Affinity labeling of GTP-binding proteins in cellular extracts.

GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins.     

Human OLA1 defines an ATPase subfamily in the Obg family of GTP-binding proteins

Purine nucleotide-binding proteins build the large family of P-loop GTPases and related ATPases, which perform essential functions in all kingdoms of life. The Obg family comprises a group of ancient GTPases belonging to the TRAFAC (for translation factors) class and can be subdivided into several distinct protein subfamilies. The founding member of one of these subfamilies is the bacterial P-loop NTPase YchF, which had so far been assumed to act as GTPase. We have biochemically characterized the human homologue of YchF and found that it binds and hydrolyzes ATP more efficiently than GTP. For this reason, we have termed the protein hOLA1, for human Obg-like ATPase 1. Further biochemical characterization of YchF proteins from different species revealed that ATPase activity is a general but previously missed feature of the YchF subfamily of Obg-like GTPases. To explain ATP specificity of hOLA1, we have solved the x-ray structure of hOLA1 bound to the nonhydrolyzable ATP analogue AMPPCP. Our structural data help to explain the altered nucleotide specificity of YchF homologues and identify the Ola1/YchF subfamily of the Obg-related NTPases as an exceptional example of a single protein subfamily, which has evolved altered nucleotide specificity within a distinct protein family of GTPases.     

GTP-binding proteins in etiolated epicotyls of Pisum sativum (Alaska) seedlings

Seven fractions of GTP-binding proteins separated by gel filtration of an extract of epicotyls of Pisum sativum seedlings were partially characterized. Seven fractions of GTP-binding proteins tentatively designated GP1 to GP7 had the capacity to be ADP-ribosylated by pertussis toxin. Pooled fractions of GP2 to GP7 showed Km values 2, 20, 50, 10, 3 and 1 nM, respectively. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented competitively in the presence of 0.1 mM GTP and also prevented in the presence of 0.1 mM ATP. Binding of [35S]GTP gamma S to the proteins produced a decrease in their molecular weights.     

DRG represents a family of two closely related GTP-binding proteins

In a previous publication we identified a novel human GTP-binding protein that was related to DRG, a developmentally regulated GTP-binding protein from the central nervous system of mouse. Here we demonstrate that both the human and the mouse genome possess two closely related drg genes, termed drg1 and drg2. The two genes share 62% sequence identity at the nucleotide and 58% identity at the protein level. The corresponding proteins appear to constitute a separate family within the superfamily of the GTP-binding proteins. The DRG1 and the DRG2 mRNA are widely expressed in human and mouse tissues and show a very similar distribution pattern. The human drg1 gene is located on chromosome 22q12, the human drg2 gene on chromosome 17p12. Distantly related species including Caenorhabditis elegans, Schizosaccharomyces pombe and Saccharomyces cerevisiae also possess two drg genes. In contrast, the genomes of archaebacteria (Halobium, Methanococcus, Thermoplasma) harbor only one drg gene, while eubacteria do not seem to contain any. The high conservation of the polypeptide sequences between distantly related organisms indicates an important role for DRG1 and DRG2 in a fundamental pathway.     

Sequence alignment and homology threading reveals prokaryotic and eukaryotic proteins similar to lactose permease

Certain prokaryotic transport proteins similar to the lactose permease of Escherichia coli (LacY) have been identified by BLAST searches from available genomic databanks. These proteins exhibit conservation of amino acid residues that participate in sugar binding and H(+) translocation in LacY. Homology threading of prokaryotic transporters based on the X-ray structure of LacY (PDB ID: 1PV7) and sequence similarities reveals a common overall fold for sugar transporters belonging to the Major Facilitator Superfamily (MFS) and suggest new targets for study. Evolution-based searches for sequence similarities also identify eukaryotic proteins bearing striking resemblance to MFS sugar transporters. Like LacY, the eukaryotic proteins are predicted to have 12 transmembrane domains (TMDs), and many of the irreplaceable residues for sugar binding and H(+) translocation in LacY appear to be largely conserved. The overall size of the eukaryotic homologs is about twice that of prokaryotic permeases with longer N and C termini and loops between TMDs III-IV and VI-VII. The human gene encoding protein FLJ20160 consists of six exons located on more than 60,000 bp of DNA sequences and requires splicing to produce mature mRNA. Cellular localization predictions suggest membrane insertion with possible proteolysis at the N terminus, and expression studies with the human protein FJL20160 demonstrate membrane insertion in both E.coli and Pichia pastoris. Widespread expression of the eukaryotic sugar transport candidates suggests an important role in cellular metabolism, particularly in brain and tumors. Homology is observed in the TMDs of both the eukaryotic and prokaryotic proteins that contain residues involved in sugar binding and H(+) translocation in LacY.     

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.     

Isoprenylation and carboxylmethylation in small GTP-binding proteins of pheochromocytoma (PC-12) cells

1. A group of 21 to 24-kDa proteins of pheochromocytoma (PC-12) cells was found in blot overlay assays to bind specifically [alpha-32P]GTP. Binding was inhibited by GTP analogues but not by ATP. Such small GTP-binding proteins were found in the cytosolic and in the particulate fraction of the cells, but they were unevenly distributed: about 75% of the small GTP-binding proteins were localized within the particulate fraction of the cells. Separation of these proteins by two-dimensional gel electrophoresis revealed the existence of seven distinct [alpha-32P]GTP-binding proteins. 2. Targeting of the small GTP-binding proteins to the particulate fraction of PC-12 cells requires modification by isoprenoids, since depleting the cells of the isoprenoid precursor mevalonic acid (MVA) by the use of lovastatin resulted in a 50% decrease in membrane-bound small GTP-binding proteins, with a proportionate increase in the cytosolic form. This blocking effect of lovastatin was reversed by exogenously added MVA. 3. In addition, metabolic labeling of PC-12 cells with [3H]MVA revealed incorporation of [3H]MVA metabolites into the cluster of 21 to 24-kDa proteins in a form typical of isoprenoids; the label was not removed from the proteins by hydroxylamine, and labeling was enhanced in cells incubated with lovastatin. The latter effect reflects a decrease in the isotopic dilution of the exogenously added [3H]MVA, as the addition of exogenous MVA reversed the effect of lovastatin on [3H]MVA-metabolite incorporation into the 21 to 24-kDa proteins. 4. Additional experiments demonstrated that isoprenylation is required not only for membrane association of small GTP-binding proteins, but also for their further modification by a methylation enzyme. This was evident in experiments in which the cells were metabolically labeled with [methyl-3H]methionine, a methylation precursor. The group of 21 to 24-kDa proteins was labeled with a methyl-3H group in a form typical of C-terminal-cysteinyl carboxylmethyl esters. Their methylation was blocked by the methylation inhibitors methylthioadenosine (MTA), 3-deazadenosine and homocysteine thiolactone as well as by lovastatin. MVA reversed the lovastatin block of methylation. 5. Two-dimensional gel analysis of the [3H]methylated proteins detected seven methylated small GTP-binding proteins that correspond to the isoprenylated proteins. Levels of the small GTP-binding proteins as well as isoprenylation and methylation were reduced by cycloheximide. 6. Distribution of the methylated proteins between particulate and cytosolic fractions was found to be similar to that of the small GTP-binding proteins (i.e., a 4:1 ratio).(ABSTRACT TRUNCATED AT 400 WORDS)     

Small GTP-binding proteins on rat liver lysosomal membranes.

GTP-binding proteins have been identified on the membranes of highly purified dextran-filled lysosomes (dextranosomes) and Triton-filled lysosomes (tritosomes) obtained from rat liver. Autoradiography of blots of lysosomal membrane proteins incubated with [alpha-32P]GTP revealed the presence of several specific GTP-binding proteins with a relative molecular mass (M(r)) predominantly in the range of 26-30 kDa. These GTP-binding proteins migrated slower in polyacrylamide gels than purified c-Ha-ras protein expressed in E. coli, whose apparent M(r) was 23 kDa in the same blot. The relative contents of GTP-binding proteins in lysosomal membranes were comparable or greater than that of plasma membranes and of microsomes. Chemical extraction showed that lysosomal GTP-binding proteins were more tightly associated with the membranes than with microsomal GTP-binding proteins. The possible involvement of lysosomal GTP-binding proteins in cellular functions including vacuolar (lysosomal) acidification and organellar dynamics are discussed.     

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.     

Partial characterization of GTP-binding proteins in Neurospora

Six fractions of GTP-binding proteins separated by gel filtration of a mycelial extract containing membrane components of Neurospora crassa were partially characterized. [35S]GTP gamma S bound to GTP-binding protein was assayed by repeated treatments with a Norit solution and centrifugation. The binding of [35S]GTP gamma S to GTP-binding proteins was competitively prevented in the presence of 0.1 to 1 mM GTP but not in the presence of ATP. These GTP-binding proteins fractionated by the gel column had Km values of 20, 7, 4, 4, 80 and 2 nM. All six fractions of these GTP-binding proteins showed the capacity to be ADP-ribosylated by pertussis toxin.     

Identification and biochemical characterization of Rap2C, a new member of the Rap family of small GTP-binding proteins

The Rap family of small GTP-binding proteins is composed by four different members: Rap1A, Rap1B, Rap2A and Rap2B. In this work we report the identification and characterization of a fifth member of this family of small GTPases. This new protein is highly homologous to Rap2A and Rap2B, binds labeled GTP on nitrocellulose, and is recognized by a specific anti-Rap2 antibody, but not by an anti-Rap1 antibody. The protein has thus been named Rap2C. Binding of GTP to recombinant purified Rap2C was Mg(2+)-dependent. However, accurate comparison of the kinetics of nucleotide binding and release revealed that Rap2C bound GTP less efficiently and possessed slower rate of GDP release compared to the highly homologous Rap2B. Moreover, in the presence of Mg(2+), the relative affinity of Rap2C for GTP was only about twofold higher than that for GDP, while, under the same conditions, Rap2B was able to bind GTP with about sevenfold higher affinity than GDP. When expressed in eukaryotic cells, Rap2C localized at the plasma membrane, as dictated by the presence of a CAAX motif at the C-terminus. We found that Rap2C represented the predominant Rap2 protein expressed in circulating mononuclear leukocytes, but was not present in platelets. Importantly, Rap2C was found to be expressed in human megakaryocytes, suggesting that the protein may be down-regulated during platelets generation. This work demonstrates that Rap2C is a new member of the Rap2 subfamily of proteins, able to bind guanine nucleotides with peculiar properties, and differently expressed by various hematopoietic subsets. This new protein may therefore contribute to the still poorly clarified cellular events regulated by this subfamily of GTP-binding proteins.     

GTP-binding proteins in a cyanobacterium Anabaena cylindrica

GTP-binding proteins were detected in a crude extract containing membrane components of Anabaena cylindrica. The crude extract was treated with 1% Lubrol PX and was fractionated by gel filtration. The binding of [35S]GTP gamma S to GTP-binding proteins was prevented in the presence of 0.1 mM GTP and in the presence of 0.1 mM ATP. Six fractions of these GTP-binding proteins, tentatively designated GA1 to GA6, were ADP-ribosylated by pertussis toxin. GA3, GA4 and GA5 had Km values of 10, 60 and 7 nM, respectively. The molecular weights of some of these GTP-binding proteins were reduced after being labelled with [35S]GTP gamma S.     

Homologs of eukaryotic Ras superfamily proteins in prokaryotes and their novel phylogenetic correlation with their eukaryotic analogs

Ras superfamily proteins are key regulators in a wide variety of cellular processes. Previously, they were considered to be specific to eukaryotes, and MglA, a group of obviously different prokaryotic proteins, were recognized as their only prokaryotic analogs or even ancestors. Here, taking advantage of quite a current accumulation of prokaryotic genomic databases, we have investigated the existence and taxonomic distribution of Ras superfamily protein homologs in a much wider prokaryotic range, and analyzed their phylogenetic correlation with their eukaryotic analogs. Thirteen unambiguous prokaryotic homologs, which possess the GDP/GTP-binding domain with all the five characteristic motifs of their eukaryotic analogs, were identified in 12 eubacteria and one archaebacterium, respectively. In some other archaebacteria, including four methanogenic archaebacteria and three Thermoplasmales, homologs were also found, but with the GDP/GTP-binding domains not containing all the five characteristic motifs. Many more MglA orthologs were identified than in previous studies mainly in delta-proteobacteria, and all were shown to have common unique features distinct from the Ras superfamily proteins. Our phylogenetic analysis indicated eukaryotic Rab, Ran, Ras, and Rho families have the closest phylogenetic correlation with the 13 unambiguous prokaryotic homologs, whereas the other three eukaryotic protein families (SRbeta, Sar1, and Arf) branch separately from them, but have a relatively close relationship with the methanogenic archaebacterial homologs and MglA. Although homologs were identified in a relative minority of prokaryotes with genomic databases, their presence in a relatively wide variety of lineages, their unique sequence characters distinct from those of eukaryotic analogs, and the topology of our phylogenetic tree altogether do not support their origin from eukaryotes as a result of lateral gene transfer. Therefore, we argue that Ras superfamily proteins might have already emerged at least in some prokaryotic lineages, and that the seven eukaryotic protein families of the Ras superfamily may have two independent prokaryotic origins, probably reflecting the 'fusion' evolutionary history of the eukaryotic cell.     

flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein

We describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are nonmotile. Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.     

Characterization of a novel Obg-like ATPase in the protozoan Trypanosoma cruzi

We characterized a gene encoding an YchF-related protein, TcYchF, potentially associated with the protein translation machinery of Trypanosoma cruzi. YchF belongs to the translation factor-related (TRAFAC) class of P-loop NTPases. The coding region of the gene is 1185 bp long and encodes a 44.3 kDa protein. BlastX searches showed TcYchF to be very similar (45-86%) to putative GTP-binding proteins from eukaryotes, including some species of trypanosomatids (Leishmania major and Trypanosoma brucei). A lower but significant level of similarity (38-43%) was also found between the predicted sequences of TcYchF and bacterial YyaF/YchF GTPases of the Spo0B-associated GTP-binding protein (Obg) family. Some of the most important features of the G domain of this family of GTPases are conserved in TcYchF. However, we found that TcYchF preferentially hydrolyzed ATP rather than GTP. The function of YyaF/YchF is unknown, but other members of the Obg family are known to be associated with ribosomal subunits. Immunoblots of the polysome fraction from sucrose gradients showed that TcYchF was associated with ribosomal subunits and polysomes. Immunoprecipitation assays showed that TcYchF was also associated with the proteasome of T. cruzi. Furthermore, inactivation of the T. brucei homolog of TcYchF by RNA interference inhibited the growth of procyclic forms of the parasite. These data suggest that this protein plays an important role in the translation machinery of trypanosomes.     

Novel GTP-binding proteins in plasma membranes of the fungus Metarhizium anisopliae

We report the existence of several families of GTP-binding proteins in plasma membranes of Metarhizium anisopliae. Two proteins (18.4 and 24 kDa) resemble mammalian Gn-proteins in their being toxin insensitive, binding [alpha-32P]GTP on nitrocellulose blots of sodium dodecyl sulfate (SDS)-polyacrylamide gels, and also in their immunological properties. Four other proteins (31-38.2 kDa) were similar except that they did not bind [alpha-32P]GTP after treatment with sodium dodecyl sulfate. An 18.2 kDa cholera toxin substrate and three toxin insensitive bands (18.6, 18.8, and 24 kDa) are novel proteins antigenically related both to mammalian G-proteins and ras gene products. An additional 23 kDa pertussis toxin substrate (the major G-protein in a crude mycelial extract) reacted strongly with antisera to G-proteins but not with anti-ras serum. Other substrates ADP ribosylated by cholera toxin or botulinum D toxin were immunologically unreactive. Analysis of the structural and functional characteristics of these multiple GTP-binding proteins will promote a better understanding of signal transduction in fungi.     

Protein length in eukaryotic and prokaryotic proteomes

We analyzed length differences of eukaryotic, bacterial and archaeal proteins in relation to function, conservation and environmental factors. Comparing Eukaryotes and Prokaryotes, we found that the greater length of eukaryotic proteins is pervasive over all functional categories and involves the vast majority of protein families. The magnitude of these differences suggests that the evolution of eukaryotic proteins was influenced by processes of fusion of single-function proteins into extended multi-functional and multi-domain proteins. Comparing Bacteria and Archaea, we determined that the small but significant length difference observed between their proteins results from a combination of three factors: (i) bacterial proteomes include a greater proportion than archaeal proteomes of longer proteins involved in metabolism or cellular processes, (ii) within most functional classes, protein families unique to Bacteria are generally longer than protein families unique to Archaea and (iii) within the same protein family, homologs from Bacteria tend to be longer than the corresponding homologs from Archaea. These differences are interpreted with respect to evolutionary trends and prevailing environmental conditions within the two prokaryotic groups.     

Remarkable sequence signatures in archaeal genomes

Complete archaeal genomes were probed for the presence of long (> or = 25 bp) oligonucleotide repeats (words). We detected the presence of many words distributed in tandem with narrow ranges of periodicity (i.e., spacer length between repeats). Similar words were not identified in genomes of non-archaeal species, namely Escherichia coli, Bacillus subtilis, Haemophilus influenzae, Mycoplasma genitalium and Mycoplasma pneumoniae. BLAST similarity searches against the GenBank nucleotide sequence database revealed that these words were archaeal species-specific, indicating that they are of a signature character. Sequence analysis and genome viewing tools showed these repeats to be restricted to non-coding regions. Thus, archaea appear to possess a non-coding genomic signature that is absent in bacterial species. The identification of a species-specific genomic signature would be of great value to archaeal genome mapping, evolutionary studies and analyses of genome complexity.     

Small GTP-binding proteins in vesicular transport

Recent recognition of the abundance of small GTP-binding proteins in eukaryotic cells has sparked off a search for the possible function of these proteins. Evidence is accumulating that SAR1, ARF, SEC4 and YPT1 in yeast and the rab and arf family in mammalian cells play a central role in the regulation of vesicle transport and organelle function.