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The TIM22 protein import pathway of the yeast mitochondrion contains several components, including a family of five proteins (Tim8p, -9p, -10p, -12p, and -13p [Tim, for translocase of inner membrane]) that are located in the intermembrane space and are 25% identical. Tim9p and Tim10p have dual roles in mediating the import of inner membrane proteins. Like the Tim8p-Tim13p complex, the Tim9p-Tim10p complex functions as a putative chaperone to guide hydrophobic precursors across the intermembrane space. Like membrane-associated Tim12p, they are members of the Tim18p-Tim22p-Tim54p membrane complex that mediates precursor insertion into the membrane. To understand the role of this family in protein import, we have used a genetic approach to manipulate the complement of the small Tim proteins. A strain has been constructed that lacks the 70-kDa soluble Tim8p-Tim13p and Tim9p-Tim10p complexes in the intermembrane space. Instead, a functional version of Tim9p (Tim9(S67C)p), identified as a second-site suppressor of a conditional tim10 mutant, maintains viability. Characterization of this strain revealed that Tim9(S67C)p and Tim10p were tightly associated with the inner membrane, the soluble 70-kDa Tim8p-Tim13p and Tim9p-Tim10p complexes were not detectable, and the rate of protein import into isolated mitochondria proceeded at a slower rate. An arrested translocation intermediate bound to Tim9(S67C)p was located in the intermembrane space, associated with the inner membrane. We suggest that the 70-kDa complexes facilitate import, similar to the outer membrane receptors of the TOM (hetero-oligomeric translocase of the outer membrane) complex, and the essential role of Tim9p and Tim10p may be to mediate protein insertion in the inner membrane with the TIM22 complex.
The primary subdivisions of the telencephalon of a characidian, Astyanax hubbsi, were extensively described and this subsequent study elucidates the organization of the secondary olfactory system based on horseradish peroxidase (HRP) and carbocyanine dye (DiI) tracing techniques. Both methods yielded similar results. Two fiber bundles project from the olfactory bulbs into the ventral rostral telencephalon: the medial olfactory tract (tom), embedded in the white matter of the ventral telencephalon (V), which gives rise to a dorsal (tom-d) and a ventral (tom-v) branch, and the lateral olfactory tract (tol). Running caudally, fibers of the tom-v synapse both ipsi- and contralaterally, after decussation in the ventral part of the anterior commissure, identified as the interbulbar commissure of Goldstein, in the medial terminal field of V, and in the preoptic terminal field. The dorsal branch (tom-d) projects bilaterally to a complex pattern of terminal fields, including the medial terminal field in V, the central terminal field in the dorsomedial forebrain (Dm), the caudal part of the lateral terminal field in the dorsoposterior telencephalon (Dp), and, finally, a hypothalamic terminal field at the lateral edge of the posterior nucleus tuberis. In addition, some fibers of tom-d and tom-v reach into the contralateral olfactory bulb. Here, both nerve terminals and cells were stained in the olfactory nucleus. The lateral olfactory tract (tol) runs along the lateral edge of the external sulcus forming a horizontal band. Extensive terminals were identified bilaterally in the lateral terminal field, which uniformly covers the medial and caudal parts of the dorsal hemispheres (Dc, Dl and Dp). Fibers decussate prominently in the dorsal part of the anterior commissure. A small number of projections, which join the stria medullaris, protrude into the diencephalon, decussate in the habenular commissure, and turn rostrally back into the telencephalon to synapse in the caudal part of the contralateral terminal field. These features resemble a simple bauplan of olfactory fiber connections in actinopterygian brains and further indicate that substantial parts of the telencephalon are not dominated by olfactory inputs.
Most mitochondrial proteins are nuclear-encoded and synthesised as preproteins on polysomes in the cytosol. They must be targeted to and translocated into mitochondria. Newly synthesised preproteins interact with cytosolic factors until their recognition by receptors on the surface of mitochondria. Import into or across the outer membrane is mediated by a dynamic protein complex coined the translocase of the outer membrane (TOM). Preproteins that are imported into the matrix or inner membrane of mitochondria require the action of one of two translocation complexes of the inner membrane (TIMs). The import pathway of preproteins is predetermined by their intrinsic targeting and sorting signals. Energy input in the form of ATP and the electrical gradient across the inner membrane is required for protein translocation into mitochondria. Newly imported proteins may require molecular chaperones for their correct folding.
Novel, very stable ruthenium and osmium containing terminal phosphinidene complexes [(eta(6)-Ar)(L)M=Mes*] (Ar=benzene, p-cymene; L=PR(3), CO, and RNC) have been prepared by dehydrohalogenation of novel [(eta(6)-Ar)MX(2)(PH(2)Mes*)] complexes in the presence of a stabilizing ligand. Xray crystal structures are reported for [(eta(6)-C(6)H(6))(PPh(3))Rud=PMes*] (9) and [(eta(6)-pCy)(PPh(3))Os=PMes*] (4). Dehydrohalogenation in the absence of a stabilizing ligand resulted in the new P-spiroannulated Ru(2)P(2)-ring structure 16. Dehydrohalogenation in the presence of but-2-yne gave a novel phosphaallyl complex [(eta(6)-Ar)Ru(eta(3)-R(2)PC(Me)CHMe)] 26, for which an X-ray crystal structure is reported. The mechanism by which 16 and 26 are obtained is presumed to involve the intermediate formation of the 16-electron (eta(6)-benzene)Rud=PMes* phosphinidene complex.
Many techniques have been described for the challenging problem of extensor mechanism rupture, including the use of synthetic grafts. In 1994 Fujikawa et al. [J. Bone Surg. 1994:76B:200-203] reported excellent results using the Leeds-Keio polyester ligament. Patients were mobilised early, had few post operative complications and minimal extensor lag. This is the first independent assessment of this technique using both subjective and objective analysis of outcome. We reviewed 11 patients (12 knees) whose extensor mechanisms were repaired as described in the original paper. At a mean of 3 years after surgery there were no infections and no re-ruptures. Good functional recovery was observed with a mean Tegner activity score of 1.7, Lysholm score of 66 and Irrgang score of 58%. Indeed, 80% of working patients returned to their former employment. However, objective tests showed peak extensor torque recovered to only half normal strength. We conclude the results of this technique of repair compare favourably with other described techniques especially in complex cases.
The preprotein translocase of the outer mitochondrial membrane (Tom) is a multi-subunit complex required for specific recognition and membrane translocation of nuclear-encoded preproteins. We have expressed and purified the cytosolic domains of three postulated import receptors, Tom20, Tom22, and Tom70. Each receptor domain is able to bind mitochondrial preproteins but with different specificity. Tom20 binds both preproteins with N-terminal presequences and preproteins with internal targeting signals; the binding is enhanced by the addition of salt. Tom22 selectively recognizes presequence-carrying preproteins in a salt-sensitive manner. Tom70 preferentially binds preproteins with internal targeting information. A chemically synthesized presequence peptide competes with preproteins for binding to Tom20 and Tom22 but not to Tom70. We conclude that each of the three import receptors binds preproteins independently and by a different mechanism. Both Tom20 and Tom22 function as presequence receptors.
Proteins destined for all submitochondrial compartments are translocated across the outer mitochondrial membrane by the TOM (translocase of the outer membrane) complex, which consists of a number of specialized receptor subunits that bind mitochondrial precursor proteins for delivery into the translocation channel. One receptor, Tom70, binds large, hydrophobic mitochondrial precursors. The current model of Tom70-mediated import involves multiple dimers of the receptor recognizing a single molecule of substrate. Here we show via a battery of biophysical and spectroscopic techniques that the cytosolic domain of Tom70 is an elongated monomer. Thermal and urea-induced denaturation revealed that the receptor, which unfolds via a multistate pathway, is a relatively unstable molecule undergoing major conformational change at physiological temperatures. The data suggest that the malleability of the monomeric Tom70 receptor is an important factor in mitochondrial import.
The mitochondrion has developed an elaborate translocation system for the import of nuclear-coded proteins and the export of proteins coded on the mitochondrial genome. Precursor proteins contain targeting and sorting information to reach the mitochondrion, whereas the translocons recognize the information and direct the precursor to the correct compartment. The outer membrane contains the TOM (translocase of the outer membrane) complex for translocation and the SAM (sorting and assembly machinery) complex for assembly of outer membrane proteins with complex topologies. At the inner membrane, the TIM23 (translocase of the inner membrane) mediates the import of mitochondrial proteins with a typical N-terminal targeting sequence, and the TIM22 complex mediates the import of polytopic inner membrane proteins. Based on its prokaryotic origin, the inner membrane also contains several components that mediate the export and assembly of proteins from within the matrix. Together the translocation and assembly complexes coordinate assembly of the mitochondrion.
We report an optimized synthesis of all canonical 2'-O-TOM protected ribonucleoside phosphoramidites and solid supports containing [13C5]-labeled ribose moieties, their sequence-specific introduction into very short RNA sequences and their use for the structure determination of two protein-RNA complexes. These specifically labeled sequences facilitate RNA resonance assignments and are essential to assign a high number of sugar-sugar and intermolecular NOEs, which ultimately improve the precision and accuracy of the resulting structures. This labeling strategy is particularly useful for the study of protein-RNA complexes with single-stranded RNA in solution, which is rapidly an increasingly relevant research area in biology.
c-Myb and the C/EBP family are transcriptional regulatory factors that act in concert to regulate the expression of myeloid-specific genes. v-Myb encoded by avian myeloblastosis virus (AMV) is a mutated form of c-Myb that contains point mutations which disrupt the cooperation with C/EBPs. To understand the mechanism of the transcriptional synergy between c-Myb and C/EBPs and the effect of the v-Myb mutations on that synergy, knowledge based on their three-dimensional structures is essential. Crystals of ternary complexes, in which various combinations of the DNA-binding domains of c-Myb or v-Myb and C/EBPalpha or C/EBPbeta are bound to a DNA fragment from tom-1A promoter, were obtained by the vapour-diffusion method. Complete diffraction data sets were obtained from each native crystal and two types of iodine-derivative crystals. A three-wavelength MAD data set was also obtained from a bromine-derivative crystal.
In plants, as in most eukaryotic cells, import of nuclear-encoded cytosolic tRNAs is an essential process for mitochondrial biogenesis. Despite its broad occurrence, the mechanisms governing RNA transport into mitochondria are far less understood than protein import. This article demonstrates by Northwestern and gel-shift experiments that the plant mitochondrial voltage-dependent anion channel (VDAC) protein interacts with tRNA in vitro. It shows also that this porin, known to play a key role in metabolite transport, is a major component of the channel involved in the tRNA translocation step through the plant mitochondrial outer membrane, as supported by inhibition of tRNA import into isolated mitochondria by VDAC antibodies and Ruthenium red. However VDAC is not a tRNA receptor on the outer membrane. Rather, two major components from the TOM (translocase of the outer mitochondrial membrane) complex, namely TOM20 and TOM40, are important for tRNA binding at the surface of mitochondria, suggesting that they are also involved in tRNA import. Finally, we show that proteins and tRNAs are translocated into plant mitochondria by different pathways. Together, these findings identify unexpected components of the tRNA import machinery and suggest that the plant tRNA import pathway has evolved by recruiting multifunctional proteins.
Recent research suggests that the tendency of paranoid individuals to attribute negative events to external personal factors and positive events to internal factors may be associated with difficulties in understanding the mental states of others, referred to as theory of mind (ToM). The present study aimed to investigate the relationship of these factors with attributional complexity and motivation. In an analogue sample, a relative ToM impairment was associated with an increased tendency to attribute negative events to external personal factors but not with differences in attributional complexity. This indicates that paranoid tendencies and mentalizing deficits are not associated with attributional simplicity, but may be related to schematic patterns of cognition.
The mitochondrial outer membrane contains a protein complex with at least eight subunits responsible for recognition and translocation of preproteins synthesized in the cytosol. Two subunits, the receptors Tom20 and Tom70, contain tetratrico peptide repeats that are thought to be involved in protein-protein interactions. We have identified Saccharomyces cerevisiae Tom72, a new Tom protein expressed at a low level. Tom72 is homologous to Tom 70, including seven tetratrico peptide repeats. Tom72 is targeted to the mitochondrial outer membrane, forms a large domain exposed to the cytosol and loosely associates with the translocase complex of the outer membrane. These results suggest that Tom72 represents a ninth, weakly expressed component of the preprotein translocase of the mitochondrial outer membrane.
Proteomic techniques provide new tools for the global analysis of protein profiles but also for the investigation of specific protein functions. The analysis of signaling cascades has traditionally been performed by the determination of enzymatic or transcription factor activities representing a certain pathway. Functional proteomics now allows more comprehensive approaches to study cellular responses induced during ligand/receptor interactions. In this study we evaluated proteomic strategies for the investigation of structure-function relationships in the erythropoietin receptor signalling complex. After expression of epidermal growth factor/erythropoietin receptor mutant molecules in an identical cellular background we characterized their potential to induce cellular activities. Using this system we focused our efforts on post-translational modifications of signalling proteins reflecting a substantial part of receptor-dependent signaling events. Although tyrosine phosphorylated proteins were enriched by immunoprecipitation the analysis using the classical approach combining two-dimensional gel electrophoresis and identification by matrix assisted laser desorption/ionization-time of flight-mass spectrometry revealed that low expressed signaling proteins cannot be detected by this technique. An alternative strategy using one-dimensional gel separation of phosphoproteins and liquid chromatography-tandem mass spectrometry, however, allowed us to identify multiple proteins involved in intracellular signalling representing already established pathways but also proteins which have not been linked to EPO-induced signaling so far. This approach offers the potential to extend functional proteomic studies to complex signaling processes.
Vesicle dispersions are useful for many applications from medicinal to consumer products. However, using these dispersions requires some knowledge of and control over their colloidal properties. Measuring interparticle interactions between vesicles should allow framing the problem in terms of Smoluchowski kinetic models and consequently anticipating time-dependent aggregation and coalescence for the dispersions. However, this can be a difficult task for many complex mixtures. A primary goal of this paper is to show that it is possible to measure interparticle potential between small vesicles by measuring the concentration-dependent collective motion using dynamic light scattering. These measurements allow determination of the second virial coefficient for the dispersion, providing a convenient platform for summing all contributions to the interaction potential over all vesicle conformations, thus making the analysis of complex mixtures more tractable. As a verification of the approach, a comparison is made to dispersions in which the stability is governed solely by electrostatics, using existing techniques to anticipate instabilities. A second goal of this paper is to build a simple potential model in which the Smoluchowski model can be used to quantitatively anticipate the aggregation behavior of the small vesicle dispersion. Together, these observations constitute a convenient approach to anticipating the behavior of vesicle (and other) dispersions in complex mixtures.