Characterization of caspase processing and activation in HL-60 cell cytosol under cell-free conditions. Nucleotide requirement and inhibitor profile.

The present studies compared caspase activation under cell-free conditions in vitro and in etoposide-treated HL-60 leukemia cells in situ. Immunoblotting revealed that incubation of HL-60 cytosol at 30 degrees C in the presence of cytochrome c and ATP (or dATP) resulted in activation of procaspases-3, -6, and -7 but not -2 and -8. Although similar selectivity was observed in intact cells, affinity labeling revealed that the active caspase species generated in vitro and in situ differed in charge and abundance. ATP and dATP levels in intact HL-60 cells were higher than required for caspase activation in vitro and did not change before caspase activation in situ. Replacement of ATP with the poorly hydrolyzable analogs 5'-adenylyl methylenediphosphate, 5'-adenylyl imidodiphosphate, or 5'-adenylyl-O-(3-thiotriphos-phate) slowed caspase activation in vitro, suggesting that ATP hydrolysis is required. Caspase activation in vitro was insensitive to phosphatase and kinase inhibitors (okadaic acid, staurosporine, and genistein) but was inhibited by Zn(2+), aurintricarboxylic acid, and various protease inhibitors, including 3,4-dichloroisocoumarin, N(alpha)-p-tosyl-L-phenylalanine chloromethyl ketone, N(alpha)-p-tosyl-L-lysine chloromethyl ketone, and N-(N(alpha)-benzyloxycarbonylphenylalanyl)alanine fluoromethyl ketone, each of which inhibited recombinant caspases-3, -6, -7, and -9. Experiments with anti-neoepitope antiserum confirmed that these agents inhibited caspase-9 activation. Collectively, these results suggest that caspase-9 activation requires nucleotide hydrolysis and is inhibited by agents previously thought to affect apoptosis by other means.

Localization of cellubrevin to the Golgi complex in pancreatic acinar cells.

Cellubrevin is the smallest (14 kDa) isoform of the synaptobrevin (VAMP) protein family and is found in a wide variety of tissues. Western blot analysis with a polyclonal antibody against the unique N-terminus of cellubrevin identified a protein of 14 kDa in rat pancreas. This protein distributed predominantly to the particulate fractions from the rat exocrine pancreas and was totally resistant to NaHCO3 washes, indicating that it is an integral membrane protein. Subcellular fractionation of pancreatic homogenates showed enrichment of this protein in the smooth microsomal fraction while negligible amounts were present in the zymogen granule membrane or the rough microsomal membrane fractions. As seen in other tissues, the 14 kDa immunoreactive form was proteolyzed by tetanus toxin. Light and electron microscopic immunocytochemistry localized cellubrevin immunoreactivity primarily to small vesicles and condensing vacuoles originating from the Golgi region, with significantly lower labeling on zymogen granules. Based on the intracellular localization of cellubrevin detected in acinar cells by immunocytochemistry and cell fractionation, we suggest that cellubrevin may be involved in the maturation of secretory granules.

v- and t-SNAREs in neuronal exocytosis: a need for additional components to define sites of release.

Synaptic vesicle recycling is a specialized form of membrane recycling which takes place in all cells between early endosomes and the plasmalemma. Synaptic vesicles exocytosis is highly regulated and occurs only at presynaptic active zones. In contrast, exocytosis of endosome-derived vesicles of the housekeeping recycling pathway takes place constitutively and throughout the cell surface. Since v- and t-SNAREs play a key role in membrane interactions leading to fusion, unique v- and t-SNAREs may be implicated in synaptic vesicle exocytosis. It was found, however, that the same v-SNAREs of the synaptobrevin family are found both on synaptic vesicles and on endosome-derived vesicles which undergo constitutive fusion. Likewise, t-SNAREs which act as plasmalemmal receptors for synaptic vesicles are not restricted to synaptic active zones. Thus, v- and t-SNAREs interactions may define which organelles can fuse with the plasmalemma, but require additional components to define properties of the exocytotic reaction which are specific for distinct classes of secretory organelles.

rbSec1A and B colocalize with syntaxin 1 and SNAP-25 throughout the axon, but are not in a stable complex with syntaxin.

rbSec1 is a mammalian neuronal protein homologous to the yeast SEC1 gene product which is required for exocytosis. Mutations in Sec1 homologues in the nervous systems of C. elegans and D. melanogaster lead to defective neurotransmitter secretion. Biochemical studies have shown that recombinant rbSec1 binds syntaxin 1 but not SNAP-25 or synaptobrevin/VAMP, the two proteins which together with syntaxin 1 form the synaptic SNARE complex. In this study we have examined the subcellular localization of rbSec1 and the degree of interaction between rbSec1 and syntaxin 1 in situ. rbSec1, which we show here to be represented by two alternatively spliced isoforms, rbSec1A and B, has a widespread distribution in the axon and is not restricted to the nerve terminal. This distribution parallels the localization of syntaxin 1 and SNAP-25 along the entire axonal plasmalemma. rbSec1 is found in a soluble and a membrane-associated form. Although a pool of rbSec1 is present on the plasmalemma, the majority of membrane-bound rbSec1 is not associated with syntaxin 1. We also show that rbSec1 is not part of the synaptic SNARE complex or of the syntaxin 1/SNAP-25 complex we show to be present in non-synaptic regions of the axon. Thus, in spite of biochemical studies demonstrating the high affinity interaction of rbSec1 and syntaxin 1, our results indicate that rbSec1 and syntaxin 1 are not stably associated. They also suggest that the function of rbSec1, syntaxin 1, and SNAP-25 is not restricted to synaptic vesicle exocytosis at the synapse.

Cellubrevin and synaptobrevins: similar subcellular localization and biochemical properties in PC12 cells.

There is strong evidence to indicate that proteins of the synaptobrevin family play a key role in exocytosis. Synaptobrevin 1 and 2 are expressed at high concentration in brain where they are localized on synaptic vesicles. Cellubrevin, a very similar protein, has a widespread tissue distribution and in fibroblasts is localized on endosome-derived, transferin receptor-positive vesicles. Since brain cellubrevin is not detectable in synaptic vesicles, we investigated whether cellubrevin and the synaptobrevins are differentially targeted when co-expressed in the same cell. We report that in the nervous system cellubrevin is expressed at significant levels only by glia and vascular cells. However, cellubrevin is coexpressed with the two synaptobrevins in PC12 cells, a neuroendocrine cell line which contains synaptic vesicle-like microvesicles. In PC12 cells, cellubrevin has a distribution very similar to that of synaptobrevin 1 and 2. The three proteins are targeted to neurites which exclude the transferrin receptor and are enriched in synaptic-like microvesicles and dense-core granules. They are recovered in the synaptic-like microvesicle peak of glycerol velocity gradients, have a similar distribution in isopycnic fractionation and are coprecipitated by anti-synaptobrevin 2 immunobeads. Finally, cellubrevin, like the synaptobrevins, interact with the neuronal t-SNAREs syntaxin 1 and SNAP-25. These results suggest that cellubrevin and the synaptobrevins have similar function and do not play a specialized role in constitutive and regulated exocytosis, respectively.

Synapsin IIa bundles actin filaments.

Synapsins are neuron-specific phosphoproteins associated with small synaptic vesicles in the presynaptic nerve terminal. Synapsin I, which has been demonstrated to bundle F-actin in vitro, has been postulated to regulate neurotransmitter release by cross-linking synaptic vesicles to the actin cytoskeleton. To investigate the possible interaction of synapsin II with actin filaments, we expressed synapsin II in Spodoptera frugiperda and High Five insect cells using a recombinant baculovirus. Purified recombinant synapsin IIa was incubated with F-actin, and bundle formation was evaluated by light scattering and electron microscopy. Synapsin IIa was found to bundle actin filaments. Dose-response curves indicated that synapsin IIa was more potent than synapsin I in bundling actin filaments. These data suggest that synapsin IIa may cross-link synaptic vesicles and actin filaments in the nerve terminal.

Interaction of Grb2 via its Src homology 3 domains with synaptic proteins including synapsin I.

Grb2 is a 25-kDa adaptor protein composed of a Src homology 2 (SH2) domain and two flanking Src homology 3 (SH3) domains. One function of Grb2 is to couple tyrosine-phosphorylated proteins (through its SH2 domain) to downstream effectors (through its SH3 domains). Using an overlay assay, we have identified four major Grb2-binding proteins in synaptic fractions. These proteins interact with wild-type Grb2 but not with Grb2 containing point mutations in each of its two SH3 domains corresponding to the loss of function mutants in the Caenorhabditis elegans Grb2 homologue sem-5. Two of the proteins, mSos and dynamin, were previously shown to bind Grb2. The third protein of 145 kDa is brain specific and to our knowledge has not been previously described. The fourth protein is synapsin I. Dynamin is required for synaptic vesicle endocytosis and synapsin I is thought to mediate the interaction of synaptic vesicles with the presynaptic cytomatrix. These data suggest that Grb2, or other proteins containing SH3 domains, may play a role in the regulation of the exo/endocytotic cycle of synaptic vesicles and therefore of neurotransmitter release.

Tetanus toxin-mediated cleavage of cellubrevin impairs exocytosis of transferrin receptor-containing vesicles in CHO cells.

Cellubrevin is a member of the synaptobrevin/VAMP family of SNAREs, which has a broad tissue distribution. In fibroblastic cells it is concentrated in the vesicles which recycle transferrin receptors but its role in membrane trafficking and fusion remains to be demonstrated. Cellubrevin, like the synaptic vesicle proteins synaptobrevins I and II, can be cleaved by tetanus toxin, a metallo-endoprotease which blocks neurotransmitter release. However, nonneuronal cells are unaffected by the toxin due to lack of cell surface receptors for its heavy chain. To determine whether cellubrevin cleavage impairs exocytosis of recycling vesicles, we tested the effect of tetanus toxin light chain on the release of preinternalized transferrin from streptolysin-O-perforated CHO cells. The release was found to be temperature and ATP dependent as well as NEM sensitive. Addition of tetanus toxin light chain, but not of a proteolytically inactive form of the toxin, resulted in a partial inhibition of transferrin release which correlated with the toxin-mediated cleavage of cellubrevin. The residual release of transferrin occurring after complete cellubrevin degradation was still ATP dependent. Our results indicate that cellubrevin plays an important role in the constitutive exocytosis of vesicles which recycle plasmalemma receptors. The incomplete inhibition of transferrin release produced by the toxin suggests the existence of a cellubrevin-independent exocytotic mechanism, which may involve tetanus toxin-insensitive proteins of the synaptobrevin/VAMP family.

Synapsin IIa: expression in insect cells, purification, and characterization.

Synapsin IIa belongs to a family of neuron-specific phosphoproteins called synapsins, which are associated with synaptic vesicles in presynaptic nerve terminals. In order to examine the biochemical properties of synapsin IIa, and ultimately its physiological function, purified protein is required. Since attempts to purify significant quantities of synapsin IIa, an isoform of the synapsins, from mammalian brain have proven difficult, we undertook the production of recombinant synapsin IIa by utilizing the baculovirus expression system. Rat synapsin IIa cDNA was introduced into the baculovirus genome via homologous recombination, and the recombinant baculovirus was purified. Spodoptera frugiperda (Sf9) cells infected with this virus expressed synapsin IIa as 5% of the total cellular protein. The recombinant protein was extracted from the particulate fraction of the infected Sf9 cells with salt and a nonionic detergent and purified by immunoaffinity chromatography. The purified synapsin IIa was phosphorylated by the catalytic subunit of cAMP-dependent protein kinase to a stoichiometry of 0.8 mol of phosphate/mol of protein. Metabolic labeling with [32P]Pi demonstrated synapsin IIa phosphorylation in infected Sf9 cells. Using a homogenate of uninfected Sf9 cells, a cAMP-dependent protein kinase activity which can phosphorylate synapsin IIa was detected. Limited proteolysis of recombinant synapsin IIa phosphorylated in vitro and in vivo resulted in identical phosphopeptide maps. Further, synapsin IIa, like synapsin I, binds with high affinity in a saturable manner to synaptic vesicles purified from rat cortex.

Synapsin I regulates glutamate release from rat brain synaptosomes.

Introduction of the dephosphorylated from of synapsin I into rat brain synaptosomes using freeze-thaw (transient) permeabilization significantly decreased the K(+)-induced release of glutamate. In contrast, introduction of synapsin I that had been phosphorylated by Ca2+/calmodulin-dependent protein kinase II was without effect on glutamate release. Addition of dephosphosynapsin I after freeze-thaw treatment also had no effect. Thus, the action of synapsin I was dependent on the phosphorylation state of synapsin I and on its entry into the synaptosomes. Our results implicate synapsin I as an important component in the regulation of neurotransmitter release in the mammalian nervous system.

Phosphorylation of Tetrahymena 22 S dynein.

Studies involving 32P labeling and wet ashing of isolated dynein reveal that isolated dynein contains approximately 6 mol of phosphate predominantly distributed over four polypeptides of molecular masses of 78, 76, 47, and 23 kDa. Dynein must, therefore, be phosphorylated to at least this extent in vivo. The catalytic subunit of cAMP-dependent protein kinase and an axonemal cAMP-dependent protein kinase contaminating the dynein preparation can further phosphorylate dynein in vitro. Each kinase can place up to 0.5 mol of phosphate on native dynein polypeptides of molecular masses of 78 and 34 kDa. Removal of two of the phosphates on isolated dynein by either acid or alkaline phosphatase results in a 28% decrease in the specific activity of dynein in the presence or absence of microtubules. Selective attenuation of the microtubule-activated ATPase, but not the uncoupled free dynein ATPase, would be indicative of a regulatory function of the phosphates. The in vivo regulation of the dynein ATPase by the two phosphates accessible to acid or alkaline phosphatase is therefore subject to question. Other phosphates on dynein must be examined for their effect on the microtubule-dynein cross-bridge cycle and motility before phosphorylation can definitively be established as a mode of dynein regulation.

Dynein structure and function.

The structure of dynein isolated from several sources follows the pattern first observed with Tetrahymena 22S dynein, which has three globular heads attached by three flexible strands to a root-like base. Recent biochemical data indicate that there is one ATPase site on each dynein head and that all three heads interact with microtubules in an ATP-sensitive manner. Accordingly, images of dynein in situ can be interpreted in terms of a model for crossbridge action where the roots of the bouquet anchor the dynein to the A-tubule and all three heads reach out to interact with the B-tubule in an ATP-dependent reaction to produce a force for sliding.