contains a mutated RNA-binding domain, effectively inhibits Rev-mediated nuclear export of RNA without blocking the nuclear export of the Rev protein, indicating that nuclear export of the carrier Rev protein can be uncoupled from nuclear export of its passenger RNA.

Isolation and Characterization of a Dual Prenylated Rab and VAMP2 Receptor

Rab GTPases have been implicated in intracellular vesicle trafficking. Using the yeast two-hybrid screen, we have isolated a rat clone that interacts with Rab3A as well as with Rab1. The gene encodes a 20.6-kDa protein with two extensive hydrophobic domains and is broadly expressed in all tissues. This protein binds to prenylated Rab GTPases but not to other small Ras-like GTPases such as the Rho/Rac family. This prenylated Rab acceptor (PRA1) also binds specifically to the synaptic vesicle protein VAMP2 (or synaptobrevin II) but shows no affinity for VAMP1 or cellubrevin in both the yeast two-hybrid system and in vitro binding assays. This specificity resides, in part, in the proline-rich domain of VAMP2 as a chimera containing this domain of VAMP2 fused to VAMP1 is able to bind to PRA1. The transmembrane domain of VAMP2 is also essential as its deletion abolished binding to PRA1. Replacement of the deleted VAMP2 transmembrane domain by a CAAX prenylation signal can not restore binding to PRA1. This interaction is therefore distinct from that required for VAMP2 binding to either syntaxin or both syntaxin and SNAP-25. Deletion analysis on PRA1 indicates that the critical Rab- and VAMP2-interacting residues reside in two regions: the amino-terminal residues 30-54 and the extreme carboxyl-terminal domain. This dual Rab and VAMP2 binding characteristic suggests that PRA1 may serve to link these two protein families in the control of vesicle docking and fusion.

A Rab GTPase Is Required for Homotypic Assembly of the Endoplasmic Reticulum

To define the requirements for the homotypic fusion of mammalian endoplasmic reticulum (ER) membranes, we have developed a quantitative in vitro enzyme-linked immunosorbent assay. This assay measures the formation of IgG (H2L2) following the fusion of ER microsomes containing either the heavy or light chain subunits. Guanine nucleotide dissociation inhibitor (GDI), a protein that extracts Rab GTPases in the GDP-bound form from membranes, potently inhibits fusion. Inhibition was not observed using GDI mutants defective in Rab binding. Kinetic analysis of the inhibitory effects of GDI revealed that Rab activation is required immediately preceding or coincident with fusion and that this step is preceded by a priming event requiring a member of the AAA ATPase family. Our results suggest that homotypic fusion of ER membranes requires Rab and that Rab activation is a transient event necessary for the formation of a fusion pore leading to the mixing of luminal contents of ER microsomes.

23] Use of two-hybrid system to identify Rab binding proteins

Publisher Summary The yeast two-hybrid system is a genetic screen that detects in vivo interactions between proteins. This system is based on the modular nature of some transcriptional factors. Such factors possess distinct DNA-binding and transcriptional activation domains. These domains can be incorporated into separate polypeptides, and they will function to activate specific gene expression if brought into close proximity. The major advantages of the yeast two-hybrid system for isolating possible effectors for Rab proteins are that (1) it is an in vivo interaction assay, which is more physiologically relevant than in vitro interaction assays, (2) it is often more sensitive than some in vitro assays, such as coimmunoprecipitations, and (3) the end product of this assay is a cDNA clone of the interacting protein. The method is, in principle, much faster than using conventional affinity purification to purify the interacting protein followed by peptide sequencing and library screening with an antibody or degenerate oligonucleotides to obtain the cDNA clone. A major disadvantage of the two-hybrid system is the possibility that the protein–protein interactions may be blocked by one of the fusion partners.