Isolation, characterization, and chromosome mapping of a human A-C1 Ha-Ras suppressor gene (HRASLS)

Recently, we cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines, embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. Mouse A-C1 has homology with a ras-responsive gene, rat Ha-rev107 (Hrasls), and modulates a Ha-ras-mediated signaling pathway. Here, we report a cDNA encoding a human homolog of mouse A-C1. The deduced amino acid sequence of human A-C1 consists of 168 amino acids, and shows 83% identity with that of mouse A-C1. Human A-C1 mRNA was expressed in skeletal muscle, testis, heart, brain, and thyroid in vivo. Moreover, expression of human A-C1 mRNA was detected at a high level in human osteosarcoma-derived U2OS cells in vitro. By FISH analysis the human A-C1 gene (HRASLS) was mapped to human chromosome 3q28--> q29.     

Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein.

We recently purified to near homogeneity a novel type of regulatory protein for smg p25A, a ras p21-like GTP-binding protein, from bovine brain cytosol. This regulatory protein, named smg p25A GDP dissociation inhibitor (GDI), regulates the GDP-GTP exchange reaction of smg p25A by inhibiting dissociation of GDP from and subsequent binding of GTP to it. In the present studies, we isolated and sequenced the cDNA of smg p25A GDI from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of purified smg p25A GDI. The cDNA has an open reading frame that encodes a protein of 447 amino acids with a calculated Mr of 50,565. This Mr is similar to those of the purified smg p25A GDI estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation, which are about 54,000 and 65,000, respectively. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits GDI activity. smg p25A GDI is hydrophilic overall, except for one hydrophobic region near the N terminus. smg p25A GDI shares low amino acid sequence homology with the Saccharomyces cerevisiae CDC25-encoded protein, which has been suggested to serve as a factor that regulates the GDP-GTP exchange reaction of the yeast RAS2-encoded protein, but not with the beta gamma subunits of GTP-binding proteins having an alpha beta gamma subunit structure, such as Gs and Gi. The smg p25A GDI mRNA was present in various tissues, including not only tissues in which smg p25A was detectable but also tissues in which it was not detectable. This fact has raised the possibility that smg p25A GDI interacts with another G protein in tissues in which smg p25A is absent.     

General role of GDP dissociation inhibitor 2 in membrane release of Rab proteins: modulations of its functional interactions by in vitro and in vivo structural modifications.

The GDP dissociation inhibitors (GDIs) represent an important class of regulatory proteins in the functional cycle and recycling of Rab GTPases. Previous studies have demonstrated that GDI-1 can operate with multiple Rab proteins. In this study we have addressed a plausible general activity of GDI-2 in supporting Rab membrane release and have analyzed the requirements of sequence-conserved vs variable regions of GDI-2 in these functional interactions. The in vitro function of expressed recombinant GDI-2 wild-type-, point-, or deletion-mutant proteins was investigated toward several Rab family members, divergent in structure, localized and operating on different membranes, including Rab2, Rab4, Rab5, Rab8, Rab9, and Rab11. We demonstrate here a general and nearly invariant ability of GDI-2(WT) to release from membranes this subset of diverse Rabs. Deletion of an 18-residue segment from the C-terminal variable region yielded a fully functional or only slightly defective GDI-2. Conversely, substitution of Met at position 250 of the conserved region markedly abrogated the activity toward all Rabs. Surprisingly, a replacement of an adjacent conserved residue (Y249V) resulted in a selective Rab-dependent response and a profound gain of function toward specific Rabs. To further test whether the endogenous GDI-2 can adopt a gain-of-function conformation, we pharmacologically stimulated intact 3T3-L1 adipocytes to induce GDI-2 tyrosine phosphorylation. We found a pronounced increase of the Rab4 soluble form and its soluble complexes with the tyrosine-phosphorylated GDI-2. Together, these results indicate that (a) GDI-2 displays a general activity to release Rabs from membranes, (b) GDI-2-conserved residues, but not the C-terminal variable region, are essential for this activity, and (c) structural modifications in GDI-2 can enhance its functional activity, directing selective interactions with individual Rabs.     

Mouse and human GTPBP2, newly identified members of the GP-1 family of GTPase

We earlier identified the GTPBP1 gene which encodes a putative GTPase structurally related to peptidyl elongation factors. This finding was the result of a search for genes, the expression of which is induced by interferon-gamma in a macrophage cell line, THP-1. In the current study, we probed the expressed sequence tag database with the deduced amino acid sequence of GTPBP1 to search for partial cDNA clones homologous to GTPBP1. We used one of the partial cDNA clones to screen a mouse brain cDNA library and identified a novel gene, mouse GTPBP2, encoding a protein consisting of 582 amino acids and carrying GTP-binding motifs. The deduced amino acid sequence of mouse GTPBP2 revealed 44.2% similarity to mouse GTPBP1. We also cloned a human homologue of this gene from a cDNA library of the human T cell line, Jurkat. GTPBP2 protein was found highly conserved between human and mouse (over 99% identical), thereby suggesting a fundamental role of this molecule across species. On Northern blot analysis of various mouse tissues, GTPBP2 mRNA was detected in brain, thymus, kidney and skeletal muscle, but was scarce in liver. Level of expression of GTPBP2 mRNA was enhanced by interferon-gamma in THP-1 cells, HeLa cells, and thioglycollate-elicited mouse peritoneal macrophages. In addition, we determined the chromosomal localization of GTPBP1 and GTPBP2 genes in human and mouse. The GTPBP1 gene was mapped to mouse chromosome 15, region E3, and human chromosome 22q12-13.1, while the GTPBP2 gene is located in mouse chromosome 17, region C-D, and human chromosome 6p21-12.     

Molecular cloning and biological activity of a novel Ha-Ras suppressor gene predominantly expressed in skeletal muscle, heart, brain, and bone marrow by differential display using clonal mouse EC cells, ATDC5.

We cloned a cDNA encoding a novel mouse protein, named A-C1, by differential display between two mouse cell lines: embryonic fibroblast C3H10T1/2 and chondrogenic ATDC5. The deduced amino acid sequence of A-C1 consists of 167 amino acids and shows 46% identity with that of a ras-responsive gene, rat Ha-rev107. Northern blot analysis showed a distinct hybridization band of 3.2 kilobases. Expression of A-C1 mRNA was detected in undifferentiated ATDC5 cells and myoblastic C2C12 cells, while none of C3H10T1/2 cells, NIH3T3 fibroblasts, Balb/c 3T3 fibroblasts, osteoblastic MC3T3-E1 cells, and ST2 bone marrow stromal cells expressed A-C1 mRNA in vitro. Moreover, A-C1 mRNA was expressed in skeletal muscle, heart, brain, and bone marrow in adult mice. By in situ hybridization, A-C1 gene expression was localized in hippocampus as well as bone marrow cells. By immunocytochemistry, A-C1 protein was detected in the cytoplasm as well as perinuclear region of the cells. Transfection of A-C1 cDNA into Ha-ras-transformed NIH3T3 cell line caused increase in the number of flat colonies and inhibition of cell growth. Our data indicate that A-C1 is expressed in some specific tissues in vivo and modulates Ha-ras-mediated signaling pathway.     

Molecular cloning and characterisation of a Rab-binding GDP-dissociation inhibitor from Medicago truncatula.

We have isolated and sequenced the full-length cDNA of a GDP-dissociation inhibitor (GDI) from the model legume Medicago truncatula L. The cDNA (MtGDI) contains an open reading frame of 1335 bp, coding for a protein of 444 amino acids with a calculated molecular mass of 49,785 kDa. The deduced amino acid sequence shows significant homology to other plant GDIs, the highest homology being found to GDI from the legume Cicer arietinum (96% identity). The MtGDI was expressed as a N-terminal FLAG-fusion protein in Escherichia coli BL21 (DE3). Its direct interaction with a small G protein of Rab type from Medicago sativa, MsRab11f, was demonstrated in vitro by co-immunoprecipitation using a peptide-specific antibody raised against MtGDI. The dissociation constant of the MtGDI-MsRab11f complex (4 muM) was determined by a surface plasmon resonance (SPR) assay. Real-time RT-PCR and Western blot analyses suggested that MtGDI is ubiquitously expressed in M. truncatula. High levels of MtGDI mRNA were detected in uninfected roots, leaves and root nodules. In etiolated seedlings and cell cultures, the amount of MtGDI mRNA was much lower. In all tissues tested, the peptide-specific anti-MtGDI antibody detected the expected 50 kDa protein in the total protein extracts. MtGDI was found in the cytosol; however, a significant fraction was associated with the intracellular membranes in seedlings and roots indicating a membrane localisation of the protein. A second immunoreactive band was detected in leaves suggesting that more than one GDI isoform exist in M. truncatula.     

Reversible phosphocholination of Rab proteins by Legionella pneumophila effector proteins

The Legionella pneumophila protein AnkX that is injected into infected cells by a Type IV secretion system transfers a phosphocholine group from CDP-choline to a serine in the Rab1 and Rab35 GTPase Switch II regions. We show here that the consequences of phosphocholination on the interaction of Rab1/Rab35 with various partner proteins are quite distinct. Activation of phosphocholinated Rabs by GTP/GDP exchange factors (GEFs) and binding to the GDP dissociation inhibitor (GDI) are strongly inhibited, whereas deactivation by GTPase activating proteins (GAPs) and interactions with Rab-effector proteins (such as LidA and MICAL-3) are only slightly inhibited. We show that the Legionella protein lpg0696 has the ability to remove the phosphocholine group from Rab1. We present a model in which the action of AnkX occurs as an alternative to GTP/GDP exchange, stabilizing phosphocholinated Rabs in membranes in the GDP form because of loss of GDI binding ability, preventing interactions with cellular GTPase effectors, which require the GTP-bound form. Generation of the GTP form of phosphocholinated Rab proteins cannot occur due to loss of interaction with cellular GEFs.     

Primary structure, genomic organization and expression of the major secretory protein of murine epididymis, ME1

The mouse cDNA and its genomic clones encoding the epididymal secretory glycoprotein ME1 were identified. The Me1 gene spans 15kb with four exons and three introns. The deduced amino-acid sequence of the ME1 cDNA revealed that it consists of 149 amino acid residues, which contain a signal peptide characteristic of secretory proteins, six cysteine residues and a proline-rich region conserved in the orthologous proteins. Northern blot analysis revealed that 1.3kb ME1 mRNA is highly expressed in the mouse epididymis. The polyclonal antibodies generated against human HE1 (ME1 orthologous protein) expressed in bacteria reacted with approximately 17 to 25kDa components in mouse epididymis crude extract. The reduction of the molecular mass of the recombinant ME1 protein with the digestion of glycopeptidase A indicated that it is modified by Asn-linked glycosylation.     

Molecular cloning of the cDNA for stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like small GTP-binding proteins) and characterization of stimulatory GDP/GTP exchange protein.

We have recently purified to near homogeneity the stimulatory GDP/GTP exchange protein for smg p21s (ras p21-like GTP-binding proteins) from bovine brain cytosol. This regulatory protein, named GDP dissociation stimulator (GDS), stimulates the GDP/GTP exchange reaction of smg p21s by stimulating the dissociation of GDP from and the subsequent binding of GTP to them. In this study, we have isolated and sequenced the cDNA of smg p21 GDS from a bovine brain cDNA library by using an oligonucleotide probe designed from the partial amino acid sequence of the purified smg p21 GDS. The cDNA has an open reading frame encoding a protein of 558 amino acids with a calculated Mr value of 61,066, similar to the Mr of 53,000 estimated for the purified smg p21 GDS by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sucrose density gradient ultracentrifugation. The isolated cDNA is expressed in Escherichia coli, and the encoded protein exhibits smg p21 GDS activity. smg p21 GDS is overall hydrophilic, but there are several short hydrophobic regions. The smg p21 GDS mRNA is present in bovine brain and various rat tissues. smg p21 GDS has low amino acid sequence homology with the yeast CDC25 and SCD25 proteins, which may regulate the GDP/GTP exchange reaction of the yeast RAS2 protein, but not with ras p21 GTPase-activating protein, the inhibitory GDP/GTP exchange proteins (GDP dissociation inhibitor) for smg p25A and rho p21s, and the beta gamma subunits of heterotrimeric GTP-binding proteins such as Gs and Gi.     

A structural model of the GDP dissociation inhibitor rab membrane extraction mechanism

Rab GDP dissociation inhibitors (GDI)-facilitated extraction of prenylated Rab proteins from membranes plays an important role in vesicular membrane trafficking. The investigated thermodynamic properties of yeast Rab.GDI and Rab.MRS6 complexes demonstrated differences in the Rab binding properties of the closely related Rab GDI and MRS6 proteins, consistent with their functional diversity. The importance of the Rab C terminus and its prenylation for GDI/MRS6 binding was demonstrated using both biochemical and structural data. The presented structures of the apo-form yeast Rab GDI and its two complexes with unprenylated Rab proteins, together with the earlier published structures of the prenylated Ypt1.GDI, provide evidence of allosteric regulation of the GDI lipid binding site opening, which plays a key role in the proposed mechanism of GDI-mediated Rab extraction. We suggest a model for the interaction of GDI with prenylated Rab proteins that incorporates a stepwise increase in affinity as the three different partial interactions are successively formed.     

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.     

Molecular cloning of cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum

We have cloned and sequenced cDNA encoding the Ca2+ release channel (ryanodine receptor) of rabbit cardiac muscle sarcoplasmic reticulum. The cDNA, 16,532 base pairs in length, encodes a protein of 4,969 amino acids with a Mr of 564,711. The deduced amino acid sequence is 66% identical with that of the skeletal muscle ryanodine receptor, but analysis of predicted secondary structures and hydropathy plots suggests that the two isoforms exhibit the same topology in both transmembrane and cytoplasmic domains. A potential ATP binding domain was identified at residues 2619-2652, a potential phosphorylation site at residue 2809, and potential calmodulin binding sites at residues 2775-2807, 2877-2898, and 2998-3016. We suggest that a modulator binding domain in the protein lies between residues 2619 and 3016. Northern blot analysis of mRNA from a variety of tissues demonstrated that the cardiac isoform is expressed in heart and brain, while the skeletal muscle isoform is expressed in both fast- and slow-twitch muscle. No ryanodine receptor mRNA was detected in extracts from smooth muscle or any other non-muscle tissue examined. The two receptors are clearly the products of separate genes, and the gene encoding the cardiac muscle ryanodine receptor was localized to chromosome 1.     

cDNA clone and expression analysis of rodent fast and slow skeletal muscle troponin I mRNAs

We have characterized the structure and expression of rodent mRNAs encoding the fast and slow skeletal muscle isoforms of the contractile regulatory protein, troponin I (TnIfast and TnIslow). TnIfast and TnIslow cDNA clones were isolated from mouse and rat muscle cDNA clone libraries and were used as isoform-specific probes in Northern blot and in situ hybridization studies. These studies showed that the TnIfast and TnIslow mRNAs are expressed in skeletal muscle, but not cardiac muscle or other tissues, and that they are differentially expressed in individual muscle fibers. Fiber typing on the basis of in situ hybridization analysis of TnI isoform mRNA content showed an excellent correlation with fiber type as assessed by myosin ATPase histochemistry. These results directly demonstrate that the differential expression of skeletal muscle TnI isoforms in the various classes of vertebrate striated muscle cells is based on gene regulatory mechanisms which control the abundances of specific TnI mRNAs in individual muscle cells. Both TnIfast and TnIslow mRNAs are expressed, at comparable levels, in differentiated cultures of rat L6 and mouse C2 muscle cell lines. Thus, although neuronal input has been shown to be an important factor in determining fast versus slow isoform-specific expression in skeletal muscle, both TnIfast and TnIslow genes can be expressed in muscle cells in the absence of nerve. Comparison of the deduced rodent TnI amino acid sequences with previously determined rabbit protein sequences showed that residues with potential fast/slow isoform-specific function are present in several discrete clusters, two of which are located near previously identified actin and troponin C binding sites.     

Molecular dissection of guanine nucleotide dissociation inhibitor function in vivo. Rab-independent binding to membranes and role of Rab recycling factors.

Guanine nucleotide dissociation inhibitor (GDI) is an essential protein required for the recycling of Rab GTPases mediating the targeting and fusion of vesicles in the exocytic and endocytic pathways. Using site-directed mutagenesis of yeast GDI1, we demonstrate that amino acid residues required for Rab recognition in vitro are critical for function in vivo in Saccharomyces cerevisiae. Analysis of the effects of Rab-binding mutants on function in vivo reveals that only a small pool of recycling Rab protein is essential for growth, and that the rates of recycling of distinct Rabs are differentially sensitive to GDI. Furthermore, we find that membrane association of Gdi1p is Rab-independent. Mutant Gdi1 proteins unable to bind Rabs were able to associate with cellular membranes as efficiently as wild-type Gdi1p, yet caused a striking loss of the endogenous cytosolic Gdi1p-Rab pools leading to dominant inhibition of growth when expressed at levels of the normal, endogenous pool. These results demonstrate a potential role for a new recycling factor in the retrieval of Rab-GDP from membranes, and illustrate the importance of multiple effectors in regulating GDI function in Rab delivery and retrieval from membranes.     

The novel skeletal muscle sarcoplasmic reticulum JP-45 protein. Molecular cloning,tissue distribution,developmental expression,and interaction with alpha 1.1 subunit of the voltage-gated calcium channel

JP-45 is a novel integral protein constituent of the skeletal muscle sarcoplasmic reticulum junctional face membrane. We identified its primary structure from a cDNA clone isolated from a mouse skeletal muscle cDNA library. Mouse skeletal muscle JP-45 displays over 86 and 50% identity with two hypothetical NCBI data base protein sequences from mouse tongue and human muscle, respectively. JP-45 is predicted to have a cytoplasmic domain, a single transmembrane segment followed by an intralumenal domain enriched in positively charged amino acids. Northern and Western blot analyses reveal that the protein is mainly expressed in skeletal muscle. The mRNA encoding JP-45 appears in 17-day-old mouse embryos; expression of the protein peaks during the second month of postnatal development and then decreases approximately 3-fold during aging. Double immunofluorescence of adult skeletal muscle fibers demonstrates that JP-45 co-localizes with the sarcoplasmic reticulum calcium release channel. Co-immunoprecipitation experiments with a monoclonal antibody against JP-45 show that JP-45 interacts with the alpha1.1 subunit voltage-gated calcium channel and calsequestrin. These results are consistent with the localization of JP-45 in the junctional sarcoplasmic reticulum and with its involvement in the molecular mechanism underlying skeletal muscle excitation-contraction coupling.     

Binding of Rab3A to synaptic vesicles

Prenylated Rab GTPases cycle between membrane-bound and soluble forms. Membrane-bound GDP-Rabs interact with GDP dissociation inhibitor (GDI), resulting in the dissociation of a Rab.GDI complex, which in turn serves as a precursor for the membrane re-association of Rabs. We have now characterized the binding of Rab3A to synaptic vesicles in vitro using either purified complexes or rat brain cytosol as source for GDI.Rab3A. Binding of Rab3A results in the immediate release of GDI from the membrane. Furthermore, binding does not require the presence of additional guanine nucleotides (GDP or GTP) or of cytosolic factors. Although nucleotide exchange follows binding, binding is initially reversible, suggesting that binding of GDP-Rab3A and nucleotide exchange are separate and independent events. Comparison with the binding of Rab1B revealed that both Rab proteins bind preferentially to their respective resident membranes although some promiscuity was observable. Binding is saturable and involves a protease-sensitive binding site that is tightly associated with the vesicle membrane.     

Structural insights into the dual nucleotide exchange and GDI displacement activity of SidM/DrrA

GDP‐bound prenylated Rabs, sequestered by GDI (GDP dissociation inhibitor) in the cytosol, are delivered to destined sub‐cellular compartment and subsequently activated by GEFs (guanine nucleotide exchange factors) catalysing GDP‐to‐GTP exchange. The dissociation of GDI from Rabs is believed to require a GDF (GDI displacement factor). Only two RabGDFs, human PRA‐1 and Legionella pneumophila SidM/DrrA, have been identified so far and the molecular mechanism of GDF is elusive. Here, we present the structure of a SidM/DrrA fragment possessing dual GEF and GDF activity in complex with Rab1. SidM/DrrA reconfigures the Switch regions of the GTPase domain of Rab1, as eukaryotic GEFs do toward cognate Rabs. Structure‐based mutational analyses show that the surface of SidM/DrrA, catalysing nucleotide exchange, is involved in GDI1 displacement from prenylated Rab1:GDP. In comparison with an eukaryotic GEF TRAPP I, this bacterial GEF/GDF exhibits high binding affinity for Rab1 with GDP retained at the active site, which appears as the key feature for the GDF activity of the protein.     

Regulation of the superoxide-generating NADPH oxidase by a small GTP-binding protein and its stimulatory and inhibitory GDP/GTP exchange proteins.

The superoxide-generating NADPH oxidase system in phagocytes consists of at least membrane-associated cytochrome b558 and three cytosolic components named SOCI/NCF-3/sigma 1/C1, SOCII/NCF-1/p47-phox, and SO-CIII/NCF-2/p67-phox. p47-phox and p67-phox were isolated, and their primary structures were determined, but SOCI has not been well characterized. In the present study, we first purified SOCI to homogeneity from the cytosol fraction of the differentiated HL-60 cells. The purified SOCI was a small GTP-binding protein (G protein) with a M(r) of about 22,000. The guanosine 5'-(3-O-thio)triphosphate-bound form, but not the GDP-bound form, of this small G protein showed the SOCI activity. The partial amino acid sequence of SOCI thus far determined was identical to the amino acid sequence deduced from the cDNA encoding rac2 p21. None of the purified small G proteins, including Ki-ras p21, smg p21B/rap1B p21, rhoA p21, and rac1 p21, showed the SOCI activity. These results indicate that SOCI is a small G protein very similar, if not identical, to rac2 p21. The GDP/GTP exchange reaction of SOCI was stimulated and inhibited by stimulatory and inhibitory GDP/GTP exchange proteins for small G proteins, named smg GDS and rho GDI, respectively. The NADPH oxidase activity was also stimulated and inhibited by smg GDS and rho GDI, respectively. These results indicate that the superoxide-generating NADPH oxidase system is regulated by both smg GDS and rho GDI through rac2 p21 or the rac2-related small G protein in phagocytes.     

An Arabidopsis gene isolated by a novel method for detecting genetic interaction in yeast encodes the GDP dissociation inhibitor of Ara4 GTPase.

The Arabidopsis Ara proteins belong to the Rab/Ypt family of small GTPases, which are implicated in intracellular vesicular traffic. To understand their specific roles in the cell, it is imperative to identify molecules that regulate the GTPase cycle. Such molecules have been found and characterized in animals and yeasts but not in plants. Using a yeast system, we developed a novel method of functional screening to detect interactions between foreign genes and identified this Rab regulator in plants. We found that the expression of the ARA4 gene in yeast ypt mutants causes exaggeration of the mutant phenotype. By introducing an Arabidopsis cDNA library into the ypt1 mutant, we isolated a clone whose coexpression overcame the deleterious effect of ARA4. This gene encodes an Arabidopsis homolog of the Rab GDP dissociation inhibitor (GDI) and was named AtGDI1. The expression of AtGDI1 complemented the yeast sec19-1 (gdi1) mutation. AtGDI1 is expressed almost ubiquitously in Arabidopsis tissues. The method described here indicates the physiological interaction of two plant molecules, Ara4 and GDI, in yeast and should be applicable to other foreign genes.