Identification of the ral and rac1 gene products, low molecular mass GTP-binding proteins from human platelets

Identification of the GTP-binding proteins from human platelet particulate fractions was attained by their purification via successive column chromatography steps followed by amino acid sequencing. To enhance the likelihood of identifying the GTP-binding proteins, two assays were employed to monitor GTP-binding activities: (i) guanosine 5'-(3-O-[35S]thio)triphosphate (GTP gamma S)-binding followed by rapid filtration and ii) [alpha-32P]GTP-binding following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotting onto nitrocellulose membranes. The latter assay permitted the isolation of a 28-kDa GTP-binding protein that bound [alpha-32P]GTP prominently but was only poorly detected with the GTP gamma S-binding assay. The amino acid sequences of three peptide fragments derived from the 28-kDa protein were identical to regions of the amino acid sequence deduced from a simian ral cDNA with the exception of one conservative substitution (Asp147----Glu). A full length human ral cDNA was isolated from a placental cDNA library, and its deduced amino acid sequence, compared with simian ral, also contained the Asp----Glu substitution along with two other substitutions and an additional three NH2-terminal amino acids. In addition to the 28-kDa protein, two distinct 25-kDa GTP-binding proteins were purified from platelets. One of these proteins has been previously characterized as G25K, an abundant low molecular mass GTP-binding protein. Partial amino acid sequence obtained from the second unidentified 25-kDa protein indicates that it is the product of the rac1 gene; a member of a newly identified gene family which encode for low molecular mass GTP-binding proteins (Didsbury, J., Weber, R.F., Bokoch, G. M., Evans, T., and Snyderman, R. (1989) J. Biol. Chem. 264, 16378-16382). These results identify two new GTP-binding proteins in human platelets, ral, the major protein that binds [alpha-32P]GTP on nitrocellulose transfers, and rac1, a substrate for botulinum C3 ADP-ribosyltransferase.     

Detection of 23-27 kDa GTP-binding proteins in platelets and other cells.

Membrane proteins from rabbit and human platelets were separated by SDS/polyacrylamide-gel electrophoresis and the resolved polypeptides blotted on nitrocellulose. A family of GTP-binding proteins, termed Gn proteins, was detected by incubation of these blots with [alpha-32P]GTP in the presence of Mg2+. A major Gn protein with a molecular mass of 27 kDa (Gn27) and lesser amounts of 23, 24 and 25 kDa Gn proteins were observed in platelet membranes; much smaller amounts were in the platelet soluble fraction. Binding of [alpha-32P]GTP by platelet Gn proteins was blocked by GDP, GTP or guanosine 5'-[gamma-thio]triphosphate, but not by GMP or adenosine 5'-[beta gamma-imido]triphosphate. Rabbit and human red-cell membranes contained only Gn27. When rat tissues were analysed for Gn proteins, the largest amounts were found in brain, which contained two membrane-bound forms (Gn27 and Gn26) and a soluble form (Gn26).     

Identification and isoprenylation of plant GTP-binding proteins

To identify isoprenylated plant GTP-binding proteins,Arabidopsis thaliana andNicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [³²P]GTPin vitro. ATGB2, anArabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTPin vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a-GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC,-XCXC, or-CCXX).In vitro geranylgeranylation of anArabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence contirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified byin vitro GTP binding, includingArabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDaArabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein -subunit superfamilies).     

Babesia gibsoni: molecular cloning and characterization of Rab6 and Rab11 homologues

Members of the Rab subfamily of GTPases have been implicated as important components in vesicle trafficking in the eukaryotes, individual Rab proteins have a remarkable degree of specific subcellular localization. As a first step towards developing a set of compartment specific probes for studying protein trafficking in Babesia-infected erythrocyte, here we describe the cloning and characterization of Rab6 and Rab11 gene homologues in Babesia gibsoni (BgRab6 and BgRab11). The deduced amino acid sequence of both BgRab6 and BgRab11 contained the highly conserved GTP-binding consensus sequence and C-terminal cysteines. Northern blotting analysis of total RNA hybridized a 1.3 kb band on both BgRab6 and BgRab11 probed blots consistent with the expected size. Using a GTP-binding assay we demonstrated that Escherichia coli expressed recombinant BgRab6 and BgRab11 were able to bind GTP. BgRab6 and BgRab11 represent the first two molecular markers of B. gibsoni.     

Expression of Rab small GTPases in epithelial Caco-2 cells: Rab21 is an apically located GTP-binding protein in polarised intestinal epithelial cells

Rab proteins belong to a subfamily of small GTP-binding protein genes of the Ras superfamily and play an important role in intracellular vesicular targeting. The presence of members of this protein family was examined in Caco-2 cells by a PCR-based strategy. Twenty-five different partial cDNA sequences were isolated, including 18 Rab protein family members. Seven novel human sequences, representing Rab2B, Rab6A', Rab6B, Rab10, Rab19B, Rab21 and Rab22A, were identified. For one clone, encoding Rab21, full-length cDNA was isolated from a Caco-2 cDNA library. Northern blot analysis showed a ubiquitous expression pattern of Rab21. To study Rab21 protein expression in Caco-2 cells, polyclonal antibodies were raised against GST-Rab21 fusion protein and characterised. The antibodies recognised Rab21 as a protein of approximately 25 kDa. Interestingly, the protein shows a general ER-like staining in nonpolarised Caco-2 cells in contrast to an apically located vesicle-like staining in polarised Caco-2 cells. Furthermore, immunohistochemical staining on human jejunal tissue showed a predominant expression of Rab21 in the epithelial cell layer with high expression levels in the apical region, whereas stem cells in the crypts were negative. We therefore suggest an alternative role for Rab21 in the regulation of vesicular transport in polarised intestinal epithelial cells.     

Detection of low molecular mass GTP-binding proteins in chromaffin granules and other subcellular fractions of chromaffin cells

A homogenate of purified chromaffin cells was fractionated, after removal of the nuclear fraction, by sucrose density gradient ultracentrifugation. The presence and subcellular localization of low molecular mass GTP-binding proteins was explored by incubation of blots of proteins from different subcellular fractions with [alpha-32P]GTP in the presence of Mg2+. The fractions enriched in intact chromaffin granule markers, i.e. catecholamines, chromogranin A, chromogranin B and cytochrome b-561 were also enriched in labelled GTP-binding proteins. Two major labelled components of 23 and 29 kDa were rapidly detected by autoradiography. Traces of 26 and 27 kDa components were also present. These components were detectable in both plasma and granule membranes. In addition to these components, the cytosolic fraction contained another GTP-binding protein of about 20 kDa. Binding of [alpha-32P]GTP was specific and dependent on Mg2+. By analogy to the findings reported in non-mammalian systems, the observations described here suggest the involvement of low molecular mass GTP-binding proteins in the chromaffin cell secretory process.     

Properties of Rab5 N-terminal domain dictate prenylation of C-terminal cysteines.

Rab5 is a Ras-related GTP-binding protein that is post-translationally modified by prenylation. We report here that an N-terminal domain contained within the first 22 amino acids of Rab5 is critical for efficient geranylgeranylation of the protein's C-terminal cysteines. This domain is immediately upstream from the "phosphate binding loop" common to all GTP-binding proteins and contains a highly conserved sequence recognized among members of the Rab family, referred to here as the YXYLFK motif. A truncation mutant that lacks this domain (Rab5(23-215) fails to become prenylated. However, a chimeric peptide with the conserved motif replacing cognate Rab5 sequence (MAYDYLFKRab5(23-215) does become post-translationally modified, demonstrating that the presence of this simple six amino acid N-terminal element enables prenylation at Rab5's C-terminus. H-Ras/Rab5 chimeras that include the conserved YXYLFK motif at the N-terminus do not become prenylated, indicating that, while this element may be necessary for prenylation of Rab proteins, it alone is not sufficient to confer properties to a heterologous protein to enable substrate recognition by the Rab geranylgeranyl transferase. Deletion analysis and studies of point mutants further reveal that the lysine residue of the YXYLFK motif is an absolute requirement to enable geranylgeranylation of Rab proteins. Functional studies support the idea that this domain is not required for guanine nucleotide binding since prenylation-defective mutants still bind GDP and are protected from protease digestion in the presence of GTP gamma S. We conclude that the mechanism of Rab geranylgeranylation involves key elements of the protein's tertiary structure including a conserved N-terminal amino acid motif (YXYLFK) that incorporates a critical lysine residue.     

Molecular and biochemical analyses of OsRab7, a rice Rab7 homolog

Rab7 is a small GTP-binding protein important in early to late endosome/lysosome vesicular transport in mammalian cells. We have isolated a Rab7 cDNA clone, OsRab7, from a cold-treated rice cDNA library by the subtraction screening method. The cDNA encodes a polypeptide of 206 amino acids with a calculated molecular mass of about 23 kDa. Its predicted amino acid sequence shows significantly high identity with the sequences of other Rab7 proteins. His-tagged OsRab7 bound to radiolabeled GTPgammaS in a specific and stoichiometric manner. Biochemical and structural properties of the Rab7 wild type (WT) protein were compared to those of Q67L and T22N mutants. The detergent 3-([3-cholamidopropyl]dimethylammonio)-1-propane sulfonate (CHAPS) increased the guanine nucleotide binding and hydrolysis activities of Rab7WT. The OsRab7Q67L mutant showed much lower GTPase activity compared to the WT protein untreated with CHAPS, and the T22N mutant showed no GTP binding activity at all. The OsRab7Q67L mutant was constitutively active for guanine nucleotide binding while the T22N mutant (dominant negative) showed no guanine nucleotide binding activity. When bound to GTP, the Rab7WT and the Q67L mutants were protected from tryptic proteolysis. The cleavage pattern of the Rab7T22N mutant, however, was not affected by GTP addition. Northern and Western blot analyses suggested that OsRab7 is distributed in various tissues of rice. Furthermore, expression of a rice Rab7 gene was differentially regulated by various environmental stimuli such as cold, NaCl, dehydration, and ABA. In addition, subcellular localization of OsRab7 was investigated in the Arabidopsis protoplasts by a double-labeling experiment using GFP-fused OsRab7 and FM4-64. GFP-OsRab7 is localized to the vacuolar membrane, suggesting that OsRab7 is implicated in a vesicular transport to the vacuole in plant cells.     

Small GTP-binding proteins associated with secretory vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20-30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras-like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [alpha-32P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22-31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Small GTP-binding Proteins Associated with Secretory Vesicles of Paramecium

GTP-binding proteins act as molecular switches in a variety of membrane-associated processes, including secretion. One group of GTP-binding proteins, 20–30 kDa, is related to the product of the ras proto-oncogene. In Saccharomyces cerevisiae, ras -like GTP-binding proteins regulate vesicular traffic in secretion. The ciliate protist Paramecium tetraurelia contains secretory vesicles (trichocysts) whose protein contents are released by regulated exocytosis. Using [α-³²P]GTP and an on-blot assay for GTP-binding, we detected at least seven GTP-binding proteins of low molecular mass (22–31 kDa) in extracts of Paramecium tetraurelia. Subcellular fractions contained characteristic subsets of these seven; cilia were enriched for the smallest (22 kDa). The pattern of GTP-binding proteins was altered in two mutants defective in the formation or discharge of trichocysts. Trichocysts isolated with their surrounding membranes intact contained two minor GTP-binding proteins (23.5 and 29 kDa) and one major GTP-binding protein (23 kDa) that were absent from demembranated trichocysts. This differential localization of GTP-binding proteins suggests functional specialization of specific GTP-binding proteins in ciliary motility and exocytosis.     

Gn-proteins are distinct from ras p21 and other known low molecular mass GTP-binding proteins in the platelet

The 27 kDa platelet membrane protein (Gn27) that binds [alpha-32P]GTP on nitrocellulose blots of SDS-polyacrylamide gels [(1987) Biochem. J. 245, 617-620] was compared with other low molecular mass GTP-binding proteins. Platelet membranes also contained 21 kDa proteins that bound anti-ras p21 antibody and 22-23 kDa proteins that could be ADP-ribosylated by botulinum neurotoxin type D. These groups of proteins were resolved electrophoretically from each other and from Gn27. A low molecular mass GTP-binding protein from bovine brain [(1987) Biochem. J. 246, 431-439] was also resolved from Gn27. At the levels normally present in cell membranes, only Gn-proteins bound significant amounts of [32P]GTP after transfer of protein from SDS-polyacrylamide gels to nitrocellulose.     

Presence of guanine nucleotide-binding proteins in Catharanthus roseus transformed roots

Biochemical analysis revealed the presence of GTP-binding proteins (G-proteins) in Catharanthus roseus hairy root cultures. In a microsomal fraction, several proteins, with molecular masses of 17, 21, 38, 42, 65, and 79 kDa were substrates for ADP-ribosylation by cholera toxin. Antisera raised against a conserved amino-acid sequence (GTSNSGKSTIVKQMK) of mammalian G α subunits recognized three proteins of 42, 50, and 79 kDa. Incubation of nitrocellulose blots with [ α -³²P]-GTP also indicated the presence of several proteins (17, 21, 50, and 79 kDa) that could bind GTP. In this system, we previously identified a phosphatidylinositol 4,5-bisphosphate-phospholipase C (PLC, EC 3.1.4.11) activity. As the activation of PLC by G-proteins was described, we decided to see whether, in our system, G-protein activators, such as guanosine 5- o -(3-thiotriphosphate) (GTP Γ S) and sodium fluoride ions, were able to regulate PLC activity in C. roseus transformed roots. Our results show that these agents regulated PLC activity in an inhibitory fashion and that this effect is dose-dependent. GTP was ineffective in producing either stimulation or inhibition of PLC activity. Our results demonstrate that non-hydrolyzable guanine nucleotides and fluoride ions exert an inhibitory effect on membrane PLC activity. In summary, a set of proteins of 17, 21, 38, 42, 50, and 79 kDa present in C. roseus transformed roots possessed at least two of the three main characteristics of a GTP-binding protein, and one of these proteins may be involved in the regulation of PLC activity in C. roseus transformed roots.     

Primary structure and possible origin of the non-glycosylated basic proline-rich protein of human submandibular/sublingual saliva.

As a first step in determining the molecular mechanism of membrane fusion stimulated by GTP in rough endoplasmic reticulum (RER), we have looked for GTP-binding proteins. Rough microsomes from rat liver were treated for the release of ribosomes, and the membrane proteins were separated by SDS/polyacrylamide-gel electrophoresis. The polypeptides were then blotted on to nitrocellulose sheets and incubated with [alpha-32P]GTP [Bhullar & Haslam (1987) Biochem. J. 245, 617-620]. A doublet of polypeptides (23 and 24 kDa) was detected in the presence of 2 microM-MgCl2. Binding of [alpha-32P]GTP was blocked by 1-5 mM-EDTA, 10-10,000 nM-GTP or 10 microM-GDP. Either guanosine 5'-[gamma-thio]triphosphate or guanosine 5'-[beta gamma-imido]triphosphate at 100 nM completely inhibited binding, but ATP, CTP or UTP at 10 mciroM did not. Pretreatment of microsomes by mild trypsin treatment (0.5-10 micrograms of trypsin/ml, concentrations known not to affect microsomal permeability) led to inhibition of [alpha-32P]GTP binding, suggesting a cytosolic membrane orientation for the GTP-binding proteins. Two-dimensional gel-electrophoretic analysis revealed the 23 and 24 kDa [alpha-32P]GTP-binding proteins to have similar acid isoelectric points. [alpha-32P]GTP binding occurred to similar proteins of rough microsomes from rat liver, rat prostate and dog pancreas, as well as to a 23 kDa protein of rough microsomes from frog liver, but occurred to distinctly different proteins in a rat liver plasma-membrane-enriched fraction. Thus [alpha-32P]GTP binding has been demonstrated to two low-molecular-mass (approx. 21 kDa) proteins in the rough endoplasmic reticulum of several varied cell types.     

Detection and characterization of GTP-binding proteins in the chloroplast envelope of spinach (Spinacia oleracea)

Proteins binding guanosine triphosphate (GTP) have emerged as important regulators in several cellular processes in plants. To investigate any role of such proteins in chloroplast functions, we subjected envelope, stroma and thylakoid fractions isolated from spinach chloroplasts to two different GTP-binding assays. With both methods, we detected GTP-specific binding only in the envelope fraction. Two chloroplast envelope proteins with the apparent molecular weights of 30.5 and 33.5 kDa, respectively, bound [α-³²P]GTP after SDS-PAGE followed by electroblotting onto a PVDF-membrane and renaturation. Both proteins were intrinsic proteins located in the outer chloroplast envelope. Also, when the fractions were incubated with [α-³²P]GTP, followed by periodate oxidation and borohydride reduction to cross-link GTP to proteins, two proteins in the envelope fraction, of apparent molecular weights of 28 and 39 kDa, appeared to specifically bind GTP. When agents that stimulate heterotrimeric G-proteins, cholera toxin or the mastoparan analogue mas7, were added to isolated chloroplast envelope, the binding of radiolabelled GTP to the 39 kDa protein, a protein of the inner chloroplast envelope, was stimulated, whereas GTP-binding of the 28 kDa protein, a protein of the outer envelope, was unchanged. Mas7 also stimulated synthesis of monogalactosyl diacylglycerol in isolated chloroplast envelope. The occurrence and regulation of GTP-binding proteins in the chloroplast envelope suggests that GTP-binding proteins could be involved in communication with the extraplastidic compartment during chloroplast biogenesis and development.     

GTP-binding proteins in luminal and basolateral membranes from pars convoluta and pars recta of rabbit kidney proximal tubule.

The GTP-binding proteins on luminal and basolateral membrane vesicles from outer cortex (pars convoluta) and outer medulla (pars recta) of rabbit proximal tubule have been examined. The membrane vesicles were highly purified, as ascertained by electron microscopy, by measurements of marker enzymes, and by investigating segmental-specific transport systems. The [35S]GTP gamma S binding to vesicles, and to sodium cholate-extracted proteins from vesicles, indicated that the total content of GTP-binding proteins were equally distributed on pars convoluta, pars recta luminal and basolateral membranes. The membranes were ADP-ribosylated with [32P]NAD+ in the presence of pertussis toxin and cholera toxin. Gel electrophoresis revealed, for all preparations, the presence of cholera toxin [32P]ADP-ribosylated 42 and 45 kDa G alpha s proteins, and pertussis toxin [32P]ADP-ribosylated 41 kDa G alpha i1, 40 kDa G alpha i2 and 41 kDa G alpha i3 proteins. The 2D electrophoresis indicated that Go's were not present in luminal nor in basolateral membranes of pars convoluta or pars recta of rabbit proximal tubule.     

Immunodetection of ralA and ralB GTP-binding proteins in various rat tissues and platelets

Polyclonal antibodies were generated against a synthetic peptide corresponding to the C-terminal (amino acids 192-204) region of ralA and ralB GTP-binding proteins. The ralA and ralB antibodies recognized a 27 kDa protein in the human platelet particulate fraction. Incubation of ralA antibodies with ralB immunizing peptide and ralB antibodies with ralA immunizing peptide prior to Western blotting did not abolish the ability of antibodies to recognize the 27 kDa protein in human platelet particulate fraction. However, when antibodies were incubated with the respective immunizing peptide prior to Western blotting, the 27 kDa human platelet protein was no longer recognized by the antibodies. Incubation of nitrocellulose blots containing polypeptides separated using SDS-PAGE with [-³²P]GTP demonstrated the presence of GTP-binding proteins of molecular mass between 23-27 kDa in rat platelets and the various tissues tested. Analysis using subtype specific antibodies demonstrated that both ralA and ralB GTP-binding proteins were expressed in rat platelets and the various tissues tested. The protein recognized by the ralA and ralB antibodies in rat tissues and platelets had mobility on SDS-PAGE identical to that of the human platelet ral protein. Varying amounts of these proteins were detected in all the tissues tested except white muscle which contained very low level of ralB protein. The widespread distribution of ralA and ralB GTP-binding proteins suggests that they may participate in a common pathway in mammalian cells and tissues.     

Characterization of a Rab11 homologue, EoRab11a, in Euplotes octocarinatus

Rab GTPases are crucial in the regulation of intracellular vesicular trafficking. A novel Rab GTPase gene, EoRab11a (GenBank accession no. EF061065 ), was isolated and identified from Euplotes octocarinatus cells in this study. It contains an ORF of 696-bp nucleotides, encoding 231 amino acids with a calculated molecular weight of 26.8 kDa. Alignment of EoRab11a with other Rab11 proteins from other eukaryotes demonstrated that these proteins shared 53–61% identity at the amino acid level. The recombinant EoRab11a was expressed in Escherichia coli and purified by immobilized metal chelate affinity chromatography and iron chromatography. The GTPase activity of EoRab11a was 0.0024 min⁻¹ detected by HPLC at 30 °C. Three mutations were generated at amino acids Ser21 and Gly22 positions in the G1 domain of EoRab11a. All three mutants, S21P, S21G and G22R, increased the GTPase activity in vitro . Immunofluorescence microscopy results indicated that EoRab11a was localized on the phagosomal membrane during phagocytosis of E. octocarinatus . These data show that EoRab11a possesses GTP hydrolysis activity and may participate in vesicle transport events during phagocytosis of E. octocarinatus .     

Cloning of two splicing variants of the novel Ras-related GTPase Rab29 which is predominately expressed in kidney

cDNA of a novel Ras-related GTP-binding protein was isolated from rat tissue by a PCR-based cloning approach, and was designated Rab29 because its deduced amino acid sequence (204 aa) is remotely similar to that of members of the Rab family (30% identity with Rab1). mRNA of Rab29 was found predominately in kidney. Recombinant Rab29 exhibited rapid exchange of bound guanine nucleotides for radiolabeled GTP but lacked a detectable intrinsic GTPase activity. A second cDNA clone was isolated which contained a 287 bp in-frame insertion with characteristics of an intron sequence; this insertion introduces a stop codon after arginine 167. The recombinant protein (Rab29Δ37) derived from the cDNA carrying the insertion was loaded with GTP during biosynthesis, but showed almost no exchange of the nucleotide for radiolabeled GTP. Thus, the C-terminus of Rab29 appears to harbor a structural element which is essential for the nucleotide exchange of the protein.