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G Batelier

Publications and source records attributed to G Batelier.

12 recordsLinked to original sources
PubMed2000

The nitrogen source-dependent 126-kDa protein from Synechococcus PCC 7942 plasmalemma: a trimer of the NrtA nitrate-binding protein.

Expression of a 126-kDa protein in the plasmalemma (cytoplasmic membrane) from Synechococcus PCC 7942 is dependent on the nitrogen source. Polyclonal antibody raised against the NrtA protein reacted with the 126-kDa protein. Two peptide sequences from the 126-kDa protein were retrieved in NrtA. FPLC purification of plasmalemma solubilised in Triton X-100 gave a fraction consisting mainly of a 126-kDa component (72.6%), as shown by sedimentation velocity. Equilibrium sedimentation of the same fraction gave evidence of the existence of an oligomeric structure (Ka = 2 x 10(3) and 2.9 subunits). Thus, the 126-kDa protein is considered as a trimeric arrangement of the 45-kDa protein in the plasmalemma.

Amino Acid Sequence↗
PubMed1999

Dynamic localization of CLIP-170 to microtubule plus ends is coupled to microtubule assembly.

CLIP-170 is a cytoplasmic linker protein that localizes to plus ends of microtubules in vivo. In this study, we have characterized the microtubule-binding properties of CLIP-170, to understand the mechanism of its plus end targeting. We show that the NH2-terminal microtubule-interacting domain of CLIP-170 alone localizes to microtubule plus ends when transfected into cells. Association of CLIP-170 with newly-formed microtubules was observed in cells microinjected with biotinylated tubulin, used as a tracer for growing microtubules. Using in vitro assays, association of CLIP-170 with recently polymerized tubulin is also seen. Cross-linking and sedimentation velocity experiments suggest association of CLIP-170 with nonpolymerized tubulin. We conclude from these experiments that the microtubule end targeting of CLIP-170 is closely linked to tubulin polymerization.

Centrosome↗
PubMed1999

RNA:pseudouridine synthetase Pus1 from Saccharomyces cerevisiae: oligomerization property and stoichiometry of the complex with yeast tRNA(Phe).

Yeast RNA:pseudouridine synthetase Pus1 catalyzes the formation of pseudouridines in tRNAs. We report here the quaternary structure of purified recombinant Pus1 in solution. At low concentration, in the absence of tRNA, Pus1 oligomerizes while at high concentration it precipitates. This oligomerization/aggregation can be prevented by addition of dodecyl-beta-D-maltoside or of yeast tRNA(Phe). The detergent does not significantly interfere with substrate binding or with activity of Pus1. The stoichiometry of the Pus1/tRNA(Phe) complex is 1/1. We conclude that the detergent covers an hydrophobic region of the RNA binding pocket responsible for Pus1 aggregation.

Biopolymers↗
PubMed1997

Structure-function studies on small heat shock protein oligomeric assembly and interaction with unfolded polypeptides.

The small heat shock protein (smHSP) and alpha-crystallin genes encode a family of 12-43-kDa proteins which assemble into large multimeric structures, function as chaperones by preventing protein aggregation, and contain a conserved region termed the alpha-crystallin domain. Here we report on the structural and functional characterization of Caenorhabditis elegans HSP16-2, a 16-kDa smHSP produced only under stress conditions. A combination of sedimentation velocity, size exclusion chromatography, and cross-linking analyses on wild-type HSP16-2 and five derivatives demonstrate that the N-terminal domain but not most of the the C-terminal extension which follows the alpha-crystallin domain is essential for the oligomerization of the smHSP into high molecular weight complexes. The N terminus of HSP16-2 is found to be buried within complexes which can accommodate at least an additional 4-kDa of heterologous sequence per subunit. Studies on the interaction of HSP16-2 with fluorescently-labeled and radiolabeled actin and tubulin reveal that this smHSP possesses a high affinity for unfolded intermediates which form early on the aggregation pathway, but has no apparent substrate specificity. Furthermore, both wild-type and C-terminally-truncated HSP16-2 can function as molecular chaperones by suppressing the thermally-induced aggregation of citrate synthase. Taken together, our data on HSP16-2 and a unique 12.6-kDa smHSP we have recently characterized demonstrate that multimerization is a prerequisite for the interaction of smHSPs with unfolded protein as well as for chaperone activity.

Actins↗
PubMed1997

Cytoplasmic chaperonin containing TCP-1: structural and functional characterization.

Actin and tubulin polypeptide chains acquire their native conformation in the presence of the cytoplasmic chaperonin containing TCP-1 (CCT, also called TRiC) and, in the case of alpha- and beta-tubulin, additional protein cofactors. It has been previously demonstrated that nucleotide exchange and ATP hydrolysis act to switch CCT between conformations that interact either strongly or weakly with unfolded substrates [Melki, R., & Cowan, N.J. (1994) Mol. Cell. Biol. 14, 2895-2904]. The present study further documents the conformational changes and function of CCT. It is first shown, by the use of a range of labeled denatured substrate proteins and a radiolabeled total soluble HeLa cell extract, that CCT in the absence of nucleotides can bind any of a large number of proteins in vitro with high affinity. Second, by the use of denatured labeled beta-actin and beta-tubulin as model substrates for binding to CCT, we demonstrate that the CCT particle can contain two substrate protein chains simultaneously. Third, by electron microscopy, sedimentation velocity, and intrinsic fluorescence measurements, we document the conformational difference between CCT in its ATP- and ADP-bound forms, as well as the change that results from binding of substrate protein. A model summarizes substrate association with CCT and the role of the nucleotide in regulating the affinity of CCT for target proteins.

Actins↗
PubMed1997

Unique structural features of a novel class of small heat shock proteins.

Small heat shock proteins (smHSPs) and alpha-crystallins constitute a family of related molecular chaperones that exhibit striking variability in size, ranging from 16 to 43 kDa. Structural studies on these proteins have been hampered by their tendency to form large, often dynamic and heterogeneous oligomeric complexes. Here we describe the structure and expression of HSP12.6, a member of a novel class of smHSPs from the nematode Caenorhabditis elegans. Like other members of its class, HSP12.6 possesses a conserved alpha-crystallin domain but has the shortest N- and C-terminal regions of any known smHSP. Expression of HSP12.6 is limited to the first larval stage of C. elegans and is not significantly up-regulated by a wide range of stressors. Unlike other smHSPs, HSP12.6 does not form large oligomeric complexes in vivo. HSP12.6 was produced in Escherichia coli as a soluble protein and purified. Cross-linking and sedimentation velocity analyses indicate that the recombinant HSP12.6 is monomeric, making it an ideal candidate for structure determination. Interestingly, HSP12.6 does not function as a molecular chaperone in vitro, since it is unable to prevent the thermally induced aggregation of a test substrate. The structural and functional implications of these findings are discussed.

Amino Acid Sequence↗
PubMed1996

Structural properties of UMP-kinase from Escherichia coli: modulation of protein solubility by pH and UTP.

UMP-kinase from Escherichia coli, unlike the analogous enzyme from eukaryotic organisms, is an oligomeric protein subjected to complex regulatory mechanisms in which UTP and GTP act as allosteric effectors. While the enzyme has an unusually low solubility at neutral pH (< or = 0.1 mg of protein/ mL), its solubility increases markedly above pH 8 and below pH 4. Furthermore, the solubility of the bacterial UMP-kinase at neutral pH is greatly enhanced in the presence of Mg-free UTP. Thermal denaturation experiments have demonstrated that UTP also increases the stability of the protein. Fourier-transform infrared spectroscopy and circular dichroism show that the secondary structure of the protein is the same at neutral and at alkaline pH. These data indicate that variations in enzyme solubility must be related to subtle changes in the tertiary and/or quaternary structure which modulate the exposure of hydrophobic surfaces in the protein molecule. A variant of UMP-kinase, obtained by site-directed mutagenesis (Asp159Asn), which is similar to the wild-type enzyme in its stability and kinetic properties, has a much increased water solubility (> 5 mg protein/mL) even at neutral pH. This suggests that salt bridges may be involved in the equilibrium between the soluble and aggregated forms of the wild-type enzyme, and that conformational changes induced upon binding of UTP increase the protein solubility by disrupting these salt bridges.

Allosteric Regulation↗
PubMed1995

Self-association of the molecular chaperone HSC70.

The self-association properties of the molecular chaperone HSC70 have been analyzed by a wide range of biochemical and biophysical techniques. Nondenaturing gel electrophoresis and cross-linking studies show the presence of multiple species going from monomer to at least trimer. Size-exclusion chromatography gives two overlapping peaks, a major one corresponding to species having the molecular mass of monomer (70 kDa) and a minor broad one corresponding to species with a molecular mass range of 150-300 kDa. Progressive dilution of the protein leads to an increase in the size of the monomer peak at the expense of that of the oligomeric peak, thus indicating a concentration-dependent chemical equilibrium. Sedimentation velocity reveals the presence of three species, whose proportions were dependent on concentration, but whose sedimentation coefficients, s20,w, of 4.3, 6.6, and 8.5 S did not vary with concentration, indicative of a slowly equilibrating system. Sedimentation equilibrium studies confirmed these results and showed a dissociation into monomers at low concentrations and an association into dimers and trimers at high concentrations. The multiple sedimentation equilibrium datasets, obtained at various initial loading concentrations as well as different rotor speeds, were fitted to a single set of equilibrium constants by a monomer-dimer-trimer association model in which the association constants for the monomer-dimer and dimer-trimer equilibrium were respectively K1-2 = 1.1 x 10(5) M-1 and K2-3 = 0.9 x 10(5) M-1. Interestingly, an isodesmic, indefinite type of association describes the data almost equally well with a single constant of 1.2 x 10(5) M-1. (ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Triphosphate↗
PubMed1981

Mechanism of tubulin assembly: role of rings in the nucleation process and of associated proteins in the stabilization of microtubules.

Several types of experiments were designed to elucidate the mechanism of initiation of tubulin assembly in the presence of microtubule-associated proteins (MAPs): (1) The evolution of the proportion of the double ring species with temperature was examined in the ultracentrifuge, under nonpolymerizing conditions (guanosine diphosphate = 0.5 mM). A net dissociation of rings into dimers occurred when temperature increased. The transition took place above 20 degrees C. (2) The kinetic parameters of the exchange at equilibrium between free tubulin dimers and tubulin in rings were studied at different temperatures, using the technique of isotopic exchange at equilibrium with radioactively labeled tubulin. The values found for the half-time of the exchange reaction varied between or approximately 100 min at 0 degrees C and 10 min at 2 degrees C. (3) The relative participation of unlabeled rings and labeled dimers to microtubules in the time course of assembly was studied at 22 degrees C. The time dependence of microtubule specific radioactivity showed that tubulin incorporated in the initial stages of assembly came predominantly from rings (85-90%). This result indicates that oligomers directly issued from rings are the first intermediates in the assembly process and suggests that at the beginning of polymerization incorporation of isomers or fragments of rings proceeds at a faster rate than their dissociation into dimers. (4) Polymerization experiments in the presence of MAPs with increasing concentrations of tubulin dimers indicated that MAPs are in rapid equilibrium with the microtubules and are distributed along the wall in a more or less loose lattice depending on the relative concentrations of tubulin and MAPs in the solution.

Animals↗