Tools to discriminate between targets of CK2 vs PLK2/PLK3 acidophilic kinases.

While the great majority of Ser/Thr protein kinases are basophilic or proline directed, a tiny minority is acidophilic. The most striking example of such "acidophilic" kinases is CK2, whose sites are specified by numerous acidic residues surrounding the target one. However PLK2 and PLK3 kinases recognize an acidic consensus similar to CK2 when tested on peptide libraries. Here we describe optimal buffer conditions for PLK2 and 3 kinase activity assays and tools such as using GTP as a phosphate donor and the specific inhibitors CX-4945 and BI 2536, useful to discriminate between acidic phosphosites generated either by CK2 or by PLK2/PLK3.

TDP-43 accumulation in inclusion body myopathy muscle suggests a common pathogenic mechanism with frontotemporal dementia.

TAR DNA binding protein-43 (TDP-43) is found in ubiquitinated inclusions (UBIs) in some frontotemporal dementias (FTD-U). One form of FTD-U, due to mutations in the valosin containing protein (VCP) gene, occurs with an inclusion body myopathy (IBMPFD). Since IBMPFD brain has TDP-43 in UBIs, we looked for TDP-43 inclusions in IBMPFD muscle. In normal muscle, TDP-43 is present in nuclei. In IBMPFD muscle, TDP-43 is additionally present as large inclusions within UBIs in muscle cytoplasm. TDP-43 inclusions were also found in 78% of sporadic inclusion body myositis (sIBM) muscles. In IBMPFD and sIBM muscle, TDP-43 migrated with an additional band on immunoblot similar to that reported in FTD-U brains. This study adds sIBM and hereditary inclusion body myopathies to the growing list of TDP-43 positive inclusion diseases.

Perinuclear biogenesis of mutant torsin-A inclusions in cultured cells infected with tetracycline-regulated herpes simplex virus type 1 amplicon vectors.

TorsinA is a novel protein identified in the search for mutations underlying the human neurologic movement disorder, early onset torsion dystonia. Relatively little is understood about the normal function of torsinA or the physiological effects of the codon deletion associated with most cases of disease. Overexpression of wild-type torsinA in cultured cells by DNA transfection results in a reticular distribution of immunoreactive protein that co-localizes with endoplasmic reticulum resident chaperones, while the dystonia-related mutant form accumulates within concentric membrane whorls and nuclear-associated membrane stacks. In this study we examined the biogenesis of mutant torsinA-positive membrane inclusions using tetracycline-regulated herpes simplex virus amplicon vectors. At low expression levels, mutant torsinA was localized predominantly around the nucleus, while at high levels it was also concentrated within cytosolic spheroid inclusions. In contrast, the distribution of wild-type torsinA did not vary, appearing diffuse and reticular at all expression levels. These observations are consistent with descriptions of inducible membrane synthesis in other systems in which cytosolic membrane whorls are derived from multilayered membrane stacks that first form around the nuclear envelope. These results also suggest that formation of mutant torsinA-positive inclusions occurs at high expression levels in culture, whereas the perinuclear accumulation of the mutant protein is present even at low expression levels that are more likely to resemble those of the endogenous protein. These nuclear-associated membrane structures enriched in mutant torsinA may therefore be of greater relevance to understanding how the dystonia-related mutation compromises cellular physiology.

Binding of the beta2 adrenergic receptor to N-ethylmaleimide-sensitive factor regulates receptor recycling.

Following agonist stimulation, most G protein-coupled receptors become desensitized and are internalized, either to be degraded or recycled back to the cell surface. What determines the fate of a specific receptor type after it is internalized is poorly understood. Here we show that the rapidly recycling beta2 adrenergic receptor (beta2AR) binds via a determinant including the last three amino acids in its carboxyl-terminal tail to the membrane fusion regulatory protein, N-ethylmaleimide-sensitive factor (NSF). This is documented by in vitro overlay assays and by cellular coimmunoprecipitations. Receptors bearing mutations in any of the last three residues fail to interact with NSF. After stimulation with the agonist isoproterenol, a green fluorescent protein fusion of NSF colocalizes with the wild type beta2AR but not with a tail-mutated beta2AR. The beta2AR-NSF interaction is required for efficient internalization of the receptors and for their recycling to the cell surface. Mutations in the beta2AR tail that ablate NSF binding reduce the efficiency of receptor internalization upon agonist stimulation. Upon subsequent treatment of cells with the antagonist propranolol, wild type receptors return to the cell surface, while tail-mutated receptors remain sequestered. Thus, the direct binding of the beta2AR to NSF demonstrates how, after internalization, the fate of a receptor is reliant on a specific interaction with a component of the cellular membrane-trafficking machinery.

TorsinA: movement at many levels.

TorsinA is the causative protein in the human neurologic disease early onset torsin dystonia, a movement disorder involving dysfunction in the basal ganglia without apparent neurodegeneration. Most cases result from a dominantly acting three-base pair deletion in the TOR1A gene causing loss of a glutamic acid near the carboxyl terminus of torsinA. Torsins are members of the AAA(+) superfamily of ATPases and are present in all multicellular organisms. Initial studies suggest that torsinA is an ER protein involved in chaperone functions and/or membrane movement.

Constitutive calcium-independent release of Toxoplasma gondii dense granules occurs through the NSF/SNAP/SNARE/Rab machinery.

The signals and the molecular machinery mediating release of dense matrix granules from pathogenic protozoan parasites are unknown. We compared the secretion of the endogenous dense granule marker GRA3 in Toxoplasma gondii with the release of a stably transfected foreign reporter, beta-lactamase, that localizes to parasite dense granules. Both proteins were released constitutively in a calcium-independent fashion, as shown using both intact and streptolysin O-permeabilized parasites. N-Ethylmaleimide and recombinant bovine Rab-guanine dissociation inhibitor inhibited beta-lactamase secretion in permeabilized parasites, whereas recombinant hamster N-ethylmaleimide-sensitive fusion protein and bovine alpha-SNAP augmented release. Guanosine 5'-3-O-(thio)triphosphate, but not cAMP, augmented secretion in the presence but not in the absence of ATP. The T. gondii NSF/SNAP/SNARE/Rab machinery participates in dense granule release using parasite protein components that can interact functionally with their mammalian homologues.

Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex.

In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod approximately 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.

Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP.

N-ethylmaleimide-sensitive factor (NSF) is a hexameric ATPase which primes and/or dissociates SNARE complexes involved in intracellular fusion events. Each NSF protomer contains three domains: an N-terminal domain required for SNARE binding and two ATPase domains, termed D1 and D2, with D2 being required for oligomerization. We have determined the 1.9 A crystal structure of the D2 domain of NSF complexed with ATP using multi-wavelength anomalous dispersion phasing. D2 consists of a nucleotide binding subdomain with a Rossmann fold and a C-terminal subdomain, which is structurally unique among nucleotide binding proteins. There are interactions between the ATP moiety and both the neighboring D2 protomer and the C-terminal subdomain that may be important for ATP-dependent oligomerization. Of particular importance are three well-ordered and conserved lysine residues that form ionic interactions with the beta- and gamma-phosphates, one of which likely contributes to the low hydrolytic activity of D2.

The AMPA receptor GluR2 C terminus can mediate a reversible, ATP-dependent interaction with NSF and alpha- and beta-SNAPs.

In this study, we demonstrate specific interaction of the GluR2 alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptor subunit C-terminal peptide with an ATPase N-ethylmaleimide-sensitive fusion protein (NSF) and alpha- and beta-soluble NSF attachment proteins (SNAPs), as well as dendritic colocalization of these proteins. The assembly of the GluR2-NSF-SNAP complex is ATP hydrolysis reversible and resembles the binding of NSF and SNAP with the SNAP receptor (SNARE) membrane fusion apparatus. We provide evidence that the molar ratio of NSF to SNAP in the GluR2-NSF-SNAP complex is similar to that of the t-SNARE syntaxin-NSF-SNAP complex. NSF is known to disassemble the SNARE protein complex in a chaperone-like interaction driven by ATP hydrolysis. We propose a model in which NSF functions as a chaperone in the molecular processing of the AMPA receptor.

A revised model for the oligomeric state of the N-ethylmaleimide-sensitive fusion protein, NSF.

The N-ethylmaleimide-sensitive fusion protein (NSF) is an ATPase that plays an essential role in intracellular membrane trafficking. Previous reports have concluded that NSF forms either a tetramer or a trimer in solution, and that assembly of the oligomer is essential for efficient activity in membrane transport reactions. However, in recent electron microscopic analyses NSF appears as a hexagonal cylinder similar in size to related ATPases known to be hexamers. We have therefore reevaluated NSF's oligomeric state using a variety of quantitative biophysical techniques. Sedimentation equilibrium and sedimentation velocity analytical ultracentrifugation, transmission electron microscopy with rotational image analysis, scanning transmission electron microscopy, and multiangle light scattering all demonstrate that, in the presence of nucleotide, NSF is predominantly a hexamer. Sedimentation equilibrium results further suggest that the NSF hexamer is held together by oligomerization of its D2 domains. The sedimentation coefficient, s20,w0, of 13.4 (+/-0. 1) S indicates that NSF has unusual hydrodynamic characteristics that cannot be solely explained by its shape. The demonstration that NSF is a hexameric oligomer highlights structural similarities between it and several related ATPases which act by switching the conformational states of their protein substrates in order to activate them for subsequent reactions.

Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy.

Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 x 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 x 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with alpha-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.

Acid-induced dissociation of VacA, the Helicobacter pylori vacuolating cytotoxin, reveals its pattern of assembly.

In this study, we describe the ultrastructural changes associated with acid activation of Helicobacter pylori vacuolating cytotoxin (VacA). Purified VacA molecules imaged by deep-etch electron microscopy form approximately 30-nm hexagonal "flowers," each composed of an approximately 15-nm central ring surrounded by six approximately 6-nm globular "petals." Upon exposure to acidic pH, these oligomeric flowers dissociate into collections of up to 12 teardrop-shaped subunits, each measuring approximately 6 x 14 nm. Correspondingly, glycerol density gradient centrifugation shows that at neutral pH VacA sediments at approximately 22 S, whereas at acidic pH it dissociates and sediments at approximately 5 S. Immunoblot and EM analysis of the 5-S material demonstrates that it represents approximately 90-kD monomers with 6 x 14-nm "teardrop" morphology. These data indicate that the intact VacA oligomer consists of 12 approximately 90-kD subunits assembled into two interlocked six-membered arrays, overlap of which gives rise to the flower-like appearance. Support for this interpretation comes from EM identification of small numbers of relatively "flat" oligomers composed of six teardrop-shaped subunits, interpreted to be halves of the complete flower. These flat forms adsorb to mica in two different orientations, corresponding to hexameric surfaces that are either exposed or sandwiched inside the dodecamer, respectively. This view of VacA structure differs from a previous model in which the flowers were interpreted to be single layers of six monomers and the flat forms were thought to be proteolysed flowers. Since acidification has been shown to potentiate the cytotoxic effects of VacA, the present results suggest that physical disassembly of the VacA oligomer is an important feature of its activation.

Neurotransmitter release - four years of SNARE complexes.

Exocytosis in neurons requires proteins known as SNAREs, membrane proteins that have now been implicated in many intracellular fusion events. SNAREs assemble into stable ternary complexes that are dissociated by the ATPase NSF (N-ethylmaleimide-sensitive factor), working together with SNAPs (soluble NSF attachment proteins). Recent results have shed new light on the mechanisms underlying SNARE (SNAP receptor) complex assembly and disassembly, and suggest changes in models that relate these reactions to vesicle docking and fusion.

Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles.

The synaptic membrane proteins synaptobrevin, syntaxin, and SNAP-25 form a ternary complex that can be disassembled by the ATPase N-ethylmaleimide-sensitive factor (NSF) in the presence of soluble cofactors (SNAP proteins). These steps are thought to represent molecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complementary approaches, we now report that such ternary complexes form in the membrane of highly purified and monodisperse synaptic vesicles in the absence of the plasma membrane. Furthermore, the complexes are reversibly dissociated by NSF and SNAP proteins. Thus, ternary complexes can be assembled and disassembled while all three proteins are anchored as neighbors in the same membrane, suggesting that NSF is involved in priming synaptic vesicles for exocytosis.

Ca2+ regulates the interaction between synaptotagmin and syntaxin 1.

While there is compelling evidence that the synaptic vesicle protein synaptotagmin serves as the major Ca2+ sensor for regulated exocytosis, it is not known how Ca2+ binding initiates membrane fusion. Here we report that Ca2+ increases the affinity, by approximately 2 orders of magnitude, between synaptotagmin and syntaxin 1, a component of the synaptic fusion apparatus. This effect is specific for divalent cations which can stimulate exocytosis of synaptic vesicles (Ca2+ > Ba2+, Sr2+ >> Mg2+). The Ca(2+)-dependence of the interaction was composed of two components with EC50 values of 0.7 and 180 microM Ca2+. The interaction is mediated by the carboxyl-terminal region of syntaxin 1 (residues 194-288) and is regulated by a novel Ca(2+)-binding site(s) which does not require phospholipids and is not disrupted by mutations that abolish Ca(2+)-dependent phospholipid binding to synaptotagmin. We propose that this interaction constitutes an essential step in excitation-secretion coupling.

Poisoning by botulinum neurotoxin A does not inhibit formation or disassembly of the synaptosomal fusion complex.

We investigated the effect of poisoning rat brain synaptosomes with botulinum neurotoxin A on the NSF-mediated disassembly of a complex consisting of syntaxin, SNAP-25 and synaptobrevin (fusion complex). Botulinum neurotoxin A specifically removes 9 amino acids from the C-terminus of SNAP-25 and efficiently blocks KCl-evoked glutamate release from synaptosomes. We report that truncated SNAP-25 is incorporated into the fusion complex of poisoned synaptosomes. The presence of truncated SNAP-25 does not interfere with the NSF-induced disassembly of the fusion complex. Also, the release of truncated SNAP-25 from the fusion complex is similar to that of the native SNAP-25. Since neither the formation of the complex nor its disassembly seems to be affected by the SNAP-25 fragment, this fragment is likely to block exocytosis by disrupting events between disassembly of the synaptosomal fusion complex and membrane fusion itself.