Loss of synaptotagmin IV results in a reduction in synaptic vesicles and a distortion of the Golgi structure in cultured hippocampal neurons.

Fusion of synaptic vesicles with the plasma membrane is mediated by the SNARE (soluble NSF attachment receptor) proteins and is regulated by synaptotagmin (syt). There are at least 17 syt isoforms that have the potential to act as modulators of membrane fusion events. Synaptotagmin IV (syt IV) is particularly interesting; it is an immediate early gene that is regulated by seizures and certain classes of drugs, and, in humans, syt IV maps to a region of chromosome 18 associated with schizophrenia and bipolar disease. Syt IV has recently been found to localize to dense core vesicles in hippocampal neurons, where it regulates neurotrophin release. Here we have examined the ultrastructure of cultured hippocampal neurons from wild-type and syt IV -/- mice using electron tomography. Perhaps surprisingly, we observed a potential synaptic vesicle transport defect in syt IV -/- neurons, with the accumulation of large numbers of small clear vesicles (putative axonal transport vesicles) near the trans-Golgi network. We also found an interaction between syt IV and KIF1A, a kinesin known to be involved in vesicle trafficking to the synapse. Finally, we found that syt IV -/- synapses exhibited reduced numbers of synaptic vesicles and a twofold reduction in the proportion of docked vesicles compared to wild-type. The proportion of docked vesicles in syt IV -/- boutons was further reduced, 5-fold, following depolarization.

Phylogenetic conservation of disulfide-linked, dimeric acetylcholine receptor pentamers in southern ocean electric rays.

Intact acetylcholine receptors have been purified on a novel affinity resin from three electric fish endemic to Australian waters. Their binding properties and morphology are compared with those of their northern hemisphere homolog, Torpedo marmorata. All four exhibit apparent dissociation constants, Kd, in the nanomolar range for the snake neurotoxin alpha-bungarotoxin and have a distinctive rosette-like appearance when viewed in negative stain under the electron microscope. Furthermore, these rosettes are paired, indicating that acetylcholine receptors from southern ocean electric fish exist as dimers, in the same fashion as their northern hemisphere counterparts. The cDNAs of the receptor's four subunits were sequenced from Hypnos monopterigium and the northern hemisphere counterpart, Torpedo marmorata, while cDNAs from only two subunits, alpha and delta, were able to be sequenced from Narcine tasmaniensis. The penultimate amino acid in the delta subunit of each of the newly sequenced fish species is a cysteine residue. Its conservation suggests that the mechanism for the observed dimerization of acetylcholine receptors is disulfide bond formation between the delta subunit of adjacent receptors, analogous to acetylcholine receptor dimers observed in other electric fish. It appears that this mechanism for receptor clustering is unique to acetylcholine receptors packed and organized in the specialized organs of electric fish. Alignment of the deduced protein sequences with the equivalent sequences from Torpedo californica and humans reveals a high degree of homology.

GTPase activity of dynamin and resulting conformation change are essential for endocytosis.

Dynamin is a large GTPase with a relative molecular mass of 96,000 (Mr 96K) that is involved in clathrin-mediated endocytosis and other vesicular trafficking processes. Although its function is apparently essential for scission of newly formed vesicles from the plasma membrane, the nature of dynamin's role in the scission process is still unclear. It has been proposed that dynamin is a regulator (similar to classical G proteins) of downstream effectors. Here we report the analysis of several point mutants of dynamin's GTPase effector (GED) and GTPase domains. We show that oligomerization and GTP binding alone, by dynamin, are not sufficient for endocytosis in vivo. Rather, efficient GTP hydrolysis and an associated conformational change are also required. These data argue that dynamin has a mechanochemical function in vesicle scission.

Nucleotide-dependent conformational changes in dynamin: evidence for a mechanochemical molecular spring.

The GTPase dynamin plays an essential part in endocytosis by catalysing the fission of nascent clathrin-coated vesicles from the plasma membrane. Using preformed phosphatidylinositol-4,5-bisphosphate-containing lipid nanotubes as a membrane template for dynamin self-assembly, we investigate the conformational changes that arise during GTP hydrolysis by dynamin. Electron microscopy reveals that, in the GTP-bound state, dynamin rings appear to be tightly packed together. After GTP hydrolysis, the spacing between rings increases nearly twofold. When bound to the nanotubes, dynamin's GTPase activity is cooperative and is increased by three orders of magnitude compared with the activity of unbound dynamin. An increase in the Kcat (but not the K(m) of GTP hydrolysis accounts for the pronounced cooperativity. These data indicate that a novel, lengthwise ('spring-like') conformational change in a dynamin helix may participate in vesicle fission.

Macromolecular structure determination by electron microscopy: new advances and recent results.

Electron microscopy is undergoing a mini-renaissance, as a number of biological systems are yielding to higher resolution analysis as a result of advances in instrumentation, specimen preparation and image-processing technology. The atomic structure of tubulin has now been solved, crucial elements of secondary structure have recently been revealed in several membrane proteins (rhodopsin, gap junctions, aquaporin, and Ca2+ and H+ ATPases) and in a virus particle, and macromolecular complexes are being seen in increasingly fine detail. This growth has been enhanced further by the ability to combine structures of macromolecular complexes derived by electron microscopy with X-ray structures of their components, in order to reconstruct molecular machines and large multiprotein complexes in immense detail.

The functional significance of multimerization in ion channels.

Recently, the allosteric behavior of ion channels has been investigated by recording the individual steps leading to the complete activation of a cyclic nucleotide-gated ion channel. This information, in combination with recent studies on nicotinic acetylcholine receptor mutants, necessitates a modification of our current theories of the allosteric mechanisms of ion channels and gives new insights into the functional significance of multimerization in ion channels.

Electron paramagnetic resonance studies of succinate:ubiquinone oxidoreductase from Paracoccus denitrificans. Evidence for a magnetic interaction between the 3Fe-4S cluster and cytochrome b.

Electron paramagnetic resonance (EPR) studies of succinate:ubiquinone oxidoreductase (SQR) from Paracoccus denitrificans have been undertaken in the purified and membrane-bound states. Spectroscopic "signatures" accounting for the three iron-sulfur clusters (2Fe-2S, 3Fe-4S, and 4Fe-4S), cytochrome b, flavin, and protein-bound ubisemiquinone radicals have been obtained in air-oxidized, succinate-reduced, and dithionite-reduced preparations at 4-10 K. Spectra obtained at 170 K in the presence of excess succinate showed a signal typical of that of a flavin radical, but superimposed with another signal. The superimposed signal originated from two bound ubisemiquinones, as shown by spectral simulations. Power saturation measurements performed on the air-oxidized enzyme provided evidence for a weak magnetic dipolar interaction operating between the oxidized 3Fe-4S cluster and the oxidized cytochrome b. Power saturation experiments performed on the succinate- and dithionite-reduced forms of the enzyme demonstrated that the 4Fe-4S cluster is coupled weakly to both the 2Fe-2S and the 3Fe-4S clusters. Quantitative interpretation of these power saturation experiments has been achieved through redox calculations. They revealed that a spin-spin interaction between the reduced 3Fe-4S cluster and the cytochrome b (oxidized) may also exist. These findings form the first direct EPR evidence for a close proximity (

Light-induced structural changes in photosynthetic reaction center: implications for mechanism of electron-proton transfer.

High resolution x-ray diffraction data from crystals of the Rhodobacter sphaeroides photosynthetic reaction center (RC) have been collected at cryogenic temperature in the dark and under illumination, and the structures were refined at 2.2 and 2.6 angstrom resolution, respectively. In the charge-separated D+QAQB- state (where D is the primary electron donor (a bacteriochlorophyll dimer), and QA and QB are the primary and secondary quinone acceptors, respectively), QB- is located approximately 5 angstroms from the QB position in the charge-neutral (DQAQB) state, and has undergone a 180 degrees propeller twist around the isoprene chain. A model based on the difference between the two structures is proposed to explain the observed kinetics of electron transfer from QA-QB to QAQB- and the relative binding affinities of the different ubiquinone species in the QB pocket. In addition, several water channels (putative proton pathways) leading from the QB pocket to the surface of the RC were delineated, one of which leads directly to the membrane surface.

Redox-dependent structural changes in the nitrogenase P-cluster.

The structure of the nitrogenase MoFe-protein from Azotobacter vinelandii has been refined to 2.0 A resolution in two oxidation states. EPR studies on the crystals indicate that the structures correspond to the spectroscopically assigned oxidized (P(OX)/M(OX)) and the native or dithionite-reduced (P(N)/M(N)) forms of the enzyme. Both MoFe-protein structures are essentially identical, with the exception of the P-cluster. The MoFe-protein P-cluster in each state is found to contain eight Fe and seven S atoms. Interconversion between the two redox states involves movement of two Fe atoms and an exchange of protein coordination for ligands supplied by a central S atom. In the oxidized P(OX) state, the cluster is coordinated by the protein through six cysteine ligands, Ser-beta188 O gamma, and the backbone amide of Cys-alpha88. In the native P(N) state, Ser-beta188 O gamma and the amide N of Cys-alpha88 no longer coordinate the cluster due to movement of their coordinated Fe atoms toward the central sulfur. Consequently, this central sulfur adopts a distorted octahedral environment with six surrounding Fe atoms. A previously described model of the P-cluster containing 8Fe-8S likely reflects the inappropriate modeling of a single structure to a mixture of these two P-cluster redox states. These observed redox-mediated structural changes of the P-cluster suggest a role for this cluster in coupling electron transfer and proton transfer in nitrogenase.

Uncompetitive substrate inhibition and noncompetitive inhibition by 5-n-undecyl-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) and 2-n-nonyl-4-hydroxyquinoline-N-oxide (NQNO) is observed for the cytochrome bo3 complex: implications for a Q(H2)-loop proton translocation mechanism.

The cytochrome bo3 ubiquinol oxidase complex from Escherichia coli contains two binding sites for ubiquinone(ol) (UQ(H2)). One of these binding sites, the ubiquinol oxidation site, is clearly in dynamic equilibrium with the UQ(H2) pool in the membrane. The second site has a high affinity for ubiquinone (UQ), stabilizes a semiquinone species, and is located physically close to the low-spin heme b component of the enzyme. The UQ molecule in this site has been proposed to remain strongly bound to the enzyme during enzyme turnover and to act as a cofactor facilitating the transfer of electrons from the substrate ubiquinol to heme b [Sato-Watanabe et al. (1994) J. Biol. Chem. 269, 28908-28912]. In this paper, the steady-state turnover of the enzyme is examined in the presence and absence of inhibitors (UHDBT and NQNO) that appear to be recognized as ubisemiquinone analogs. It is found that the kinetics are accounted for best by a noncompetitive inhibitor binding model. Furthermore, at high concentrations, the substrates ubiquinol-1 and ubiquinol-2 inhibit turnover in an uncompetitive fashion. Together, these observations strongly suggest that there must be at least two UQ(H2) binding sites that are in rapid equilibrium with the UQ(H2) pool under turnover conditions. Although these data do not rule out the possibility that a strongly bound UQ molecule functions to facilitate electron transfer to heme b, they are more consistent with the behavior expected if the two UQ(H2) binding sites were to function in a Q(H2)-loop mechanism (similar to that of the cytochrome bc1 complex) as originally proposed by Musser and co-workers [(1993) FEBS Lett. 327, 131-136]. In this model, ubiquinol is oxidized at one site and ubiquinone is reduced at the second site. While the structural similarities of the heme-copper ubiquinol and cytochrome c oxidase complexes suggest the possibility that these two families of enzymes translocate protons by similar mechanisms, the current observations indicate that the Q(H2)-loop proton translocation mechanism for the heme-copper ubiquinol oxidase complexes should be further investigated and experimentally tested.

Cytochrome c oxidase: chemistry of a molecular machine.

The plethora of proposed chemical models attempting to explain the proton pumping reactions catalyzed by the CcO complex, especially the number of recent models, makes it clear that the problem is far from solved. Although we have not discussed all of the models proposed to date, we have described some of the more detailed models in order to illustrate the theoretical concepts introduced at the beginning of this section on proton pumping as well as to illustrate the rich possibilities available for effecting proton pumping. It is clear that proton pumping is effected by conformational changes induced by oxidation/reduction of the various redox centers in the CcO complex. It is for this reason that the CcO complex is called a redox-linked proton pump. The conformational changes of the proton pump cycle are usually envisioned to be some sort of ligand-exchange reaction arising from unstable geometries upon oxidation/reduction of the various redox centers. However, simple geometrical rearrangements, as in the Babcock and Mitchell models are also possible. In any model, however, hydrogen bonds must be broken and reformed due to conformational changes that result from oxidation/reduction of the linkage site during enzyme turnover. Perhaps the most important point emphasized in this discussion, however, is the fact that proton pumping is a directed process and it is electron and proton gating mechanisms that drive the proton pump cycle in the forward direction. Since many of the models discussed above lack effective electron and/or proton gating, it is clear that the major difficulty in developing a viable chemical model is not formulating a cyclic set of protein conformational changes effecting proton pumping (redox linkage) but rather constructing the model with a set of physical constraints so that the proposed cycle proceeds efficiently as postulated. In our discussion of these models, we have not been too concerned about which electron of the catalytic cycle was entering the site of linkage, but merely whether an ET to the binuclear center played a role. However, redox linkage only occurs if ET to the activated binuclear center is coupled to the proton pump. Since all of the models of proton pumping presented here, with the exception of the Rousseau expanded model and the Wikström model, have a maximum stoichiometry of 1 H+/e-, they inadequately explain the 2 H+/e- ratio for the third and fourth electrons of the dioxygen reduction cycle (see Section V.B). One way of interpreting this shortfall of protons is that the remaining protons are pumped by an as yet undefined indirectly coupled mechanism. In this scenario, the site of linkage could be coupled to the pumping of one proton in a direct fashion and one proton in an indirect fashion for a given electron. For a long time, it was assumed that at least some elements of such an indirect mechanism reside in subunit III. While recent evidence argues against the involvement of subunit III in the proton pump, subunit III may still participate in a regulatory and/or structural capacity (Section II.E). Attention has now focused on subunits I and II in the search for residues intimately involved in the proton pump mechanism and/or as part of a proton channel. In particular, the role of some of the highly conserved residues of helix VIII of subunit I are currently being studied by site directed mutagenesis. In our opinion, any model that invokes heme alpha 3 or CuB as the site of linkage must propose a very effective means by which the presumedly fast uncoupling ET to the dioxygen intermediates is prevented. It is difficult to imagine that ET over the short distance from heme alpha 3 or CuB to the dioxygen intermediate requires more than 1 ns. In addition, we expect the conformational changes of the proton pump to require much more than 1 ns (see Section V.B).

Shedding light on the use of heat to treat HIV infections.

Considering the lack of effectiveness of current therapies to treat HIV disease, the authors present observations that provide a strong cogent argument for critically designed and meticulously performed clinical trials employing whole body hyperthermia with or without other therapeutic modalities. Only as a result of such clinical trials will it be possible to fairly evaluate the role of hyperthermia as a potential therapy for treatment of chronic HIV infection.

Comparison of ubiquinol and cytochrome c terminal oxidases. An alternative view.

There have been numerous instances in the recent literature where the properties of ubiquinol and cytochrome c terminal oxidases are compared. Here we specifically examine the cytochrome bo3-type ubiquinol oxidase from Escherichia coli and the cytochrome aa3-type cytochrome c oxidases. A second redox-active copper site (CuA) is present only in the cytochrome c oxidases and the physiological electron donors for the two enzymes are different (ubiquinol-8 vs. ferrocytochrome c). In our opinion, these differences are significant and most likely indicate that distinct turnover mechanisms are operative in the two enzymes.