Putative GTPase Research Articles

Rapid identification of diverse Campylobacter lari strains isolated from mussels and oysters using a reverse hybridization line probe assay.

Campylobacters isolated from mussels and oysters in The Netherlands were analysed by a novel assay, based on DNA amplification with primers, based on semiconserved GTP-binding sites of a putative GTPase gene. Polymerase chain reaction (PCR) was followed by a single step reverse hybridization line probe assay (PCR-LiPA). This permits identification of Campylobacter jejuni, C. coli, C. lari and C. upsaliensis. Among a group of 44 isolates, three C. jejuni, one C. upsaliensis, one double infection with C. jejuni and C. coli, and 38 C. lari strains were identified. These results were in complete agreement with conventional identification methods and whole cell protein analysis. One C. hyointestinalis isolate was not identified by the PCR-LiPA, since the reverse hybridization assay does not comprise specific probes for this particular species. PCR products from 36 C. lari isolates were sequenced and phylogenetic analysis revealed the presence of two major C. lari subgroups: one comprised 11 highly homologous sequences, whereas the 25 sequences in the other subgroup were more heterogeneous. This confirmed earlier findings that C. lari isolates comprise a more heterogeneous group of isolates as compared with C. jejuni, C. coli and C. upsaliensis. Based on the sequence information, a novel PCR-LiPA was developed that permits specific and rapid detection of the different C. lari variants.

RagA is a functional homologue of S. cerevisiae Gtr1p involved in the Ran/Gsp1-GTPase pathway.

Human RagA and RagB is reported to be 52% identical to a putative GTPase of Saccharomyces cerevisiae, Gtr1p. According to the reported nucleotide sequence, we amplified human RagA and RagBs cDNAs from the human B cell cDNA library with PCR. Both cDNAs rescued a cold sensitivity of S. cerevisiae, gtr1-11. Furthermore, we introduced into the cloned human RagA cDNA, the mutation 'T21L' corresponding to the gtr1-11 mutation which has been reported to suppress not only all of rcc1-, temperature-sensitive mutants of Ran/Gsp1p GTPase GDP/GTP-exchanging factor, but also rna1-1, a temperature-sensitive mutant of Ran/Gsp1p GTPase-activating protein. The resulting RagAgtr1-11 cDNA partially, but significantly, suppressed both rcc1- and rna1-1 mutations. These results indicated that RagA and RagBs are functional homologues of S. cervisiae Gtr1p. Interestingly, while wild-type human RagA and RagBs were localized within the cytoplasm, similar to S. cerevisiae Gtr1p, the mutated human RagAgtr1-11 corresponding to a dominant negative form of RagA was distributed in discrete speckles in the nucleus, being localized side by side with SC-35, a non-snRNP of the splicing complex. In contrast, a dominant positive form of RagA, Q66L was localized in the cytoplasm. Thus, RagA was suggested to shuttle between the cytoplasm and the nucleus, depending on the bound nucleotide state.

Interaction between hamartin and tuberin, the TSC1 and TSC2 gene products.

Tuberous sclerosis (TSC) is an autosomal dominant disorder caused by a mutation in either the TSC1 or TSC2 tumour suppressor gene. The disease is characterized by a broad phenotypic spectrum that can include seizures, mental retardation, renal dysfunction and dermatological abnormalities. TSC2 encodes tuberin, a putative GTPase activating protein for rap1 and rab5. The TSC1 gene was recently identified and codes for hamartin, a novel protein with no significant homology to tuberin or any other known vertebrate protein. Here, we show that hamartin and tuberin associate physically in vivo and that the interaction is mediated by predicted coiled-coil domains. Our data suggest that hamartin and tuberin function in the same complex rather than in separate pathways.

Regulation of transducin GTPase activity by human retinal RGS.

The intrinsic GTPase activity of transducin controls inactivation of the effector enzyme, cGMP phosphodiesterase (PDE), during turnoff of the visual signal. The inhibitory gamma-subunit of PDE (Pgamma), an unidentified membrane factor and a retinal specific member of the RGS family of proteins have been shown to accelerate GTP hydrolysis by transducin. We have expressed a human homologue of murine retinal specific RGS (hRGSr) in Escherichia coli and investigated its role in the regulation of transducin GTPase activity. As other RGS proteins, hRGSr interacted preferentially with a transitional conformation of the transducin alpha-subunit, GtalphaGDPAlF4-, while its binding to GtalphaGTPgammaS or GtalphaGDP was weak. hRGSr and Pgamma did not compete for the interaction with GtalphaGDPAlF4-. Affinity of the Pgamma-GtalphaGDPAlF4- interaction was modestly enhanced by addition of hRGSr, as measured by a fluorescence assay of GtalphaGDPAlF4- binding to Pgamma labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (PgammaBC). Binding of hRGSr to GtalphaGDPAlF4- complexed with PgammaBC resulted in a maximal approximately 40% reduction of BC fluorescence allowing estimation of the hRGSr affinity for GtalphaGDPAlF4- (Kd 35 nM). In a single turnover assay, hRGSr accelerated GTPase activity of transducin reconstituted with the urea-stripped rod outer segment (ROS) membranes by more than 10-fold to a rate of 0.23 s-1. Addition of Pgamma to the reconstituted system reduced the GTPase level accelerated by hRGSr (kcat 0.085 s-1). The GTPase activity of transducin and the PDE inactivation rates in native ROS membranes in the presence of hRGSr were elevated 3-fold or more regardless of the membrane concentrations. In ROS suspensions containing 30 microM rhodopsin these rates exceeded 0.7 s-1. Our data suggest that effects of hRGSr on transducin's GTPase activity are attenuated by Pgamma but independent of a putative membrane GTPase activating protein factor. The rate of transducin GTPase activity in the presence of hRGSr is sufficient to correlate it with in vivo turnoff kinetics of the visual cascade.

Molecular discrimination betweenCampylobacter jejuni,Campylobacter coli,Campylobacter lariandCampylobacter upsaliensisby polymerase chain reaction based on a novel putative GTPase gene

Polymerase chain reaction (PCR) mediated DNA fingerprinting has resulted in the identification of a novelCampylobacter jejunigene, encoding a GTPase protein. The gene, consisting of 383 amino acids contained semi-conserved GTP-binding sites (designated G-1 to G-4), that are characteristic for members of the GTPase protein superfamily. Remarkably, this gene fromC. jejuniappears to encode a member of a novel family of GTP-binding proteins, containing two separate putative GTP-binding domains, each comprising a series of semi-conserved GTP-binding motifs. Spacing between these motifs is highly conserved. Based on this novel gene, a general PCR strategy for the identification ofC. jejuniC. coliC. lariandC. upsaliensiswas developed. PCR primers were deduced from GTP-binding motifs G-1 and G-3 of the first GTP-binding domain. These GTP-binding sites flank a variable region of precisely 117 bp in the fourCampylobacterspp. that allowed the development of species-specific probes. This PCR-hybridization assay offers a novel tool for rapid molecular detection and specific identification of the thermophilicCampylobacterspp.

Putative GTPase Gtr1p genetically interacts with the RanGTPase cycle in Saccharomyces cerevisiae.

In order to identify a protein interacting with RCC1, a guanine nucleotide-exchange factor for the nuclear GTPase Ran, we isolated a series of cold-sensitive suppressors of mtr1-2, a temperature-sensitive mutant of the Saccharomyces cerevisiae RCC1 homologue. One of the isolated suppressor mutants was mutated in the putative GTPase Gtr1p, being designated as gtr1-11. It also suppressed other alleles of mtr1-2, srm1-1 and prp20-1 in contrast to overexpression of the S. cerevisiae Ran/TC4 homologue Gsp1p, previously reported to suppress prp20-1, but not mtr1-2 or srm1-1. Furthermore, gtr1-11 suppressed the rna1-1, temperature-sensitive mutant of the Gsp1p GTPase-activating protein, but not the srp1-31, temperature-sensitive mutant of the S. cerevisiae importin alpha homologue. mtr1-2, srm1-1 and prp20-1 were also suppressed by overexpression of the mutated Gtr1p, Gtr1-11p. In summary, Gtr1p that was localized in the cytoplasm by immunofluoresence staining was suggested to function as a negative regulator for the Ran/TC4 GTPase cycle.

Tec homology (TH) adjacent to the PH domain

The pleckstrin homology (PH) domain is extended in the Btk kinase family by a region designated the TH (Tec homology) domain, which consists of about 80 residues preceding the SH3 domain. The TH domain contains a conserved 27 amino acid stretch designated the Btk motif and a proline-rich region. Sequence similarity was found to a putative Ras GTPase activating protein and a human interferon-γ binding protein both in the PH domain and the Btk motif region. SLK1/SSP31 protein kinase and a non-catalytic p85 subunit of PI-3 kinase had similarity only with the proline rich region. The identification of a PH domain extension in some signal transduction proteins in different species suggests that this region is involved in protein—protein interactions.

The Escherichia coli hflA locus encodes a putative GTP-binding protein and two membrane proteins, one of which contains a protease-like domain.

The hflA (high frequency of lysogenization) locus of Escherichia coli governs the lysis-lysogeny decision of bacteriophage lambda by controlling stability of the phage cII protein. hflA contains three genes, hflX, hflK, and hflC, encoding polypeptides of 50, 46, and 37 kDa, respectively. We have determined the nucleotide sequence of 3843 base pairs containing hflA and have found three large open reading frames corresponding to hflX, hflK, and hflC. HflX contains the three sequence motifs typical of GTP-binding proteins and appears to be a member of a distinct family of putative GTPases. HflC and HflK appear to be integral membrane proteins which show some similarity to each other and to a human membrane protein. The C-terminal region of HflC contains a domain resembling the catalytic domain of ClpP, a bacterial ATP-dependent protease. We hypothesize that HflK and HflC constitute a distinct membrane-bound protease whose activity may be modulated by HflX GTPase.

Growth-regulated expression of rhoG, a new member of the ras homolog gene family.

Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.

Growth-regulated expression of rhoG, a new member of the ras homolog gene family.

Cellular transition from the resting state to DNA synthesis involves master switches genes encoding transcriptional factors (e.g., fos, jun, and egr genes), whose targets remain to be fully characterized. To isolate coding sequences specifically accumulated in late G1, a differential screening was performed on a cDNA library prepared from hamster lung fibroblasts stimulated for 5 h with serum. One of the positive clones which displayed a sevenfold induction, turned out to code for a protein sharing homology to Ras-like products. Cloning and sequence analysis of the human homolog revealed that this putative new small GTPase, referred to as rhoG, is more closely related to the rac, CDC42, and TC10 members of the rho (ras homolog) gene family and might have diverged very early during evolution. rhoG mRNA accumulates in proportion to the mitogenic strength of various purified growth factors used for the stimulation, as a consequence of transcriptional activation. G1-specific RNA accumulation is impaired upon addition of antimitogenic cyclic AMP and is enhanced when protein synthesis is inhibited, mainly as a result of RNA stabilization. rhoG mRNA expression is observed in a wide variety of human organs but reaches a particularly high level in lung and placental tissues.

A putative Ras GTPase activating protein acts as a negative regulator of signaling by the Sevenless receptor tyrosine kinase

A Drosophila gene with similarity to the mammalian Ras GTPase activating protein has been isolated in screens for mutations that affect eye development. Inactivation of the locus, Gap1, mimics constitutive activation of the Sevenless receptor tyrosine kinase and eliminates the need for a functional Sevenless protein in the R7 cell. Our results suggest that Gap 1 acts as a negative regulator of signaling by Sevenless by down-regulating the activity of the Ras1 protein, which has been shown to be a key element in signaling by Sevenless.