Biogenesis of the protein storage vacuole crystalloid.

We identify new organelles associated with the vacuolar system in plant cells. These organelles are defined biochemically by their internal content of three integral membrane proteins: a chimeric reporter protein that moves there directly from the ER; a specific tonoplast intrinsic protein; and a novel receptor-like RING-H2 protein that traffics through the Golgi apparatus. Highly conserved homologues of the latter are expressed in animal cells. In a developmentally regulated manner, the organelles are taken up into vacuoles where, in seed protein storage vacuoles, they form a membrane-containing crystalloid. The uptake and preservation of the contents of these organelles in vacuoles represents a unique mechanism for compartmentalization of protein and lipid for storage.

The protein storage vacuole: a unique compound organelle.

Storage proteins are deposited into protein storage vacuoles (PSVs) during plant seed development and maturation and stably accumulate to high levels; subsequently, during germination the storage proteins are rapidly degraded to provide nutrients for use by the embryo. Here, we show that a PSV has within it a membrane-bound compartment containing crystals of phytic acid and proteins that are characteristic of a lytic vacuole. This compound organization, a vacuole within a vacuole whereby storage functions are separated from lytic functions, has not been described previously for organelles within the secretory pathway of eukaryotic cells. The partitioning of storage and lytic functions within the same vacuole may reflect the need to keep the functions separate during seed development and maturation and yet provide a ready source of digestive enzymes to initiate degradative processes early in germination.

Autoantibodies to glutamate receptor GluR3 in Rasmussen's encephalitis.

Rasmussen's encephalitis is a progressive childhood disease of unknown cause characterized by severe epilepsy, hemiplegia, dementia, and inflammation of the brain. During efforts to raise antibodies to recombinant glutamate receptors (GluRs), behaviors typical of seizures and histopathologic features mimicking Rasmussen's encephalitis were found in two rabbits immunized with GluR3 protein. A correlation was found between the presence of Rasmussen's encephalitis and serum antibodies to GluR3 detected by protein immunoblot analysis and by immunoreactivity to transfected cells expressing GluR3. Repeated plasma exchanges in one seriously ill child transiently reduced serum titers of GluR3 antibodies, decreased seizure frequency, and improved neurologic function. Thus, GluR3 is an autoantigen in Rasmussen's encephalitis, and an autoimmune process may underlie this disease.

PEST sequences and regulation by proteolysis.

In 1986, we proposed that polypeptide sequences enriched in proline (P), glutamic acid (E), serine (S) and threonine (T) target proteins for rapid destruction. For much of the past decade there were only sporadic experimental tests of the hypothesis. This situation changed markedly during the past two years with a number of papers providing strong evidence that PEST regions do, in fact, serve as proteolytic signals. Here, we briefly review the properties of PEST regions and some interesting examples of the conditional nature of such signals. Most of the article, however, focuses on recent experimental support for the hypothesis and on mechanisms responsible for the rapid degradation of proteins that contain PEST regions.

Exploring dinosaur neuropaleobiology: viewpoint computed tomography scanning and analysis of an Allosaurus fragilis endocast.

The unique opportunity to examine an exceptionally well-preserved natural endocranial cast (endocast) from a carnivorous dinosaur of the late Jurassic period, Allosaurus fragilis, was afforded this neurobiologist. The endocast exhibits numerous surface features including the complete vestibular apparatus. Spiral computed tomography scanning revealed multiple internal features including putative blood vessels, connective tissue-like arrays, and a prominent symmetrical density consistent with the putative brain or its cast. The evidence suggests that this organism's neurobiology resembled closely that of modern crocodylian species and should be included for consideration when examining ideas of Allosaurus evolution, behavior, and eventual extinction.

Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site.

Two rabbits immunized with a portion of glutamate receptor (GluR) subunit GluR3 (amino acids 245-457) exhibited seizure-like behaviors, suggesting that antibodies to GluR3 may modulate neuronal excitability. Using whole-cell recording, rabbit GluR3 antisera were tested on cultured fetal mouse cortical neurons. In a subset of kainate-responsive neurons, miniperfusion of antisera and IgG evoked currents that were blocked by CNQX. Immunoreactivity to synthetic peptides prepared to subregions GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395) was present in both rabbit sera. Peptide GluR3B, but not GluR3A, specifically blocked antisera- and IgG-evoked currents. Similar receptor activation and anti-GluR3 reactivity was present in sera from patients with active Rasmussen's encephalitis, an intractable pediatric epilepsy. Thus, antibodies to GluR3 define a region involved in agonist binding and specific receptor activation. These data suggest that antibodies to neuronal receptors can function as agonists and that autoantibodies to GluRs may be highly specific neurotoxicants in some neurological diseases.

Selective RNA editing and subunit assembly of native glutamate receptors.

RNA editing and subunit assembly of ionotropic glutamate receptors (GluRs) were examined in an oligodendrocyte progenitor cell line, CG4, which expresses GluR2-GluR4, GluR6, GluR7, KA1, and KA2. AMPA-evoked currents rapidly desensitize, whereas kainate-evoked currents contain a steady-state component with a nearly linear current-voltage relation and a fast desensitizing component that is inwardly rectifying. The Q/R site is edited > 95% to the arginine codon in GluR2(Q607) mRNA, and < 5% in GluR6(Q621) mRNA. Immunoprecipitation experiments demonstrate that GluR6 and/or GluR7 subunits assemble with KA2, but not with GluR2-GluR4. These results indicate that oligodendrocyte progenitor cells selectively edit and assemble glutamate receptors into at least two functionally and structurally distinct heteromeric channels.

Pharmacological and functional diversity of neuronal nicotinic acetylcholine receptors.

Recent molecular cloning studies have identified several genes encoding alpha and beta subunits of the nicotinic acetylcholine receptor. These genes have distinct, although overlapping, patterns of expression in the brain and peripheral ganglia. Multiple nicotinic receptors with distinct pharmacological and functional properties can be made in oocytes by pairwise combination of different alpha-type subunits with different beta-type subunits. Both alpha and beta subunits contribute to the pharmacological and functional diversity. Evan Deneris and colleagues explain how oocyte expression studies, in concert with immunological and electrophysiological analysis in vivo, are beginning to reveal the subunit compositions of different neuronal nicotinic receptor subtypes.

RNA editing (Q/R site) and flop/flip splicing of AMPA receptor transcripts in young and old brains.

The effects of aging on the efficiency of RNA processing of AMPA glutamate receptor (GluR) subunits GluR1, GluR2, and GluR 3 was examined for RNA editing at the Q/R site of GluR2 and for alternative splicing of the flip or flop exons for GluR1-3. RNA isolated from six young (3 months old) and old (22-23 months old) animals was reverse-transcribed for PCR and restriction endonuclease analyses to distinguish between edited forms of GluR2 and flip/flop isoforms of GluR1-3. Unedited transcripts of GluR2 at the Q/R site (which controls calcium permeability) were not detected (at the limit of detection of >/= 2.5%) from the corticies and hippocampi of young and old animals. Distribution of flop/flip isoforms in the cortex, hippocampus, hypothalamus, and striatum varied between GluR subunits and brain region, with GluR2 showing the greatest differences. However, no differences in alternative splicing of GluRs 1-3 were observed between young and old animals, suggesting that the fidelity of GluR transcript processing remains intact in the brains of aged animals.

KEKE motifs. Proposed roles in protein-protein association and presentation of peptides by MHC class I receptors.

A stretch of 28 'alternating' lysine (K) and glutamate (E) residues is found in an activator of the multicatalytic protease. Such 'KEKE sequences' are also present in subunits of the multicatalytic protease, in subunits of the 26S protease and in a variety of chaperonins. We propose that KEKE regions promote association between protein complexes. Furthermore, they may contribute to the selection of peptides presented on MHC Class I receptors.

Autoantibodies to glutamate receptor subunit GluR2 in nonfamilial olivopontocerebellar degeneration.

We describe a 63-year-old man with a 5-year history of progressive sporadic olivopontocerebellar atrophy (OPCA) who exhibits high serum titers of IgM autoantibodies to the neuronal glutamate receptor subunit GluR2. Immunohistochemistry revealed intense staining of mouse cerebellar Purkinje cells and cells in the pontine nuclei and olivary complex. Glutamate receptor currents were activated in a subset of cultured mouse neurons by an anti-GluR2 IgM fraction, and they were blocked by the competitive AMPA-type glutamate receptor antagonist CNQX and by a synthetic peptide to a specific epitope region of GluR2 (AA 369-393). The patient was treated with nine courses of plasmapheresis with little improvement of symptomatology. However, IgM titers to GluR2 decreased approximately 8-fold and the serum functional activity decreased proportionally. These findings may suggest a role for autoimmunity to glutamate receptors in the pathophysiology of certain forms of progressive nervous system degeneration.

Antibodies prepared to neuronal glutamate receptor subunit3 bind IFNalpha-receptors: implications for an autoimmune process.

Anti-glutamate receptor (GluR) agonist-like antibodies prepared in rabbits that are similar to autoantibodies found in Rasmussen's encephalitis (RE) were found to exhibit greater immunoreactivity towards human interferon alpha receptor (IFNAR-1) than to GluR3. Since antibodies prepared to a defined region of GluR3 can react preferentially to an unrelated sequence in the human IFNAR-1, we propose that the IFNAR-1 may be an heteroclitic antigen of GluR3. These results suggest that differential reactivity toward heteroclitic antigens may contribute to variable clinical characteristics of certain autoimmune diseases.

Organization and quantitative analysis of kainate receptor subunit GluR5-7 immunoreactivity in monkey hippocampus.

A monoclonal antibody specific for GluR5-7 (mAb-4F5) has been used to characterize the distribution of kainate class glutamate receptor subunits in monkey hippocampus. Immunolabeled neurons were present in all subfields of the hippocampus as well as the dentate gyrus and subiculum. Quantitative immunofluorescence analysis by confocal microscopy demonstrated differential levels of immunoreactivity such that the highest intensities were in neurons within CA1 and subiculum as compared with those within CA3 or dentate gyrus. The regional differences in levels of subunit immunoreactivity correlate with the relative vulnerability of hippocampal neurons in several neurodegenerative disorders.

Stimulus complexity and rate of alternation in binocular rivalry.

Pairs of random figures representing four levels of complexity were presented in a binocular-rivalry situation to 12 student volunteer observers. The effect of different levels of complexity on the rate at which complete alternations of the rivalry stimuli occurred was assessed. Rate of alternation was significantly inversely related to the level of stimulus complexity.

Allosaurus, crocodiles, and birds: evolutionary clues from spiral computed tomography of an endocast.

Because the brain does not usually leave direct evidence of its existence in the fossil record, our view of this structure in extinct species has relied upon inferences drawn from comparisons between parts of the skeleton that do fossilize or with modern-day relatives that survived extinction. However, soft-tissue structure preservation may indeed occasionally occur, particularly in the endocranial space. By applying modern imaging and analysis methods to such natural cranial "endocasts," we can now learn more than ever thought possible about the brains of extinct species. I will discuss one such example in which spiral computed tomography (CT) scanning analysis has been successfully applied to reveal preserved internal structures of a naturally occurring endocranial cast of Allosaurus fragilis, the dominant carnivorous dinosaur of the late Jurassic period. The ability to directly examine the neuroanatomy of an extinct dinosaur, whose modern-day relatives are birds and crocodiles, has exciting implications about Allosaurus' behavior, its adaptive responses to its environment, and its eventual extinction.

Degradation of structurally characterized proteins injected into HeLa cells. Basic measurements.

Thirty-five proteins of known x-ray structure were labeled by chloramine-T radioiodination or by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using red cell-mediated microinjection. Degradation rates of the injected proteins were then determined over the next 50 h by measuring the release of soluble isotope to the culture medium. Control experiments demonstrated that the measured rates were not compromised by proteolysis within RBCs, the presence of unfused RBCs, or degradation of protein released from RBCs to the medium. Degradation of some injected proteins was faster during the first 12 h after fusion than at later times, apparently a response of HeLa cells to trypsinization. However, all proteins exhibited first-order degradation rates between 24 and 48 h post injection. Except for seven proteins, stabilities measured during this interval were unaffected by the labeling procedure. Reductive methylation was used to choose among the seven discordant values, and half-lives for the 35 proteins ranged from 16 h for lysozyme to 214 h for yeast alcohol dehydrogenase. Since half-lives for six of the injected proteins closely match values obtained by in vivo measurements, we consider our estimates of the metabolic stabilities of the injected proteins to be generally accurate. Therefore, the half-lives obtained by microinjection should prove useful in the search for relationships between protein structure and intracellular stability.

Degradation of structurally characterized proteins injected into HeLa cells. Effects of intracellular location and the involvement of lysosomes.

Thirty-five proteins of known x-ray structure were radioiodinated and injected into HeLa cells. The cells were then cultured in the presence or absence of the lysosomotropic agents, ammonium chloride and chloroquine. These compounds did not inhibit the degradation of an injected protein unless its half-life was greater than 45 h. Among the more stable proteins the extent of inhibition was proportional to their half-lives. These results indicate that all injected proteins are transferred to lysosomes at comparable rates such that the fraction of a specific protein degraded in lysosomes depends upon its rate of degradation in the cytosol. That is, basal autophagy is nonselective in HeLa cells. The intracellular location of each injected protein was measured by homogenization of injected cells in sucrose and differential sedimentation or by extraction in buffers containing Triton X-100. Solubilities of the injected proteins ranged from 6 to 89%, and stabilities of 10 proteins, originally extracellular in function, were inversely proportional to their solubility. These results illustrate the potential importance of subcellular location on protein stability in the cytosol.

Degradation of structurally characterized proteins injected into HeLa cells. Tests of hypotheses.

We have compared sequence and structural features of 35 proteins to their metabolic stabilities in HeLa cells. No relationship was observed between the half-life of an injected protein and its subunit molecular weight, isoelectric point, hydrophobicity, thermostability, surface charge density, or N-terminal residue. Other properties, including susceptibility to oxidation, specific combinations of amino acids, secondary structure composition, and solvent exposed residues, also failed to correlate with protein stability. Although a weak inverse correlation was obtained when stability was compared to asparagine and glutamine content, we conclude that the degradation of an injected protein is unlikely to be related to any single structural parameter. Rather, we hypothesize that it results from an interplay between subcellular location and still poorly defined surface features of the injected proteins.