TRAP1 and the proteasome regulatory particle TBP7/Rpt3 interact in the endoplasmic reticulum and control cellular ubiquitination of specific mitochondrial proteins

Tumor necrosis factor receptor-associated protein-1 (TRAP1) is a mitochondrial (MITO) antiapoptotic heat-shock protein. The information available on the TRAP1 pathway describes just a few well-characterized functions of this protein in mitochondria. However, our group's use of mass-spectrometric analysis identified TBP7, an AAA-ATPase of the 19S proteasomal subunit, as a putative TRAP1-interacting protein. Surprisingly, TRAP1 and TBP7 colocalize in the endoplasmic reticulum (ER), as demonstrated by biochemical and confocal/electron microscopic analyses, and interact directly, as confirmed by fluorescence resonance energy transfer analysis. This is the first demonstration of TRAP1's presence in this cellular compartment. TRAP1 silencing by short-hairpin RNAs, in cells exposed to thapsigargin-induced ER stress, correlates with upregulation of BiP/Grp78, thus suggesting a role of TRAP1 in the refolding of damaged proteins and in ER stress protection. Consistently, TRAP1 and/or TBP7 interference enhanced stress-induced cell death and increased intracellular protein ubiquitination. These experiments led us to hypothesize an involvement of TRAP1 in protein quality control for mistargeted/misfolded mitochondria-destined proteins, through interaction with the regulatory proteasome protein TBP7. Remarkably, expression of specific MITO proteins decreased upon TRAP1 interference as a consequence of increased ubiquitination. The proposed TRAP1 network has an impact in vivo, as it is conserved in human colorectal cancers, is controlled by ER-localized TRAP1 interacting with TBP7 and provides a novel model of the ER-mitochondria crosstalk.     

Molecular Events Initiating Exit of a Copper-transporting ATPase ATP7B from the Trans-Golgi Network

The copper-transporting ATPase ATP7B has a dual intracellular localization: the trans-Golgi network (TGN) and cytosolic vesicles. Changes in copper levels, kinase-mediated phosphorylation, and mutations associated with Wilson disease alter the steady-state distribution of ATP7B between these compartments. To identify a primary molecular event that triggers ATP7B exit from the TGN, we characterized the folding, activity, and trafficking of the ATP7B variants with mutations within the regulatory N-terminal domain (N-ATP7B). We found that structural changes disrupting the inter-domain contacts facilitate ATP7B exit from the TGN. Mutating Ser-340/341 in the N-ATP7B individually or together to Ala, Gly, Thr, or Asp produced active protein and shifted the steady-state localization of ATP7B to vesicles, independently of copper levels. The Ser340/341G mutant had a lower kinase-mediated phosphorylation under basal conditions and no copper-dependent phosphorylation. Thus, negative charges introduced by copper-dependent phosphorylation are not obligatory for ATP7B trafficking from the TGN. The Ser340/341A mutation did not alter the overall fold of N-ATP7B, but significantly decreased interactions with the nucleotide-binding domain, mimicking consequences of copper binding to N-ATP7B. We propose that structural changes that specifically alter the inter-domain contacts initiate exit of ATP7B from the TGN, whereas increased phosphorylation may be needed to maintain an open interface between the domains.     

Maintenance of Golgi structure and function depends on the integrity of ER export.

The Golgi apparatus comprises an enormous array of components that generate its unique architecture and function within cells. Here, we use quantitative fluorescence imaging techniques and ultrastructural analysis to address whether the Golgi apparatus is a steady-state or a stable organelle. We found that all classes of Golgi components are dynamically associated with this organelle, contrary to the prediction of the stable organelle model. Enzymes and recycling components are continuously exiting and reentering the Golgi apparatus by membrane trafficking pathways to and from the ER, whereas Golgi matrix proteins and coatomer undergo constant, rapid exchange between membrane and cytoplasm. When ER to Golgi transport is inhibited without disrupting COPII-dependent ER export machinery (by brefeldin A treatment or expression of Arf1[T31N]), the Golgi structure disassembles, leaving no residual Golgi membranes. Rather, all Golgi components redistribute into the ER, the cytoplasm, or to ER exit sites still active for recruitment of selective membrane-bound and peripherally associated cargos. A similar phenomenon is induced by the constitutively active Sar1[H79G] mutant, which has the additional effect of causing COPII-associated membranes to cluster to a juxtanuclear region. In cells expressing Sar1[T39N], a constitutively inactive form of Sar1 that completely disrupts ER exit sites, Golgi glycosylation enzymes, matrix, and itinerant proteins all redistribute to the ER. These results argue against the hypothesis that the Golgi apparatus contains stable components that can serve as a template for its biogenesis. Instead, they suggest that the Golgi complex is a dynamic, steady-state system, whose membranes can be nucleated and are maintained by the activities of the Sar1-COPII and Arf1-coatomer systems.     

Role of parthenogenetic natality and emergence from diapausing eggs in the dynamics of some rotifer populations

There are few quantitative data on the role of emergence from diapausing eggs in population dynamics of natural populations of zooplankton species; to our knowledge, all these concern copepods and cladocerans. We present here direct estimates of emergence from bottom resting eggs for another important category of freshwater zooplankton, namely rotifers. Three populations of rotifers of the genus Brachionus were studied in a lake. During the study period 10 population increases, each corresponding to an individual sampling interval, were detected. For each interval, emergence from immediately hatching, parthenogenetic eggs calculated on the basis of the Edmondson-Paloheimo model and emergence from diapausing bottom eggs determined in short-term experiments were estimated and compared to each other. We found that three of the population increases observed are entirely explained by parthenogenetic natality. In contrast, emergence from diapausing eggs can, on its own, account for none of population increases. For two population increases, however, it accounts for that part of population growth which remains unexplained by the parthenogenetic natality. For rotifer populations studied, emergence from diapausing eg eggs is generally less important than parthenogenetic births, when both are regarded as an immediate cause of population growth. This is in sharp contrast to the data available for some crustaceans (De Stasio, 1990) where the role of emergence from diapausing eggs in population dynamics has been clearly shown.     

Spectrum and territorial distribution of hereditary diseases in the population of the Krasnodar Krai]

The analysis of the spectrum of hereditary diseases in the population of the Krasnodar province is performed and the influence of the population dynamics factors on the spectrum is discussed. More than 130 nosological forms were discovered in the population of approx. 200,000. Among these, there are 63 autosomal dominant, 49 autosomal recessive and 17 X-linked recessive forms. Of the most frequent autosomal dominant diseases (more than 1 per 50,000) autosomal recessive and X-linked recessive disorders 13, 7 and 7 forms, respectively, were picked up. The coefficient of diversity of hereditary diseases (the number of nosological forms per 10 inhabitants) with different types of inheritance is higher in the Krasnodar population, as compared with the Kostroma population. The problem of similarity of the "nucleus" of autosomal-recessive disorders in Russian populations is discussed.     

The structure of postsynaptic densities isolated from dog cerebral cortex: I. overall morphology and protein composition

A postsynaptic density (PSD) fraction, including some adherent subsynaptic web material, has been isolated from dog cerebral cortex by a short-procedure modification of methods of Davis and Bloom (21, 22) and Cotman and Taylor (20), using Triton X-100. The fraction has been visualized by thin-section, replica, and negative (phosphotungstic acid) staining electron microscopy and its proteins separated by high-resoltuion SDS gel electrophoresis. Morphologically, the preparation seems to be quite pure, with very little membrane contamination. The density is composed of protein, no nuclei acids, and very little phospholipids being detectable. The fraction had no ATPase or GTPase activity, but it did have a very small amount of cytochrome c oxidase activity (of a specific activity less than 0.5 percent that of a mitochondrial fraction) and a small amount of 5'- nucleotidase activity (of a specific activity between 6 and 7 percent that of a synaptic membrane fraction). Electron micrographs reveal cup-shaped structures approximately 400nm long and approximately 40nm wide, made up of apparent particles 13-28nm in diameter. However, en face views, and particularly micrographs of replicas and PTA-stained preparations, reveal a disk-shaped structure, outside diameter approximately 400 nm, in which filaments are seen to extend from the central part of the density. High resolution gel electrophoresis studies indicated some 15 major proteins and perhaps 10 or more minor ones; the predominant protein had a mol wt of 51,000, followed by ones at 45,000, 40,000, 31,000, 26,000, and several at 100,000. A comparison by gel electrophoresis of density fraction proteins with those of a lysed synaptosomal membrane fraction containing some adherent densities indicated some comigrating proteins, but the major membrane fraction protein, mol wt 52,000, was not found in the density fraction. Antibodies raised against the density fraction reacted with a preparation of solubilized synaptic membrane proteins. By both these criteria, it was considered that the density and the synaptic membrane have some proteins in common. By separately mixing (125)I-labeled myelin, synaptic vesicle, and mitochondrial fraction proteins with synaptosomes, and then isolating the density fraction from the mixture, it was concluded that a major 26,000 mol wt density fraction protein was common to both mitochondria and density, that none of the proteins of the density were contaminants from the mitochondrial fraction, that a minor approximately 150,000 band was a contaminant from the synaptic vesicle fraction, and that the moderately staining PSD fraction protein of 17,000 mol wt band was the result of contamination by the major basic protein of myelin. On the basis of the marker enzymatic assays and the mixing experiments, it is considered that the density fraction is moderately pure biochemically, and that its protein composition, aside from a few exceptions noted above, reflects its in situ character.     

The structure of postsynaptic densities isolated from dog cerebral cortex: II. characterization and arrangement of some of the major proteins within the structure

An attempt was made to identify some of the proteins of the postsynaptic density (PSD) fraction isolated from dog cerebral cortex. The major protein has been tentatively labeled "neurofilament" protein, on the basis of its 51,000 mol wt correspondence to a protein found in neurofilament preparations. Other proteins are akin to some dog myofibrillar proteins, on the basis if immunological crossreaction and equal sodium dodecyl sulfate (SDS)-gel electrophoretic mobilities. While a protein similar to dog muscle myosin is not present in the PSD fraction, a major protein present is actin, as evident from reactivity with antiactin serum, from SDS-gel mobility, and from amino acid composition. Only very little tubulin may be present in the PSD fraction, as determined by gel electrophoresis. Various treatments of the PSD fraction were attempted in order to extract some proteins, as revealed by gel electrophoresis, and to observe the structural changes of the PSD fraction residue after extraction of these proteins. The PSD is remarkably resistant to various extraction conditions, with only 4 M guanidine being found to extract most of the proteins, except the 51,000 mol wt protein. Disulfide reducing agents such as dithiothreitol (DTT), blocking agents such as p-chloromercuribenzoate (PCMB) (both in the presence of deoxycholate [DOC]), a Ca++ extractor, ethylene glycol-bis (beta- aminoethyl ether) N,N,N',N'-tetraacetate (EGTA), and guanidine caused an opening up of the native dense PSD structure, revealing approximately 10-nm filaments, presumably consisting of "neurofilament" protein. Both DTT-DOC and PCMB-DOC removed chiefly actin but also some other proteins. EGTA, in greatly opening up the structure, as observed in the electron microscope, revealed both 10-nm and 3- to 5-nm filaments; the later could be composed of actin, since actin was still in the residue after the treatment. EGTA removed a major 18,000 mol wt component and two minor proteins of 68,000 and 73,000 mol wt. Based on the morphological and biochemical evidence, a picture is presented of the PSD as a structure partly made up of 10-nm and 3- to 5-nm filaments, held together through Ca++ interaction and by bonds amendable to breakage by sulfhydrylblocking and disulfide-reducing reagents; either removal of Ca++ and/or rupture of these disulfide bonds opens up the structure. On the basis of the existence of filamentous proteins and the appearance of the PSD after certain treatments as a closed or open structure, a theory is presented with envisages the PSD to function as a modulator in the conduction of the nerve impulse, by movements of its protein relative.