Revisiting the GroEL-GroES reaction cycle via the symmetric intermediate implied by novel aspects of the GroEL(D398A) mutant

The Escherichia coli chaperonin GroEL is a double-ring chaperone that assists in protein folding with the aid of GroES and ATP. It is believed that GroEL alternates the folding-active rings and that the substrate protein (and GroES) can bind to the open trans-ring only after ATP in the cis-ring is hydrolyzed. However, we found that a substrate protein prebound to the trans-ring remained bound during the first ATP cycle, and this substrate was assisted by GroEL-GroES when the second cycle began. Moreover, a slow ATP-hydrolyzing GroEL mutant (D398A) in the ATP-bound form bound a substrate protein and GroES to the trans-ring. The apparent discrepancy with the results from an earlier study (Rye, H. S., Roseman, A. M., Chen, S., Furtak, K., Fenton, W. A., Saibil, H. R., and Horwich, A. L. (1999) Cell 97, 325-338) can be explained by the previously unnoticed fact that the ATP-bound form of the D398A mutant exists as a symmetric 1:2 GroEL-GroES complex (the "football"-shaped complex) and that the substrate protein (and GroES) in the medium is incorporated into the complex only after the slow turnover. In light of these results, the current model of the GroEL-GroES reaction cycle via the asymmetric 1:1 GroEL-GroES complex deserves reexamination.     

The structure of ClpB: a molecular chaperone that rescues proteins from an aggregated state

Molecular chaperones assist protein folding by facilitating their "forward" folding and preventing aggregation. However, once aggregates have formed, these chaperones cannot facilitate protein disaggregation. Bacterial ClpB and its eukaryotic homolog Hsp104 are essential proteins of the heat-shock response, which have the remarkable capacity to rescue stress-damaged proteins from an aggregated state. We have determined the structure of Thermus thermophilus ClpB (TClpB) using a combination of X-ray crystallography and cryo-electron microscopy (cryo-EM). Our single-particle reconstruction shows that TClpB forms a two-tiered hexameric ring. The ClpB/Hsp104-linker consists of an 85 A long and mobile coiled coil that is located on the outside of the hexamer. Our mutagenesis and biochemical data show that both the relative position and motion of this coiled coil are critical for chaperone function. Taken together, we propose a mechanism by which an ATP-driven conformational change is coupled to a large coiled-coil motion, which is indispensable for protein disaggregation.     

Morphometric analysis of photoreceptive,neuronal and endocrinal cell differentiation of avian pineal cells: an in vitro immunohistochemical study on the developmental transition from neuronal to photo-endocrinal property

Little is known about the developmental origin, determination and differentiation of different pineal immunoreactive cells in the avian group, and an experimental establishment is then required to explain the differentiation of cell types (i.e. photosensory, neural and secretory types). The present in vitro study suggests that the avian pineal organ is made up of multiple types of cells with different immunoreactivity at the ontogenic state (from embryonic day 9 to day 14), before it acquires the final photoendocrinal nature of the mature state. The morphometric analysis suggests that the developmental changes in the morphology of the quail pinealocytes appear to represent a condensed expression of the phylogenic development in the ontogeny. Several types of immunoreactive cells from a neuronal line were suppressed with maturation of developing pineal glands, while other cell types such as photoreceptive and endocrinal lines became more prominent. The melatonin level in the culture medium presented a high value up to 72 hr of culture, followed by a decrease as well as dampening of the level at the end of the culture possibly because the cultures were maintained in dark. The results of the present study, a combined analysis of morphometry and RIA, open a new line for research into the pineal development and cell differentiation.     

Regulatory interplay between proton motive force, ADP, phosphate, and subunit epsilon in bacterial ATP synthase

ATP synthase couples transmembrane proton transport, driven by the proton motive force (pmf), to the synthesis of ATP from ADP and inorganic phosphate (P(i)). In certain bacteria, the reaction is reversed and the enzyme generates pmf, working as a proton-pumping ATPase. The ATPase activity of bacterial enzymes is prone to inhibition by both ADP and the C-terminal domain of subunit epsilon. We studied the effects of ADP, P(i), pmf, and the C-terminal domain of subunit epsilon on the ATPase activity of thermophilic Bacillus PS3 and Escherichia coli ATP synthases. We found that pmf relieved ADP inhibition during steady-state ATP hydrolysis, but only in the presence of P(i). The C-terminal domain of subunit epsilon in the Bacillus PS3 enzyme enhanced ADP inhibition by counteracting the effects of pmf. It appears that these features allow the enzyme to promptly respond to changes in the ATP:ADP ratio and in pmf levels in order to avoid potentially wasteful ATP hydrolysis in vivo.     

Change in the developmental fate of the chick optic vesicle from the neural retina to the telencephalon

The forebrain develops into the telencephalon, diencephalon, and optic vesicle (OV). The OV further develops into the optic cup, the inner and outer layers of which develop into the neural retina and retinal pigmented epithelium (RPE), respectively. We studied the change in fate of the OV by using embryonic transplantation and explant culture methods. OVs excised from 10-somite stage chick embryos were freed from surrounding tissues (the surface ectoderm and mesenchyme) and were transplanted back to their original position in host embryos. Expression of neural retina-specific genes, such as Rax and Vsx2 (Chx10), was downregulated in the transplants. Instead, expression of the telencephalon-specific gene Emx1 emerged in the proximal region of the transplants, and in the distal part of the transplants close to the epidermis, expression of an RPE-specific gene Mitf was observed. Explant culture studies showed that when OVs were cultured alone, Rax was continuously expressed regardless of surrounding tissues (mesenchyme and epidermis). When OVs without surrounding tissues were cultured in close contact with the anterior forebrain, Rax expression became downregulated in the explants, and Emx1 expression became upregulated. These findings indicate that chick OVs at stage 10 are bi-potential with respect to their developmental fates, either for the neural retina or for the telencephalon, and that the surrounding tissues have a pivotal role in their actual fates. An in vitro tissue culture model suggests that under the influence of the anterior forebrain and/or its surrounding tissues, the OV changes its fate from the retina to the telencephalon.     

Neural retinal regeneration in the anuran amphibian Xenopus laevis post-metamorphosis: transdifferentiation of retinal pigmented epithelium regenerates the neural retina

In urodele amphibians like the newt, complete retina and lens regeneration occurs throughout their lives. In contrast, anuran amphibians retain this capacity only in the larval stage and quickly lose it during metamorphosis. It is believed that they are unable to regenerate these tissues after metamorphosis. However, contrary to this generally accepted notion, here we report that both the neural retina (NR) and lens regenerate following the surgical removal of these tissues in the anuran amphibian, Xenopus laevis, even in the mature animal. The NR regenerated both from the retinal pigment epithelial (RPE) cells by transdifferentiation and from the stem cells in the ciliary marginal zone (CMZ) by differentiation. In the early stage of NR regeneration (5-10 days post operation), RPE cells appeared to delaminate from the RPE layer and adhere to the remaining retinal vascular membrane. Thereafter, they underwent transdifferentiation to regenerate the NR layer. An in vitro culture study also revealed that RPE cells differentiated into neurons and that this was accelerated by the presence of FGF-2 and IGF-1. The source of the regenerating lens appeared to be remaining lens epithelium, suggesting that this is a kind of repair process rather than regeneration. Thus, we show for the first time that anuran amphibians retain the capacity for retinal regeneration after metamorphosis, similarly to urodeles, but that the mode of regeneration differs between the two orders. Our study provides a new tool for the molecular analysis of regulatory mechanisms involved in retinal and lens regeneration by providing an alternative animal model to the newt, the only other experimental model.     

Coupling of rotation and catalysis in F(1)-ATPase revealed by single-molecule imaging and manipulation

F(1)-ATPase is a rotary molecular motor that proceeds in 120 degrees steps, each driven by ATP hydrolysis. How the chemical reactions that occur in three catalytic sites are coupled to mechanical rotation is the central question. Here, we show by high-speed imaging of rotation in single molecules of F(1) that phosphate release drives the last 40 degrees of the 120 degrees step, and that the 40 degrees rotation accompanies reduction of the affinity for phosphate. We also show, by single-molecule imaging of a fluorescent ATP analog Cy3-ATP while F(1) is forced to rotate slowly, that release of Cy3-ADP occurs at approximately 240 degrees after it is bound as Cy3-ATP at 0 degrees . This and other results suggest that the affinity for ADP also decreases with rotation, and thus ADP release contributes part of energy for rotation. Together with previous results, the coupling scheme is now basically complete.     

Rocky shore turfs dominated by Corallina (Corallinales,Rhodophyta) in northern Japan†

Three intertidal sites dominated by Corallina turfs were investigated in Hokkaido, Japan. The sites (A, B and C) differed in slope, wave exposure and length of time exposed to air during tidal cycles. Monthly samples were analyzed for frond morphology and other features. Site A, the most wave-exposed site, was dominated by Corallina sp. X, an unknown species, and sites B and C by Corallina pilulifera Postels et Ruprecht. At the different sites the populations differed in conceptacle abundance, coverage by epiphytic Titanoderma corallinae (P. Crouan et H. Crouan) Woelkerling, Chamberlain et Silva, amount of contained sediment, numbers of axes per quadrat, numbers of branch fusions, branch entanglement, frond dryweight, frond length, amount of adventitious branching, numbers of epiphytes (exclusive of T. corallinae), and numbers of animal species. Ninety-one animal species were recorded from the turfs. Corallina is affected morphologically by conditions inherent in its microhabitat, including desiccation, epiphyte loading and the abundance of herbivores.     

Roles of the two ATP binding sites of ClpB from Thermus thermophilus.

As a member of molecular chaperone Hsp100/Clp family, TClpB from Thermus thermophilus has two nucleotide binding domains, NBD1 and NBD2, in a single polypeptide, each containing WalkerA and WalkerB consensus motifs. To probe their roles, mutations were introduced into the WalkerA or WalkerB motifs of each or both of the NBDs. The results are as follows. 1) For each of the NBDs, the ability of nucleotide binding is lost by mutations in the WalkerA motif but is retained by mutations in the WalkerB motif. 2) Each NBD has a casein-stimulatable small basic ATPase activity that is lost when the WalkerB motif is mutated. 3) TClpB assembles into a uniform 580-kDa oligomer when ATP is present at 55 degrees C, and only the mutants in the WalkerA motif in NBD1 fail to assemble, indicating that ATP binding to NBD1 but not hydrolysis is necessary and sufficient for the assembly. 4) Chaperone function of TClpB was lost when the WalkerA motif in each of the NBDs was mutated. Mutants in the WalkerB motifs of each NBD retained some chaperone activity.     

Reconstitution in vitro of V1 complex of Thermus thermophilus V-ATPase revealed that ATP binding to the A subunit is crucial for V1 formation

Vacuolar-type H(+)-ATPase (V-ATPase or V-type ATPase) is a multisubunit complex comprised of a water-soluble V(1) complex, responsible for ATP hydrolysis, and a membrane-embedded V(o) complex, responsible for proton translocation. The V(1) complex of Thermus thermophilus V-ATPase has the subunit composition of A(3)B(3)DF, in which the A and B subunits form a hexameric ring structure. A central stalk composed of the D and F subunits penetrates the ring. In this study, we investigated the pathway for assembly of the V(1) complex by reconstituting the V(1) complex from the monomeric A and B subunits and DF subcomplex in vitro. Assembly of these components into the V(1) complex required binding of ATP to the A subunit, although hydrolysis of ATP is not necessary. In the absence of the DF subcomplex, the A and B monomers assembled into A(1)B(1) and A(3)B(3) subcomplexes in an ATP binding-dependent manner, suggesting that ATP binding-dependent interaction between the A and B subunits is a crucial step of assembly into V(1) complex. Kinetic analysis of assembly of the A and B monomers into the A(1)B(1) heterodimer using fluorescence resonance energy transfer indicated that the A subunit binds ATP prior to binding the B subunit. Kinetics of binding of a fluorescent ADP analog, N-methylanthraniloyl ADP (mant-ADP), to the monomeric A subunit also supported the rapid nucleotide binding to the A subunit.