Signal peptide determinants of SecA binding and stimulation of ATPase activity

A signal peptide is required for entry of a preprotein into the secretory pathway, but how it functions in concert with the other transport components is unknown. In Escherichia coli, SecA is a key component of the translocation machinery found in the cytoplasm and at membrane translocation sites. Synthetic signal peptides corresponding to the wild type alkaline phosphatase signal sequence and three sets of model signal sequences varying in hydrophobicity and amino-terminal charge were generated. These were used to establish the requirements for interaction with SecA. Binding to SecA, modulation of SecA conformations sensitive to protease, and stimulation of SecA-lipid ATPase activity occur with functional signal sequences but not with transport-incompetent ones. The extent of SecA interaction is directly related to the hydrophobicity of the signal peptide core region. For signal peptides of moderate hydrophobicity, stimulation of the SecA-lipid ATPase activity is also dependent on amino-terminal charge. The results demonstrate unequivocally that the signal peptide, in the absence of the mature protein, interacts with SecA in aqueous solution and in a lipid bilayer. We show a clear parallel between the hierarchy of signal peptide characteristics that promote interaction with SecA in vitro and the hierarchy of those observed for function in vivo.     

Activation of sea urchin sperm flagellar dynein ATPase activity by salt-extracted axonemes

When 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I.R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsin-treated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KCl. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.     

A protein recognized by antibodies to Asp-Asp-Asp-Glu-Asp shows specific binding activity to heterogeneous nuclear transport signals

Nuclear proteins contain a signal, termed the nuclear transport signal, that specifies their selective transport into the nucleus. Previously we reported that antibodies to Asp-Asp-Asp-Glu-Asp (DDDED) inhibited nuclear transport of nuclear proteins in vivo. We therefore tried to detect a cellular receptor of nuclear transport signals as a protein that reacted with both anti-DDDED antibody and nuclear transport signal sequences. Using two steps of affinity chromatography, anti-DDDED-Sepharose and nucleoplasmin-Sepharose, we obtained a protein of 69 kDa (p69) from the nuclear pore fraction that showed these characters. This p69 recognized by anti-DDDED antibody interacted specifically with SV40 large T antigen and nucleoplasmin transport signals.     

Stator assembly and activation mechanism of the flagellar motor by the periplasmic region of MotB

Torque generation in the Salmonella flagellar motor is coupled to translocation of H⁺ ions through the proton-conducting channel of the Mot protein stator complex. The Mot complex is believed to be anchored to the peptidoglycan (PG) layer by the putative peptidoglycan-binding (PGB) domain of MotB. Proton translocation is activated only when the stator is installed into the motor. We report the crystal structure of a C-terminal periplasmic fragment of MotB (MotB_C) that contains the PGB domain and includes the entire periplasmic region essential for motility. Structural and functional analyses indicate that the PGB domains must dimerize in order to form the proton-conducting channel. Drastic conformational changes in the N-terminal portion of MotB_C are required both for PG binding and the proton channel activation.     

Rotation and switching of the flagellar motor assembly in Halobacterium halobium.

Halobacterium halobium swims with a polarly inserted motor-driven flagellar bundle. The swimming direction of the cell can be reserved by switching the rotational sense of the bundle. The switch is under the control of photoreceptor and chemoreceptor proteins that act through a branched signal chain. The swimming behavior of the cells and the switching process of the flagellar bundle were investigated with a computer-assisted motion analysis system. The cells were shown to swim faster by clockwise than by counterclockwise rotation of the flagellar bundle. From the small magnitude of speed fluctuations, it is concluded that the majority, if not all, of the individual flagellar motors of a cell rotate in the same direction at any given time. After stimulation with light (blue light pulse or orange light step-down), the cells continued swimming with almost constant speed but then slowed before they reversed direction. The cells passed through a pausing state during the change of the rotational sense of the flagellar bundle and then exhibited a transient acceleration. Both the average length of the pausing period and the transient acceleration were independent of the stimulus size and thus represent intrinsic properties of the flagellar motor assembly. The average length of the pausing period of individual cells, however, was not constant. The time course of the probability for spontaneous motor switching was calculated from frequency distribution and shown to be independent of the rotational sense. The time course further characterizes spontaneous switching as a stochastic rather than an oscillator-triggered event.     

Structural basis of assembly and torque transmission of the bacterial flagellar motor

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Ultrastable pRNA hexameric ring gearing hexameric phi29 DNA-packaging motor by revolving without rotating and coiling

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ATP-dependent hexameric assembly of the heat shock protein Hsp101 involves multiple interaction domains and a functional C-proximal nucleotide-binding domain

Members of the Hsp100 family of heat stress proteins are present in species throughout the bacterial, plant, and fungal kingdoms. Most Hsp100 proteins are composed of five domains that include two nucleotide-binding domains required for their ATP-dependent oligomerization. Mutations within the first but not the second nucleotide-binding site disrupt self-assembly of bacterial Hsp100, whereas the reverse is true for yeast Hsp104. We have examined the functional requirements for oligomerization of plant Hsp101 and have found that Hsp101 resembles Hsp104 in that it assembles into a hexameric complex in an ATP-dependent manner. Self-assembly of Hsp101 involves at least three distinct interaction domains located in the N-proximal domain and in the first and second nucleotide-binding domains. The interaction domain in the second nucleotide-binding domain included the Walker A motif, and mutations within this element disrupted self-assembly of Hsp101. In contrast, mutations affecting conserved residues of the Walker A motif within the first nucleotide-binding site did not affect self-assembly. No interaction between Hsp101 and Hsp104 was observed. These results suggest that plant Hsp101 self-assembly involves multiple evolutionarily diverged interaction domains as well as an evolutionarily conserved requirement for a functional C-proximal nucleotide-binding site.     

TrwD, the hexameric traffic ATPase encoded by plasmid R388, induces membrane destabilization and hemifusion of lipid vesicles

TrwD, a hexameric ATP hydrolase encoded by plasmid R388, is a member of the PulE/VirB11 protein superfamily of traffic ATPases. It is essential for plasmid conjugation, particularly for expression of the conjugative W pilus. In the present study, we analyzed the effects that TrwD produced on unilamellar vesicles consisting of cardiolipin and phosphatidylcholine in equimolar amounts. TrwD induced dose-dependent vesicle aggregation and intervesicular mixing of the lipids located in the outer monolayers in the presence of calcium. It also induced extensive leakage of the vesicular aqueous contents. A point mutant of TrwD with a mutation in the P loop of the nucleotide-binding region (K203Q) that lacks both ATPase activity and the ability to support conjugation showed the same behavior as native TrwD in all of these processes, which were independent of the presence of ATP. Structure prediction methods revealed a close similarity to Helicobacter pylori protein HP0525, another member of the PulE/VirB11 family, whose crystal structure is known. The interpretation of our data in the light of this structure is that TrwD interacts with the lipid bilayer through hydrophobic regions in its N-terminal domain, which leads to a certain degree of membrane destabilization. TrwD appears to be a part of the conjugation machinery that interacts with the membranous systems in order to facilitate DNA transfer in bacteria.     

The anion-stimulated ATPase ArsA shows unisite and multisite catalytic activity.

ArsA, an anion-stimulated ATPase, consists of two nucleotide binding domains, A1 in the N terminus and A2 in the C terminus of the protein, connected by a linker. The A1 domain contains a high affinity ATP binding site, whereas the A2 domain has low affinity and it requires the allosteric ligand antimonite for binding ATP. ArsA is known to form a UV-activated adduct with [alpha-(32)P]ATP in the linker region. This study shows that on addition of antimonite, much more adduct is formed. Characterization of the nature of the adduct suggests that it is between ArsA and ADP, instead of ATP, indicating that the adduct formation reflects hydrolysis of ATP. The present study also demonstrates that the A1 domain is capable of carrying out unisite catalysis in the absence of antimonite. On addition of antimonite, multisite catalysis involving both A1 and A2 sites occurs, resulting in a 40-fold increase in ATPase activity. Studies with mutant proteins suggest that the A2 site may be second in the sequence of events, so that its role in catalysis is dependent on a functional A1 site. It is also proposed that ArsA goes through an ATP-bound and an ADP-bound conformation, and the linker region, where ADP binds under both unisite and multisite catalytic conditions, may play an important role in the energy transduction process.     

Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.     

Role of the cytoplasmic C terminus of the FliF motor protein in flagellar assembly and rotation

Twenty-six FliF monomers assemble into the MS ring, a central motor component of the bacterial flagellum that anchors the structure in the inner membrane. Approximately 100 amino acids at the C terminus of FliF are exposed to the cytoplasm and, through the interaction with the FliG switch protein, a component of the flagellar C ring, are essential for the assembly of the motor. In this study, we have dissected the entire cytoplasmic C terminus of the Caulobacter crescentus FliF protein by high-resolution mutational analysis and studied the mutant forms with regard to the assembly, checkpoint control, and function of the flagellum. Only nine amino acids at the very C terminus of FliF are essential for flagellar assembly. Deletion or substitution of about 10 amino acids preceding the very C terminus of FliF resulted in assembly-competent but nonfunctional flagella, making these the first fliF mutations described so far with a Fla(+) but Mot(-) phenotype. Removal of about 20 amino acids further upstream resulted in functional flagella, but cells carrying these mutations were not able to spread efficiently on semisolid agar plates. At least 61 amino acids located between the functionally relevant C terminus and the second membrane-spanning domain of FliF were not required for flagellar assembly and performance. A strict correlation was found between the ability of FliF mutant versions to assemble into a flagellum, flagellar class III gene expression, and a block in cell division. Motile suppressors could be isolated for nonmotile mutants but not for mutants lacking a flagellum. Several of these suppressor mutations were localized to the 5' region of the fliG gene. These results provide genetic support for a model in which only a short stretch of amino acids at the immediate C terminus of FliF is required for flagellar assembly through stable interaction with the FliG switch protein.     

ATPase activity and ATP-dependent proton translocation in plasma membrane vesicles of turtle bladder epithelial cells

ATP-induced quenching of fluorescence of acridine orange (a pH probe) or Oxonol V (a potential difference probe) is evoked in turtle bladder membrane vesicles in suspending media of appropriate ionic composition and is insensitive to oligomycin, valinomycin, and ouabain. These effects are ascribed to a membrane-bound, ouabain-resistant ATPase which mediates an active electrogenic proton transport.     

Dynamic modulation of mitochondrial membrane physical properties and ATPase activity by diet lipid.

A longitudinal cross-over feeding design was used to investigate the relationship of dietary lipid composition to the membrane lipid environment and activity of mitochondrial ATPase in vivo. Rats were fed a polyunsaturated fatty-acid-rich oil (soya-bean oil) for 12 days, crossed-over to a monounsaturated fatty-acid-rich oil (rapeseed oil) for the next 11 days, then returned to soya-bean oil for 11 more days. Additional rats were fed either soya-bean oil or rapeseed oil throughout. Rats fed rapeseed oil had lower rates of ATPase-catalysed ATP/[32P]Pi exchange than rats fed soya-bean oil. Arrhenius plots showed higher transition temperature (Tt) and activation energy (Ea) for rats fed rapeseed oil. Switching from soya-bean oil to rapeseed oil was dynamically followed by changes in the thermotropic and kinetic properties of the mitochondrial ATPase exchange reaction. Returning to soya-bean oil reversed these changes. The rapid and reversible modulation of Tt caused by a change of the type of fat ingested suggests that membrane physicochemical properties are not under rigid intrinsic control but are continually modified by the profile of exogenously derived fatty acids. The studies suggest that in vivo the activity of mitochondrial ATPase is in part determined by dietary lipid via its influence on the microenvironment of the enzyme. The rapidity and ready reversibility of changes observed for this subcellular-membrane-bound enzyme suggest that dietary fatty-acid balance may be an important determinant of other membrane functions in the body.     

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg²⁺. The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.     

Synthesis and assembly of flagellar components by Caulobacter crescentus motility mutants

Cultures of wild-type Caulobacter crescentus and strains with fla mutations representing 24 genes were pulse-labeled with 14C-amino acids and analyzed by immunoprecipitation to study the synthesis of flagellar components. Most fla mutants synthesize flagellin proteins at a reduced rate, suggesting the existence of some mechanism to prevent the accumulation of unpolymerized flagellin subunits. Two strains contain deletions that appear to remove a region necessary for this regulation. The hook protein does not seem to be subject to this type of regulation and, in addition, appears to be synthesized as a faster-sedimenting precursor. Mutations in a number of genes result in the appearance of degradation products of either the flagellin or the hook proteins. Mutations in flaA, -X, -Y, or -Z result in the production of filaments (stubs) that contain altered ratios of the flagellin proteins. In some flaA mutants, other flagellin-related proteins were assembled into the stub structures in addition to the flagellins normally present. Taken together, these analyses have begun to provide insight into the roles of individual fla genes in flagellum biogenesis in C. crescentus.     

Regulation of KinI kinesin ATPase activity by binding to the microtubule lattice

KinI kinesins are important in regulating the complex dynamics of the microtubule cytoskeleton. They are unusual in that they depolymerize, rather than move along microtubules. To determine the attributes of KinIs that distinguish them from translocating kinesins, we examined the ATPase activity, microtubule affinity, and three-dimensional microtubule-bound structure of a minimal KinI motor domain. Together, the kinetic, affinity, and structural data lead to the conclusion that on binding to the microtubule lattice, KinIs release ADP and enter a stable, low-affinity, regulated state, from which they do not readily progress through the ATPase cycle. This state may favor detachment, or diffusion of the KinI to its site of action, the microtubule ends. Unlike conventional translocating kinesins, which are microtubule lattice-stimulated ATPases, it seems that with KinIs, nucleotide-mediated modulation of tubulin affinity is only possible when it is coupled to protofilament deformation. This provides an elegant mechanistic basis for their unique depolymerizing activity.     

Filament assembly and regulation of the actin-activated ATPase activity of thymus myosin

The effects of light chain phosphorylation on the actin-activated ATPase activity and filament assembly of calf thymus cytoplasmic myosin were examined under a variety of conditions. When unphosphorylated and phosphorylated thymus myosins were monomeric, their MgATPase activities were not activated or only very slightly activated by actin, but when they were filamentous, their MgATPase activities were stimulated by actin. The phosphorylated myosin remained filamentous at lower Mg2+ concentrations and higher KC1 concentrations than did the unphosphorylated myosin, and the myosin concentration required for filament assembly was lower for phosphorylated myosin than for unphosphorylated myosin. By varying the myosin concentration, it was possible to have under the same assay conditions mostly monomeric myosin or mostly filamentous myosin; under these conditions, the actin-activated ATPase activities of the filamentous myosins were much greater than those of the monomeric myosins. The addition of phosphorylated myosin to unphosphorylated myosin promoted the assembly of unphosphorylated myosin into filaments. These results suggest that phosphorylation may regulate the actomyosin-based motile activities in vertebrate nonmuscle cells by regulating myosin filament assembly.