The GTP-dependent restriction enzyme McrBC from Escherichia coli forms high-molecular mass complexes with DNA and produces a cleavage pattern with a characteristic 10-base pair repeat

The GTP-dependent restriction enzyme McrBC consists of two polypeptides: one (McrB) that is responsible for GTP binding and hydrolysis as well as DNA binding and another (McrC) that is responsible for DNA cleavage. It recognizes two methylated or hemimethylated RC sites (R(m)C) at a distance of approximately 30 to more than 2000 base pairs and cleaves the DNA close to one of the two R(m)C sites. This process is strictly coupled to GTP hydrolysis and involves the formation of high-molecular mass complexes. We show here using footprinting techniques, surface plasmon resonance, and scanning force microscopy experiments that in the absence of McrC, McrB binds to a single R(m)C site. If a second R(m)C site is present on the DNA, it is occupied independently by McrB. Whereas the DNA-binding domain of McrB forms 1:1 complexes with each R(m)C site and shows a clear footprint on both R(m)C sites, full-length McrB forms complexes with a stoichiometry of at least 4:1 at each R(m)C site, resulting in a slightly more extended footprint. In the presence of McrC, McrB forms high-molecular mass complexes of unknown stoichiometry, which are considerably larger than the complexes formed with McrB alone. In these complexes and when GTP is present, the DNA is cleaved next to one of the R(m)C sites at distances differing by one to five helical turns, suggesting that in the McrBC-DNA complex only a few topologically well-defined phosphodiester bonds of the DNA are accessible for the nucleolytic center of McrC.     

A mutational analysis of the PD...D/EXK motif suggests that McrC harbors the catalytic center for DNA cleavage by the GTP-dependent restriction enzyme McrBC from Escherichia coli

McrBC is a unique restriction enzyme which binds specifically to the bipartite recognition sequence R(m)CN( approximately )(30)(-)( approximately )(2000)R(m)C and in the presence of GTP translocates the DNA and cleaves both strands at multiple positions within the two R(m)C "half-sites". It is known that McrBC is composed of two subunits: McrB which binds and hydrolyzes GTP and specifically interacts with DNA and McrC whose function is not clear but which has been suspected to harbor the catalytic center for DNA cleavage. A multiple-sequence alignment of the amino acid sequence of Escherichia coli McrC and of six presumably homologous open reading frames from various bacterial species shows that a sequence motif found in many restriction enzymes, but also in other nucleases, the PD.D/EXK motif, is conserved among these sequences. A mutational analysis, in which the carboxylates (aspartic acid in McrC) of this motif were substituted with alanine or asparagine and lysine was substituted with alanine or arginine, strongly suggests that Asp244, Asp257, and Lys259 represent the catalytic center of E. coli McrC. Whereas the variants D244A (or -N), D257A (or -N), and K259A are inactive in DNA cleavage (K259R has residual DNA cleavage activity), they interact with McrB like wild-type McrC, as can be deduced from the finding that they stimulate the McrB-catalyzed GTP hydrolysis to the same extent as wild-type McrC. Thus, whereas McrC variants defective in DNA cleavage can stimulate the GTPase activity of McrB, the DNase activity of McrC is not supported by McrB variants defective in GTP hydrolysis.     

McrB: a prokaryotic protein specifically recognizing DNA containing modified cytosine residues.

Restriction of DNA by the Escherichia coli K-12 McrBC restriction endonuclease, which consists of the two subunits McrB and McrC, depends on the presence of modified cytosine residues in a special constellation. From previous work by others it was known that restriction of 5-methylcytosine-containing DNA requires two methylated 5'-PuC sites separated by approximately 40-80 non-defined base pairs. Here we show that binding of the McrBC nuclease is mediated exclusively by the McrB subunit. McrB has a low affinity for non-methylated DNA, with which it forms low molecular weight complexes. The affinity for DNA is significantly increased, with variations depending on the sequence context, by hemi- or fully methylated 5'-PuC sites. Binding to such substrates yields high molecular weight complexes, presumably involving several McrB molecules. Methylation at unique 5'-PuC sites can be sufficient to stimulate DNA binding by McrB. As such substrates are not cleaved by the nuclease, restriction apparently requires the coordinated interaction of molecules bound to neighbouring 5'-PumC sites. The binding properties of McrB exhibit some similarities to recently identified eukaryotic proteins interacting in a non-sequence-specific manner with DNA containing methylated 5'-CpG sequences and might point to a common molecular origin of these proteins. In addition to DNA, McrB also binds GTP, an essential cofactor in DNA restriction by McrBC. McrC neither binds to DNA nor modulates the DNA binding potential of McrB. As McrC is essential for restriction it appears to predominantly function in catalysis.     

Methyl-specific DNA binding by McrBC, a modification-dependent restriction enzyme

McrBC, a GTP-requiring, modification-dependent endonuclease of Escherichia coli K-12, specifically recognizes DNA sites of the form 5' R(m)C 3'. DNA cleavage normally requires translocation-mediated coordination between two such recognition elements at distinct sites. We have investigated assembly of the cleavage-competent complex with gel-shift and DNase I footprint analysis. In the gel-shift system, McrB(L) binding resulted in a fast-migrating specific shifted band, in a manner requiring both GTP and Mg(2+). The binding was specific for methylated DNA and responded to local sequence changes in the same way that cleavage does. Single-stranded DNA competed for McrB(L)-binding in a modification and sequence-specific fashion. A supershifted species was formed in the presence of McrC and GTPgammaS. DNase I footprint analysis showed modest cooperativity in binding to two sites, and a two-site substrate displayed protection in non-specific spacer DNA in addition to the recognition elements. The addition of McrC did not affect the footprint obtained. We propose that McrC effects a conformational change in the complex rather than a reorganization of the DNA:protein interface.     

The GTP-binding domain of McrB: more than just a variation on a common theme?

The methylation-dependent restriction endonuclease McrBC from Escherichia coli K12 cleaves DNA containing two R(m)C dinucleotides separated by about 40 to 2000 base-pairs. McrBC is unique in that cleavage is totally dependent on GTP hydrolysis. McrB is the GTP binding and hydrolyzing subunit, whereas MrC stimulates its GTP hydrolysis. The C-terminal part of McrB contains the sequences characteristic for GTP-binding proteins, consisting of the GxxxxGK(S/T) motif (position 201-208), followed by the DxxG motif (position 300-303). The third motif (NKxD) is present only in a non-canonical form (NTAD 333-336). Here we report a mutational analysis of the putative GTP-binding domain of McrB. Amino acid substitutions were initially performed in the three proposed GTP-binding motifs. Whereas substitutions in motif 1 (P203V) and 2 (D300N) show the expected, albeit modest effects, mutation in the motif 3 is at variance with the expectations. Unlike the corresponding EF-Tu and ras -p21 variants, the D336N mutation in McrB does not change the nucleotide specificity from GTP to XTP, but results in a lack of GTPase stimulation by McrC. The finding that McrB is not a typical G protein motivated us to perform a search for similar sequences in DNA databases. Eight microbial sequences were found, mainly from unfinished sequencing projects, with highly conserved sequence blocks within a presumptive GTP-binding domain. From the five sequences showing the highest homology, 17 invariant charged or polar residues outside the classical three GTP-binding motifs were identified and subsequently exchanged to alanine. Several mutations specifically affect GTP affinity and/or GTPase activity. Our data allow us to conclude that McrB is not a typical member of the superfamily of GTP-binding proteins, but defines a new subfamily within the superfamily of GTP-binding proteins, together with similar prokaryotic proteins of as yet unidentified function.     

The McrBC restriction endonuclease assembles into a ring structure in the presence of G nucleotides

McrBC from Escherichia coli K-12 is a restriction enzyme that belongs to the family of AAA(+) proteins and cuts DNA containing modified cytosines. Two proteins are expressed from the mcrB gene: a full-length version, McrB(L), and a short version, McrB(S). McrB(L) binds specifically to the methylated recognition site and is, therefore, the DNA-binding moiety of the McrBC endonuclease. McrB(S) is devoid of DNA-binding activity. We observed that the quaternary structure of the endonuclease depends on binding of the cofactors. In gel filtration experiments, McrB(L) and McrB(S) form high molecular weight oligomers in the presence of Mg(2+) and GTP, GDP or GTP-gamma-S. Oligomerization did not require the presence of DNA and was independent of GTP hydrolysis. Electron micrographs of negatively stained McrB(L) and McrB(S) revealed ring-shaped particles with a central channel. Mass analysis by scanning transmission electron microscopy indicates that McrB(L) and McrB(S) form single heptameric rings as well as tetradecamers. In the presence of McrC, a subunit that is essential for DNA cleavage, the tetradecameric species was the major form of the endonuclease.     

Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12.

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modified DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB(+)-dependent restriction can be divided into two sets: some modified sequences containing 5-methylcytosine are restricted by McrB+ cells even when McrC-, but most such sequences are restricted in vivo only by McrB+ McrC+ cells. The sequences restricted only by McrB+C+ include T-even bacteriophage containing 5-hydroxymethylcytosine (restriction of this phage is the RglB+ phenotype), some sequences containing N4-methylcytosine, and some sequences containing 5-methylcytosine. The sequence codes for two polypeptides of 54 (McrB) and 42 (McrC) kilodaltons, whereas in vitro translation yields four products, of approximately 29 and approximately 49 (McrB) and of approximately 38 and approximately 40 (McrC) kilodaltons. The McrB polypeptide sequence contains a potential GTP-binding motif, so this protein presumably binds the nucleotide cofactor. The deduced McrC polypeptide is somewhat basic and may bind to DNA, consistent with its genetic activity as a modulator of the specificity of McrB. At the nucleotide sequence level, the G+C content of mcrBC is very low for E. coli, suggesting that the genes may have been acquired recently during the evolution of the species.     

McrBs, a modulator peptide for McrBC activity.

McrBC is a methylation-dependent endonuclease from Escherichia coli K-12. The enzyme recognizes DNA with modified cytosines preceded by a purine. McrBC restricts DNA that contains at least two methylated recognition sites separated by 40-80 bp. Two gene products, McrBL and McrBs, are produced from the mcrB gene and one, McrC, from the mcrC gene. DNA cleavage in vitro requires McrBL, McrC, GTP and Mg2+. We found that DNA cleavage was optimal at a ratio of 3-5 McrBL per molecule of McrC, suggesting that formation of a multisubunit complex with several molecules of McrBL is required for cleavage. To understand the role of McrBs, we have purified the protein and analyzed its role in vitro. At the optimal ratio of 3-5 McrBL per molecule of McrC, McrBs acted as an inhibitor of DNA cleavage. Inhibition was due to sequestration of McrC and required the presence of GTP, suggesting that the interaction is GTP dependent. If McrC was in excess, a condition resulting in suboptimal DNA cleavage, addition of McrBs enhanced DNA cleavage, presumably due to sequestration of excess McrC. We suggest that the role of McrBs is to modulate McrBC activity by binding to McrC.     

Overproduction and purification of McrC protein from Escherichia coli K-12.

The McrC protein, encoded by one of the two genes involved in the McrB restriction system, was produced in Escherichia coli cells by using a T7 expression system. Following sequential DEAE-Sepharose and hydroxylapatite column chromatography, the protein was purified to apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified McrC protein agreed exactly with the one deduced from the DNA sequence by Ross et al. (J. Bacteriol. 171:1974-1981, 1989).     

Evidence of participation of McrB(S) in McrBC restriction in Escherichia coli K-12.

The McrBC restriction system has the ability to restrict DNA containing 5-hydroxymethylcytosine, N4-methylcytosine, and 5-methylcytosine at specific sequences. The mcrB gene produces two gene products. The complete mcrB open reading frame produces a 51-kDa protein (McrB(L)) and a 33-kDa protein (McrB(S)). The smaller McrB polypeptide is produced from an in-frame, internal translational start site in the mcrB gene. The McrB(S) sequence is identical to that of McrB(L) except that it lacks 161 amino acids present at the N-terminal end of the latter protein. It has been suggested that McrB(L) is the DNA binding restriction subunit. The function of McrB(S) is unknown, although there has been speculation that it plays some role in the modulation of McrBC restriction. Studies of the function of McrB(S) have been challenging since it is produced in frame with McrB(L). In this study, we tested the effects of underproduction (via antisense RNA) and overproduction (via gene dosage) of mcrBC gene products on restriction levels of the mcrBC+ strain JM107. Among the parameters monitored was the induction of SOS responses, which indicate of DNA damage. Evidence from this study suggests that McrB(S) is necessary for stabilization of the McrBC restriction complex in vivo.     

McrA and McrB restriction phenotypes of some E. coli strains and implications for gene cloning.

The McrA and McrB (modified cytosine restriction) systems of E. coli interfere with incoming DNA containing methylcytosine. DNA from many organisms, including all mammalian and plant DNA, is expected to be sensitive, and this could interfere with cloning experiments. The McrA and B phenotypes of a few strains have been reported previously (1-4). The Mcr phenotypes of 94 strains, primarily derived from E. coli K12, are tabulated here. We briefly review some evidence suggesting that McrB restriction of mouse-modified DNA does occur in vivo and does in fact interfere with cloning of specific mouse sequences.     

Kinetic characterization of the GTPase activity of phage lambda terminase: evidence for communication between the two "NTPase" catalytic sites of the enzyme.

The terminase enzyme from bacteriophage lambda is responsible for the insertion of viral DNA into the confined space within the capsid. The enzyme is composed of the virally encoded proteins gpA (73.3 kDa) and gpNu1 (20.4 kDa) isolated as a gpA(1).gpNu1(2) holoenzyme complex. Lambda terminase possesses a site-specific nuclease activity, an ATP-dependent DNA strand-separation activity, and an ATPase activity that must work in concert to effect genome packaging. We have previously characterized the ATPase activity of the holoenzyme and have identified catalytic active sites in each enzyme subunit [Tomka and Catalano (1993) Biochemistry 32, 11992-11997; Hwang et al. (1996) Biochemistry 35, 2796-2803]. We have noted that GTP stimulates the ATPase activity of the enzyme, and terminase-mediated GTP hydrolysis has been observed. The studies presented here describe a kinetic analysis of the GTPase activity of lambda terminase. GTP hydrolysis by the enzyme requires divalent metal, is optimal at alkaline pH, and is strongly inhibited by salt. Interestingly, while GTP can bind to the enzyme in the absence of DNA, GTP hydrolysis is strictly dependent on the presence of polynucleotide. Unlike ATP hydrolysis that occurs at both subunits of the holoenzyme, a single catalytic site is observed in the steady-state kinetic analysis of GTPase activity (k(cat) approximately 37 min(-)(1); K(m) approximately 500 microM). Moreover, while GTP stimulates ATP hydrolysis (apparent K(D) approximately 135 microM for GTP binding), all of the adenosine nucleotides examined strongly inhibit the GTPase activity of the enzyme. The data presented here suggest that the two "NTPase" catalytic sites in terminase holoenzyme communicate, and we propose a model describing allosteric interactions between the two sites. The biological significance of this interaction with respect to the assembly and disassembly of the multiple nucleoprotein packaging complexes required for virus assembly is discussed.     

ATP-stimulated hydrolysis of GTP by RecA protein: kinetic consequences of cooperative RecA protein-ATP interactions

The cooperativity of the single-stranded DNA dependent nucleoside triphosphatase activity of the recA protein was investigated by examining the influence of a good substrate (ATP) on the hydrolysis of a poor substrate (GTP). At pH 7.5 and 37 degrees C, both ATP and GTP are hydrolyzed with a turnover number of 17.5 min-1. The S0.5 for GTP (750 microM), however, is nearly 20-fold higher than the S0.5 for ATP (45 microM). Low concentrations of ATP activate the GTPase activity of the recA protein by lowering the S0.5 for GTP; in the presence of 50 microM ATP, the S0.5 for GTP is reduced from 750 microM to 200 microM. Concentrations of ATP greater than 50 microM result in competitive inhibition of the ATP-activated GTPase activity. Although GTP is a substrate for hydrolysis, it will not substitute for ATP as a high-energy cofactor in the standard recA protein promoted three-strand exchange reaction. To account for these results, a minimal kinetic model is presented in which ATP binding induces specific conformational changes in the recA protein that do not occur with GTP binding.     

Nucleotide binding domain 1 of the human retinal ABC transporter functions as a general ribonucleotidase.

Members of the ATP binding cassette (ABC) superfamily are transmembrane proteins that are found in a variety of tissues which transport substances across cell membranes in an energy-dependent manner. The retina-specific ABC protein (ABCR) has been linked through genetic studies to a number of inherited visual disorders, including Stargardt macular degeneration and age-related macular degeneration (ARMD). Like other ABC transporters, ABCR is characterized by two nucleotide binding domains and two transmembrane domains. We have cloned and expressed the 522-amino acid (aa) N-terminal cytoplasmic region (aa 854-1375) of ABCR containing nucleotide binding domain 1 (NBD1) with a purification tag at its amino terminus. The expressed recombinant protein was found to be soluble and was purified using single-step affinity chromatography. The purified protein migrated as a 66 kDa protein on SDS-PAGE. Analysis of the ATP binding and hydrolysis properties of the NBD1 polypeptide demonstrated significant differences between NBD1 and NBD2 [Biswas, E. E., and Biswas, S. B. (2000) Biochemistry 39, 15879-15886]. NBD1 was active as an ATPase, and nucleotide inhibition studies suggested that nucleotide binding was not specific for ATP and all four ribonucleotides can compete for binding. Further analysis demonstrated that NBD1 is a general nucleotidase capable of hydrolysis of ATP, CTP, GTP, and UTP. In contrast, NBD2 is specific for adenosine nucleotides (ATP and dATP). NBD1 bound ATP with a higher affinity than NBD2 (K(mNBD1) = 200 microm vs K(mNBD2) = 631 microm) but was less efficient as an ATPase (V(maxNBD1) = 28.9 nmol min(-)(1) mg(-)(1) vs V(maxNBD2) = 144 nmol min(-)(1) mg(-)(1)). The binding efficiencies for CTP and GTP were comparable to that observed for ATP (K(mCTP) = 155 microm vs K(mGTP) = 183 microm), while that observed for UTP was decreased 2-fold (K(mUTP) = 436 microm). Thus, the nucleotide binding preference of NBD1 is as follows: CTP > GTP > ATP > UTP. These studies demonstrate that NBD1 of ABCR is a general nucleotidase, whereas NBD2 is a specific ATPase.     

Co-activation of RanGTPase and inhibition of GTP dissociation by Ran-GTP binding protein RanBP1.

RCC1 (the regulator of chromosome condensation) stimulates guanine nucleotide dissociation on the Ras-related nuclear protein Ran. Both polypeptides are components of a regulatory pathway that has been implicated in regulating DNA replication, onset of and exit from mitosis, mRNA processing and transport, and import of proteins into the nucleus. In a search for further members of the RCC1-Ran signal pathway, we have identified proteins of 23, 45 and 300 kDa which tightly bind to Ran-GTP but not Ran-GDP. The purified soluble 23 kDa Ran binding protein RanBP1 does not activate RanGTPase, but increases GTP hydrolysis induced by the RanGTPase-activating protein RanGAP1 by an order of magnitude. In the absence of RanGAP, it strongly inhibits RCC1-induced exchange of Ran-bound GTP. In addition, it forms a stable complex with nucleotide-free RCC1-Ran. With these properties, it differs markedly from guanine diphosphate dissociation inhibitors which preferentially prevent the exchange of protein-bound GDP and in some cases were shown to inhibit GAP-induced GTP hydrolysis. RanBP1 is the first member of a new class of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins.     

Evidence for specific binding sites for guanine nucleotides in adipocyte and hepatocyte plasma membranes. A difference in fate of GTP and guanosine 5'-(beta, gamma-imino) triphosphate.

Binding and degradation of GTP and guanosine 5'-(beta, gamma-imino)triphosphate (Gpp(NH)p by plasma membranes from rat liver and fat cells were investigated. Gpp(NH)p is hydrolyzed predominantly by nucleotide pyrophosphohydrolases in the membranes, whereas GTP is hydrolyzed primarily by nucleotide phosphohydrolases. These enzymes are not specific for the guanine nucleotides since co-addition of the analogous adenine nucleotides spares their hydrolysis. Both Gpp(NH)p and GTP are taken up by the membranes at sites which, to the extent that high concentrations of the corresponding adenine nucleotides fail to inhibit uptake, appear to be specific for guanine nucleotides. Gpp(NH)p taken up at these sites remains essentially intact irrespective of the degree of hydrolysis of unbound Gpp(NH)p by nucleotide pyrophosphohydrolases, indicating that the binding siteis incapable of degrading Gpp(NH)p. GTP and GDP inhibit competitively the binding of Gpp(NH)p; the binding constants for the three nucleotides are similar (0.1 to 0.4 muM) and are in the same range required for their effects on adenylate cyclase activity. Binding of the nucleotides is inhibited by sulfhydryl agents, suggesting that a sulfhydryl group is involved in the binding process. In contrast to binding of Gpp(NH)p, uptake of GTP is accompanied by substantial hydrolysis, primarily to GDP, under incubation conditions (high [ATP] plus ATP regenerating system) in which [GTP] in the medium remains essentially constant. GDP bound to the membranes is progressively hydrolyzed to 5'-GMP. Thus, GTP and Gpp(NH)p, although binding to the same specific sites, are differentially susceptible to hydrolysis at their terminal phosphates when bound to these sites. These findings are discussed in terms of the markedly different potencies of GTP and Gpp(NH)p as activators of adenylate cyclase systems.     

Op18/stathmin mediates multiple region-specific tubulin and microtubule-regulating activities.

Oncoprotein18/stathmin (Op18) is a regulator of microtubule (MT) dynamics that binds tubulin heterodimers and destabilizes MTs by promoting catastrophes (i.e., transitions from growing to shrinking MTs). Here, we have performed a deletion analysis to mechanistically dissect Op18 with respect to (a) modulation of tubulin GTP hydrolysis and exchange, (b) tubulin binding in vitro, and (c) tubulin association and MT-regulating activities in intact cells. The data reveal distinct types of region-specific Op18 modulation of tubulin GTP metabolism, namely inhibition of nucleotide exchange and stimulation or inhibition of GTP hydrolysis. These regulatory activities are mediated via two-site cooperative binding to tubulin by multiple nonessential physically separated regions of Op18. In vitro analysis revealed that NH(2)- and COOH-terminal truncations of Op18 have opposite effects on the rates of tubulin GTP hydrolysis. Transfection of human leukemia cells with these two types of mutants result in similar decrease of MT content, which in both cases appeared independent of a simple tubulin sequestering mechanism. However, the NH(2)- and COOH-terminal-truncated Op18 mutants regulate MTs by distinct mechanisms as evidenced by morphological analysis of microinjected newt lung cells. Hence, mutant analysis shows that Op18 has the potential to regulate tubulin/MTs by more than one specific mechanism.     

Characterization of the DNA-dependent GTPase activity of T4 gene 41 protein, an essential component of the T4 bacteriophage DNA replication apparatus

The product specified by T4 bacteriophage gene 41 is known from genetic analyses to be essential for phage DNA replication in vivo. Correspondingly, the purified gene 41 protein is an essential component of an efficient in vitro DNA replication system reconstructed from seven purified T4 replication proteins; it is required both for the synthesis of short RNA primers (in conjunction with the T4 gene 61 protein) and for the rapid unwinding of the double-helical DNA template at a replication fork. The purified gene 41 protein exhibits a DNA-dependent GTPase (and ATPase) activity. In this report, we have used this associated GTPase activity as a biochemical probe for the analysis of the interactions between DNA and the 41 protein. Our results suggest that, upon binding GTP, the 41 protein monomer is induced to form a dimer, which can them form a tight complex with single-stranded DNA. Driven by the repeated hydrolysis of GTP molecules, the 41 protein dimer appears to run rapidly along the bound DNA chain. Studies with the synthetic GTP analogue, GTP gamma S, suggest that GTP hydrolysis is required for this 41 protein movement, but that it is not essential for the function of the 41 protein in RNA primer synthesis. In sum, our observations suggest that a 41 protein dimer runs along the lagging strand template at a DNA replication fork; from this position, it functions as a DNA helicase and simultaneously interacts with the T4 gene 61 protein to make the pentaribonucleotide primers which initiate Okazaki pieces at specific primer initiation sites.     

Kinetic analysis of the hydrolysis of GTP by p21N-ras. The basal GTPase mechanism

The rate constants have been determined for elementary steps in the basal GTPase mechanism of normal p21N-ras (Gly-12) and an oncogenic mutant (Asp-12): namely GTP binding, hydrolysis, phosphate release, and GDP release. By extrapolation from data at lower temperatures, the GTP association rate constant at 37 degrees C is 1.4 x 10(8) M-1 s-1 for the normal protein and 4.8 x 10(8) M-1 s-1 for the mutant. Other rate constants were measured directly at 37 degrees C, and three processes have similar slow values. GTP dissociation is at 1.0 x 10(-4) s-1 (normal) and 5.0 x 10(-4) s-1 (mutant). The hydrolysis step is at 3.4 x 10(-4) s-1 (normal) and 1.5 x 10(-4) s-1 (mutant). GDP dissociates at 4.2 x 10(-4) s-1 (normal) and 2.0 x 10(-4) s-1 (mutant). GDP association rate constants are similar to those for GTP, 0.5 x 10(8) M-1 s-1 for normal and 0.7 x 10(8) M-1 s-1 for mutant. Both hydrolysis and GDP release therefore contribute to rate limitation of the basal GTPase activity. There are distinct differences (up to 5-fold) between rate constants for the normal and mutant proteins at a number of steps. The values are consistent with the reduced GTPase activity for this mutant and suggest little difference between normal and mutant proteins in the relative steady-state concentrations of GTP and GDP complexes that may represent active and inactive states. The results are discussed in terms of the likely role of p21ras in transmembrane signalling.     

Role of coatomer and phospholipids in GTPase-activating protein-dependent hydrolysis of GTP by ADP-ribosylation factor-1

The binding of the coat protein complex, coatomer, to the Golgi is mediated by the small GTPase ADP-ribosylation factor-1 (ARF1), whereas the dissociation of coatomer, requires GTP hydrolysis on ARF1, which depends on a GTPase-activating protein (GAP). Recent studies demonstrate that when GAP activity is assayed in a membrane-free environment by employing an amino-terminal truncation mutant of ARF1 (Delta17-ARF1) and a catalytic fragment of the ARF GTPase-activating protein GAP1, GTP hydrolysis is strongly stimulated by coatomer (Goldberg, J., (1999) Cell 96, 893-902). In this study, we investigated the role of coatomer in GTP hydrolysis on ARF1 both in solution and in a phospholipid environment. When GTP hydrolysis was assayed in solution using Delta17-ARF1, coatomer stimulated hydrolysis in the presence of the full-length GAP1 as well as with a Saccharomyces cerevisiae ARF GAP (Gcs1) but had no effect on hydrolysis in the presence of the phosphoinositide dependent GAP, ASAP1. Using wild-type myristoylated ARF1 loaded with GTP in the presence of phospholipid vesicles, GAP1 by itself stimulated GTP hydrolysis efficiently, and coatomer had no additional effect. Disruption of the phospholipid vesicles with detergent resulted in reduced GAP1 activity that was stimulated by coatomer, a pattern that resembled Delta17-ARF1 activity. Our findings suggest that in the biological membrane, the proximity between ARF1 and its GAP, which results from mutual binding to membrane phospholipids, may be sufficient for stimulation of ARF1 GTPase activity.