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M F Carlier

Publications and source records attributed to M F Carlier.

At least 73 records · Page 4Linked to original sources
PubMed1992

Interaction between G-actin and myosin subfragment-1 probed by covalent cross-linking.

The topography of rapid equilibrium complexes formed between G-actin and myosin subfragment-1, which are the first kinetic intermediates in the polymerization process into F-acto-S1 filaments, has been probed by chemical cross-linking. In the absence of ATP, cross-linking of G-actin-S1 complexes by 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) yielded a major 165-170-kDa and a fainter 200-205-kDa doublet polypeptide. The actin:S1 molar ratio was 1 in the EDC-cross-linked complexes, using either double labeling techniques or the method combining EDC + N-hydroxysuccinimide. Chemical cleavages of the covalently cross-linked complexes by formic acid and N-hydroxylamine (Sutoh, K. (1983) Biochemistry 22, 1579-1585) showed that in the main cross-linked 165-kDa polypeptide, the 1-12 acidic N-terminal region of actin was covalently linked to the lysine-rich region connecting the central 50-kDa domain to the C-terminal 20-kDa domain of S1, as in F-acto-S1 complexes. G-actin, but not F-actin, was covalently cross-linked to S1 by N,N'-paraphenylenedimaleimide (p-PDM). A major 195-kDa and a minor 165-kDa polypeptide were obtained, could be separated from actin and S1 by DEAE-cellulose chromatography, and did not exhibit actin-activated Mg-ATPase activity. Both EDC-cross-linked and p-PDM-cross-linked complexes between G-actin and S1 could be incorporated into F-acto-S1 decorated filaments. The C-terminal cysteine 374 of actin is involved in the p-PDM cross-linked 195-kDa complex. Accordingly, a covalent photocross-linked 200-kDa conjugate was formed between S1 heavy chain and benzophenone-G-actin, obtained by covalent modification of Cys374 by benzophenonemaleimide (Tao, T., Lamkin, M., and Scheiner, C. J. (1985) Arch. Biochem. Biophys. 240, 627-634). These results demonstrate that (i) G-actin-S1 and F-actin-S1 complexes display a large similarity in the EDC-cross-linked electrostatic close contacts and (ii) a change in the environment of Cys374 is linked to the polymerization into F-actin-S1 decorated filaments.

Actins↗
PubMed1992

Nucleotide hydrolysis regulates the dynamics of actin filaments and microtubules.

Actin filaments and microtubules are major dynamic components of the cytoskeleton of eukaryotic cells. Assembly of these polymers from monomeric actin or tubulin occurs with expenditure of energy, because ATP (or GTP) tightly bound to actin (or tubulin) is irreversibly hydrolysed during polymerization. Therefore, actin filaments an microtubules are dissipative structures. Our purpose has been to understand how the dissipation of chemical energy perturbs the laws of reversible helical polymerization defined by Oosawa, and affects the dynamics of these polymers. A kinetic study has shown that nucleotide is hydrolysed on the polymer within at least two steps consecutive to the incorporation of the monomer: cleavage of the gamma-phosphoester bond followed by the slower release of Pi; only the second reaction appears reversible. Pi release, and not cleavage of the gamma-phosphate, is linked to the destabilization of protein-protein interactions in the polymer, and therefore plays the role of a conformational switch. The dynamic properties of the polymer in the NTP- and NDP-Pi intermediate states of the assembly process have been investigated using non-hydrolysable analogues of nucleotides and structural analogues of Pi, AlF4- and (BeF3-, H2O). Because nucleotide hydrolysis is uncoupled from polymerization, actin filaments and microtubules grow with a 'cap' of terminal NTP- and NDP-Pi-subunits that interact strongly, and prevent the rapid depolymerization of the unstable core of the polymer formed of NDP-subunits. The fact that the dynamic properties of the polymer are affected by bound nucleotide results in a nonlinear dependence of the rate of elongation on monomer concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Actins↗
PubMed1992

Interaction of myosin subfragment-1 with F- and G-actin in equilibrium.

The structural changes of the F-actin-myosin head (S1) complex during the cross-bridge cycle are essential in muscle contraction. Although a large body of evidence has accumulated showing that the actin: S1 stoichiometry in the decorated F-actin-S1 filament is 1:1 at saturation by S1, a recent report by Andreev and Borejdo (1991, Biochem. Biophys. Res. Comm. 177, 350-356) indicated that under some conditions, the actin: S1 stoichiometry could be 2:1 at saturation by S1. Because of the important implications of this result in the mechanism of acto-myosin motility, we have re-investigated this issue. It is shown here that evidence for the 2:1 stoichiometry was circumstantial and was only observed under conditions where 50% of the actin was F-actin, i.e. at a total actin concentration twice as large as the critical concentration. The interaction of S1 with both F- and G-actin in dynamic equilibrium is studied in detail. The present data fully support the 1:1 actin: S1 stoichiometry in the decorated filament at saturation by S1.

Actins↗
PubMed1992

Covalent modification of G-actin by pyridoxal 5'-phosphate: polymerization properties and interaction with DNase I and myosin subfragment 1.

Pyridoxal 5'-phosphate (PLP), a lysine-specific reagent, has been used to modify G-actin. At pH 7.5, PLP reacted with 1.7-2 lysines on G-actin. Limited proteolytic digestion experiments indicated that, in agreement with previous works, essentially lysine-61 was modified in a 1:1 fashion by PLP, other lysines being much less reactive. A PLP-derivatized affinity label of ATP binding sites, AMPPLP, reacted with two additional lysines that do not appear to be located in the ATP site on G-actin. PLP-G-actin did not polymerize spontaneously up to 30 microM; however, it retained other essential native properties of G-actin. PLP-actin bound to the barbed ends of actin filaments with an equilibrium dissociation constant of 4 microM and prevented dilution-induced depolymerization like a capping protein. PLP-actin copolymerized with unmodified actin. The stability of F-actin copolymers decreased with the fraction of PLP-actin incorporated, consistent with a model within which the actin-PLP-actin interactions in the copolymer are 50-fold weaker, and PLP-actin-PLP-actin interactions are 200-fold weaker than regular actin-actin interactions. PLP-actin bound DNase I with an equilibrium association constant of 2 nM-1, i.e., 10-fold lower than that of unmodified actin. PLP modification did not affect the binding of G-actin to myosin subfragment 1. However, polymerization of PLP-actin by myosin subfragment 1 was not observed in low ionic strength buffers, whereas PLP-F-actin-S1 filaments, in which the stoichiometry PLP-actin:S1 is 1:1, were formed with an apparent critical concentration of 4.5 microM in the presence of 0.1 M KCl.

Actins↗
PubMed1991

Interaction of microtubule-associated proteins with microtubules: yeast lysyl- and valyl-tRNA synthetases and tau 218-235 synthetic peptide as model systems.

The respective contributions of electrostatic interaction and specific sequence recognition in the binding of microtubule-associated proteins (MAPs) to microtubules have been studied, using as models yeast valyl- and lysyl-tRNA synthetases (VRS, KRS) that carry an exposed basic N-terminal domain, and a synthetic peptide reproducing the sequence 218-235 on tau protein, known to be part of the microtubule-binding site of MAPs. VRS and KRS bind to microtubules with a KD in the 10(-6) M range, and tau 218-235 binds with a KD in the 10(-4) M range. Binding of KRS and tau 218-235 is accompanied by stabilization and bundling of microtubules, without the intervention of an extraneous bundling protein. tau 218-235 binds to microtubules with a stoichiometry of 2 mol/mol of assembled tubulin dimer in agreement with the proposed binding sequences alpha[430-441] and beta[422-434]. Binding stoichiometries of 2/alpha beta S tubulin and 1/alpha S beta S tubulin were observed following partial or complete removal of the tubulin C-terminal regions by subtilisin, which localizes the site of subtilisin cleavage upstream residue alpha-441 and downstream residue beta-434. Quantitative measurements show that binding of MAPs, KRS, VRS, and tau 218-235 is weakened but not abolished following subtilisin digestion of the C-terminus of tubulin, indicating that the binding site of MAPs is not restricted to the extreme C-terminus of tubulin.

Amino Acid Sequence↗
PubMed1991

Myosin subfragment-1 interacts with two G-actin molecules in the absence of ATP.

The interaction between G-actin and myosin subfragment-1 (S1) has been monitored by pyrenyl-actin fluorescence and light scattering. In low ionic strength buffer and in the absence of ATP the polymerization of G-actin induced by myosin subfragment-1 is preceded by the formation of binary GS and ternary G2S complexes in which S1 interacts tightly in rapid equilibrium (K greater than 10(7) M-1) with one and two G-actin molecules, respectively. Pyrenyl fluorescence of G-actin is enhanced 4-fold in GS and 3-fold in G2S. At concentrations of G-actin and S1 in the micromolar range and above, G2S is the predominant species at G-actin/S1 ratios equal to or greater than 1. The isomer of myosin subfragment-1 carrying the A1 light chain, S1(A1), forms a tighter ternary complex than the isomer S1(A2). Actin-bound ATP is not hydrolyzed upon formation of GS and G2S. In the presence of one molar equivalent or more of myosin subfragment-1/mol of G-actin, in low ionic strength buffer containing no nucleotides, G-actin polymerizes faster in the presence of S1(A1) than in the presence of S1(A2). The interaction of S1 with G-actin is inhibited by the binding of ATP or ADP to S1, ATP having a higher affinity for S1 than ADP. The possible structural similarity of the G2S complex to the F-acto-S1 complex in the rigor state and the potential significance of a ternary (actin)2-myosin interaction for actomyosin-based motility are discussed.

Actins↗
PubMed1991

Interaction between chromium GTP and tubulin. Stereochemistry of GTP binding, GTP hydrolysis, and microtubule stabilization.

Chromium GTP (CrGTP) has been used to probe the stereochemistry of metal-GTP binding to exchangeable site of tubulin and to examine the fate and role of nucleotide-bound metal ion in GTP hydrolysis associated with microtubule assembly. The absolute stereoconfiguration of the two pairs of diastereomers of beta,gamma-bidentate CrGTP has been determined by comparison of their visible circular dichroism spectra with those of the beta,gamma-CrATP isomers whose configurations have been established (Lin, I., and Dunaway-Mariano, D. (1988) J. Am. Chem. Soc. 110, 950-956). Tubulin binds metal-GTP preferentially in the delta pseudoaxial configuration. CrGTP-tubulin shows a high propensity to undergo tubulin-tubulin interactions with associated hydrolysis of CrGTP. Hydrolysis of CrGTP in microtubule assembly develops in two consecutive steps: cleavage of the gamma-phosphate followed by release of Pi and chromium. In contrast to other NTPases (actin, hexokinase) tubulin appears able to catalyze the dissociation of the stable chromium-phosphate bonds, which implies a highly nucleophilic environment of the binding site of the metal-triphosphate moiety of GTP. Microtubules assembled from CrGTP-tubulin are made of 90% GDP subunits, and their stability is linked to a 10% proportion of CrGDP-Pi subunits, scattered along the microtubule, from which Pi does not dissociate. The possibility is evoked that some tubulin variants do not catalyze release of Pi and metal ion efficiently, and their presence could affect microtubule dynamics.

Animals↗
PubMed1991

Role of ATP-bound divalent metal ion in the conformation and function of actin. Comparison of Mg-ATP, Ca-ATP, and metal ion-free ATP-actin.

The fluorescence of N-acetyl-N'-(sulfo-1-naphthyl)ethylenediamine (AEDANS) covalently bound to Cys-374 of actin is used as a probe for different conformational states of G-actin according to whether Ca-ATP, Mg-ATP, or unchelated ATP is bound to the nucleotide site. Upon addition of large amounts (greater than 10(2)-fold molar excess) of EDTA to G-actin, metal ion-free ATP-G-actin is obtained with EDTA bound. Metal ion free ATP-G-actin is characterized by a higher AEDANS fluorescence than Mg-ATP-G-actin, which itself has a higher fluorescence than Ca-ATP-G-actin. Evidence for EDTA binding to G-actin is shown using difference spectrophotometry. Upon binding of EDTA, the rate of dissociation of the divalent metal ion from G-actin is increased (2-fold for Ca2+, 10-fold for Mg2+) in a range of pH from 7.0 to 8.0. A model is proposed that quantitatively accounts for the kinetic data. The affinity of ATP is weakened 10(6)-fold upon removal of the metal ion. Metal ion-free ATP-G-actin is in a partially open conformation, as indicated by the greater accessibility of -SH residues, yet it retains functional properties of polymerization and ATP hydrolysis that appear almost identical to those of Ca-ATP-actin, therefore different from those of Mg-ATP-actin. These results are discussed in terms of the role of the ATP-bound metal ion in actin structure and function.

Actins↗
PubMed1991

Nucleotide hydrolysis in cytoskeletal assembly.

Two major polymers of the cytoskeleton, actin filaments and microtubules, are assembled with expenditure of energy: the ATP/GTP tightly bound to actin/tubulin is irreversibly hydrolyzed to ADP/GTP during the assembly process, and liberation of Pi in the medium occurs subsequent to the incorporation of subunits in the polymer. Pi release acts as a switch, causing the destabilization of protein-protein interactions in the polymer, therefore regulating the dynamics of these fibres. An understanding of this regulation in vivo requires that progress be made in four areas: the chemistry of the NTPase reaction; the structure of the intermediates in nucleotide hydrolysis and the nature of the conformational switch; the regulation of parameters involved in dynamic instability of microtubules; and the possible involvement of nucleotide hydrolysis in the macroscopic organization of these polymers in highly concentrated solutions, compared with the simple case of a equilibrium polymers. Progress made along these lines will define trends for future investigation.

Actins↗
PubMed1990

Direct evidence for GTP and GDP-Pi intermediates in microtubule assembly.

Identification of the kinetic intermediates in GTP hydrolysis on microtubules and characterization of their assembly properties is essential in understanding microtubule dynamics. By using an improved glass filter assay that selectively traps microtubules with a dead time of 2 s and monitoring taxol-induced rapid assembly of microtubules from [gamma-32P,3H]GTP-tubulin 1:1 complex, direct evidence has been obtained for GTP- and GDP-Pi-microtubule transient states in the early stages of the polymerization process. A simple kinetic analysis of GTP hydrolysis on microtubules within two sequential pseudo-first-order processes led to apparent first-order rate constants of 0.065 s-1 for the cleavage of the gamma-phosphate and 0.02 s-1 for the liberation of Pi, assuming a simple random model. Apparent rate constants for GTP hydrolysis and Pi release were independent of the composition of the buffer used to polymerize tubulin. The significance of these values with respect to those derived from previous studies from this and other laboratories and the possibility of a vectorial model for GTP hydrolysis are discussed.

Absorption↗
PubMed1990

Actin polymerization and ATP hydrolysis.

This paper surveys several aspects of the consequences of ATP hydrolysis associated with actin polymerization, and their physiological implications. ATP hydrolysis occurs on F-actin in two subsequent reactions, cleavage of ATP followed by the slower release of Pi. The latter reaction is linked to a conformation change of the actin subunit that causes a destabilization of the actin-actin interactions in the filament, i.e., a structural change of the filament. The nature of the nucleotide bound to terminal subunits therefore affects the dynamics of actin filaments. It is shown that this regulation is different at the two ends, terminal F-ADP-Pi subunits being present at steady state at the barbed end, while F-ADP-subunits are present at the pointed end. While cleavage of ATP on F-actin is irreversible, Pi release is reversible, which allows the regulation of filament dynamics by cellular Pi. The nature of the divalent metal ion--Ca2+ or Mg2(+)--tightly bound to actin, in direct interaction with ATP, also affects the conformation of actin and the rate of ATP hydrolysis, therefore regulating actin dynamics. Finally, the rate of nucleotide exchange on G-actin is relatively slow, which allows the critical concentration to increase with the number of filaments in ATP, a property largely used by the cell via the action of severing proteins.

Actin Cytoskeleton↗
PubMed1989

Evidence for the direct interaction between tightly bound divalent metal ion and ATP on actin. Binding of the lambda isomers of beta gamma-bidentate CrATP to actin.

Competition between Ca2+ and Mg2+ for binding to a single high affinity site on actin has been confirmed. Occupancy of this site only by either Ca2+ or Mg2+ affects the conformation of actin and its ability to form nuclei and hydrolyze ATP. G-actin binds the beta gamma-bidentate CrATP, a substitution inert analog of metal-ATP complexes, and shows a high specificity for the lambda isomers. Binding of CrATP to ADP-actin is accompanied by the dissociation of tightly bound ADP and Ca2+. CrATP-actin shows a high tendency to form nuclei, like MgATP-actin. Polymerization of CrATP-actin is accompanied by cleavage of the gamma-phosphate, but subsequent Pi release cannot occur because the product of the reaction is the stable CrADP-Pi complex. All these results support the view that the divalent metal ion tightly bound to actin interacts with the beta- and gamma-phosphates of ATP in the nucleotide site.

Actins↗
PubMed1989

Characterization of the aluminum and beryllium fluoride species bound to F-actin and microtubules at the site of the gamma-phosphate of the nucleotide.

Aluminum fluoride and beryllium fluoride complexes have previously been shown to bind tightly to F-ADP-actin and GDP-microtubules in competition with Pi and to mimic the XDP-Pi transient state of the polymerization. The structure of the bound complexes is investigated here in further detail. Using a fluoride ion-specific electrode, the number of fluoride atoms per aluminum or beryllium atom in the bound complex could be determined. The results indicate that AIF-4 and either BeF2(OH)-.H2O or BeF3-.H2O are the tightly bound species in both F-actin and microtubules. The dependences of the binding on pF and pH are consistent with this conclusion. The possible geometries of aluminum and beryllium fluorides in the gamma-phosphate subsite of the nucleotide are discussed in correlation with the catalytic mechanism of nucleotide hydrolysis.

Actins↗
PubMed1989

Cold depolymerization of microtubules to double rings: geometric stabilization of assemblies.

The kinetic pathway of microtubule depolymerization at 0 degrees C has been examined. Microtubules made of MAP-containing and MAP-free tubulins were depolymerized at 0 degree C in the presence of [3H]GDP or [3H]GTP or of trace amounts of 125I dimeric tubulin. The products of depolymerization were separated on a column, their structures were identified by electron microscopy, and the time course of incorporation of 3H or 125I labels in the different components of the system was determined. Two predominant assembly states of tubulin found in the nonmicrotubule state were alpha-beta dimers and double rings. Kinetic data indicate that ring formation from disassembling microtubules does not occur by direct coiling of protofilaments as previously thought, but disassembling GDP subunits are in very rapid equilibrium with curved oligomers that are kinetic intermediates in the isodesmic assembly of GDP-tubulin. The formation of oligomers and rings from dimers, at concentrations as low as 10 microM, is much faster than nucleotide exchange on alpha-beta-tubulin. Disassembly of double rings, in contrast, is slower than nucleotide exchange on alpha-beta-tubulin, by 1 order of magnitude in the absence of MAPs and 2 orders of magnitude in the presence of MAPs. These results support the model proposed previously to explain spontaneous oscillations in microtubule assembly. They are consistent with the existence of an equilibrium between two conformations of tubulin, "straight", i.e., microtubule forming, and "curved", i.e., ring forming, under the allosteric control of bound nucleotide. The straight conformation requires the presence of two ionizable hydroxyls on the gamma-phosphate in GTP or GDP-Pi.

Animals↗
PubMed1989

Mechanism of GTP hydrolysis in tubulin polymerization: characterization of the kinetic intermediate microtubule-GDP-Pi using phosphate analogues.

Beryllium fluoride (BeF3-) has previously been shown to bind tightly to microtubules as a structural analogue of Pi and to mimic the GDP-Pi transient state in tubulin polymerization [Carlier, M.-F., Didry, D., Melki, R., Chabre, M., & Pantaloni, D. (1988) Biochemistry 27, 3555-3559]. The interaction of BeF3- with tubulin is analyzed here in greater detail. BeF3- binds to and dissociates from microtubule GDP subunits at very slow rates (k+ congruent to 100 M-1 s-1; k- congruent to 6 x 10(-4) s-1), suggesting that a slow conformation change of tubulin, linked to the stabilization of the microtubule structure, follows BeF3- binding. The possibility is evoked that BeF3- acts as a transition-state analogue in the GTPase reaction of tubulin. BeF3- does not bind to dimeric nor to oligomeric GDP-tubulin with high affinity. Substoichiometric binding of BeF3- to microtubules provides extensive stabilization of the structure. An original mechanistic model that accounts for the data is proposed. The kinetic parameters for microtubule elongation in the presence of GTP- and GDP-tubulin with and without BeF3- have been determined. Data support the following views: (i) Microtubules at steady state and in a regime of slow growth in the presence of GTP are stabilized by a cap of GDP-Pi subunits functionally similar to GDP-BeF3 subunits. (ii) In the presence of BeF3-, microtubules elongate from GDP-tubulin within the following sequence of reactions: initial nonproductive binding of GDP-tubulin to microtubule ends is followed by the binding of BeF3- and the associated conformation change allowing sustained elongation.

Animals↗
PubMed1989

RecA protein-promoted cleavage of LexA repressor in the presence of ADP and structural analogues of inorganic phosphate, the fluoride complexes of aluminum and beryllium.

Complexes formed from A13+ or Be2+ and fluoride inhibit the single-stranded DNA-dependent ATPase activity of RecA protein. In contrast, poly(dT)-RecA-ADP complexes, which are inactive for cleavage of LexA protein, become fully active in the presence of AlF4- or BeF3- ions. These data suggest that fluoride complexes of aluminum and beryllium (called herein X) convert RecA-ADP complexes, which bind weakly to single-stranded DNA, into RecA-ADP-X complexes, which bind tightly to single-stranded DNA, the ADP-X moiety behaving as a nonhydrolyzable analogue of ATP. We propose that AlF4- and BeF3- ions act as analogues of inorganic phosphate by binding to the site of the gamma-phosphate of ATP on RecA-ADP complexes, hence mimicking the single-stranded DNA-RecA-ADP-Pi transition state. We conclude that the elementary reaction that switches RecA protein from a high affinity single-stranded DNA binding state to a low affinity single-stranded DNA binding state is not ATP hydrolysis per se but Pi release.

Adenosine Diphosphate↗