Inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3-L1 adipocytes.

Lovastatin treatment caused down-regulation of the insulin-responsive glucose transporter 4 (Glut4) and up-regulation of Glut1 in 3T3-L1 adipocytes. These changes in protein expression were associated with a marked inhibition of insulin-stimulated glucose transport. Lovastatin had no effect on cell cholesterol levels, but its effects were reversed by mevalonate, demonstrating that inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3-L1 adipocytes. These findings support the notion that whole body insulin resistance may arise as a result of perturbations in general biochemical pathways, rather than primary defects in insulin signalling.

Phosphorylation of cysteine string protein by protein kinase A. Implications for the modulation of exocytosis.

Cyclic AMP-dependent protein kinase (PKA) enhances regulated exocytosis in neurons and most other secretory cells. To explore the molecular basis of this effect, known exocytotic proteins were screened for PKA substrates. Both cysteine string protein (CSP) and soluble NSF attachment protein-alpha (alpha-SNAP) were phosphorylated by PKA in vitro, but immunoprecipitation of cellular alpha-SNAP failed to detect (32)P incorporation. In contrast, endogenous CSP was phosphorylated in synaptosomes, PC12 cells, and chromaffin cells. In-gel kinase assays confirmed PKA to be a cellular CSP kinase, with phosphorylation occurring on Ser(10). PKA phosphorylation of CSP reduced its binding to syntaxin by 10-fold but had little effect on its interaction with HSC70 or G-protein subunits. Furthermore, an in vivo role for Ser(10) phosphorylation at a late stage of exocytosis is suggested by analysis of chromaffin cells transfected with wild type or non-phosphorylatable mutant CSP. We propose that PKA phosphorylation of CSP could modulate the exocytotic machinery, by selectively altering its availability for protein-protein interactions.

SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis.

Lipid rafts are microdomains present within membranes of most cell types. These membrane microdomains, which are enriched in cholesterol and glycosphingolipids, have been implicated in the regulation of certain signal transduction and membrane traffic pathways. To investigate the possibility that lipid rafts organize exocytotic pathways in neuroendocrine cells, we examined the association of proteins of the exocytotic machinery with rafts purified from PC12 cells. The target soluble N-ethylmaleimide-sensitive factor attachment protein receptor (tSNARE) proteins syntaxin 1A and synaptosomal-associated protein of 25 kDa (SNAP-25) were both found to be highly enriched in lipid rafts ( approximately 25-fold). The vesicle SNARE vesicle-associated membrane protein (VAMP)2 was also present in raft fractions, but the extent of this recovery was variable. However, further analysis revealed that the majority of VAMP2 was associated with a distinct class of raft with different detergent solubility characteristics to the rafts containing syntaxin 1A and SNAP-25. Interestingly, no other studied secretory proteins were significantly associated with lipid rafts, including SNARE effector proteins such as nSec1. Chemical crosslinking experiments showed that syntaxin1A/SNAP-25 heterodimers were equally present in raft and nonraft fractions, whereas syntaxin1A/nSec1 complexes were detected only in nonraft fractions. SDS-resistance assays revealed that raft-associated syntaxin1A/SNAP-25 heterodimers were able to interact with VAMP2. Finally, reduction of cellular cholesterol levels decreased the extent of regulated exocytosis of dopamine from PC12 cells. The results described suggest that the interaction of SNARE proteins with lipid rafts is important for exocytosis and may allow structural and spatial organization of the secretory machinery.

The synaptic vesicle protein, cysteine-string protein, is associated with the plasma membrane in 3T3-L1 adipocytes and interacts with syntaxin 4.

Adipocytes and muscle cells play a major role in blood glucose homeostasis. This is dependent upon the expression of Glut4, an insulin-responsive facilitative glucose transporter. Glut4 is localised to specialised intracellular vesicles that fuse with the plasma membrane in response to insulin stimulation. The insulin-induced translocation of Glut4 to the cell surface is essential for the maintenance of optimal blood glucose levels, and defects in this system are associated with insulin resistance and type II diabetes. Therefore, a major focus of recent research has been to identify and characterise proteins that regulate Glut4 translocation. Cysteine-string protein (Csp) is a secretory vesicle protein that functions in presynaptic neurotransmission and also in regulated exocytosis from non-neuronal cells. We show that Csp1 is expressed in 3T3-L1 adipocytes and that cellular levels of this protein are increased following cell differentiation. Combined fractionation and immunofluorescence analyses reveal that Csp1 is not a component of intracellular Glut4-storage vesicles (GSVs), but is associated with the adipocyte plasma membrane. This association is stable, and not affected by either insulin stimulation or chemical depalmitoylation of Csp1. We also demonstrate that Csp1 interacts with the t-SNARE syntaxin 4. As syntaxin 4 is an important mediator of insulin-stimulated GSV fusion with the plasma membrane, this suggests that Csp1 may play a regulatory role in this process. Syntaxin 4 interacts specifically with Csp1, but not with Csp2. In contrast, syntaxin 1A binds to both Csp isoforms, and actually exhibits a higher affinity for the Csp2 protein. The results described raise a number of interesting questions concerning the intracellular targeting of Csp in different cell types, and suggest that the composition and synthesis of GSVs may be different from synaptic and other secretory vesicles. In addition, the interaction of Csp1 with syntaxin 4 suggests that this Csp isoform may play a role in insulin-stimulated fusion of GSVs with the plasma membrane.

Cysteine-string protein: the chaperone at the synapse.

Cysteine-string protein (Csp) is a major synaptic vesicle and secretory granule protein first discovered in Drosophila and Torpedo. Csps were subsequently identified from Xenopus, Caenorhabditis elegans, and mammalian species. It is clear from the study of a null mutant in Drosophila that Csp is required for viability of the organism and that it has a key role in neurotransmitter release. In addition, other studies have directly implicated Csp in regulated exocytosis in mammalian neuroendocrine and endocrine cell types, and its distribution suggests a general role in regulated exocytosis. An early hypothesis was that Csp functioned in the control of voltage-gated Ca2+ channels. Csp, however, must have an additional function as a direct regulator of the exocytotic machinery as changes in Csp expression modify the extent of exocytosis triggered directly by Ca2+ in permeabilised cells. Csps possess a cysteine-string domain that is highly palmitoylated and confers membrane targeting. In addition, Csps have a conserved "J" domain that mediates binding to an activation of the Hsp70/ Hsc70 chaperone ATPases. This and other evidence implicate Csps as molecular chaperones in the synapse that are likely to control the correct conformational folding of one or more components of the vesicular exocytotic machinery. Targets for Csp include the vesicle protein VAMP/synaptobrevin and the plasma membrane protein syntaxin 1, the significance of which is discussed in possible models to account for current knowledge of Csp function.

Quantification of SNARE protein levels in 3T3-L1 adipocytes: implications for insulin-stimulated glucose transport.

Insulin-stimulates glucose transport in peripheral tissues by stimulating the movement ('translocation') of a pool of intracellular vesicles containing the glucose transporter Glut4 to the cell surface. The fusion of these vesicles with the plasma membrane results in a large increase in the numbers of Glut4 molecules at the cell surface and a concomitant enhancement of glucose uptake. It is well established that proteins of the VAMP- (synaptobrevin) and syntaxin-families play a fundamental role in the insulin-stimulated fusion of Glut4-containing vesicles with the plasma membrane. Studies have identified key roles for vesicle associated membrane protein-2 (VAMP2) and syntaxin-4 in this event, and more recently have also implicated SNAP-23 and Munc18c in this process. In this study, we have quantified the absolute levels of expression of these proteins in murine 3T3-L1 adipocytes, with the objective of determining the stoichiometry of these proteins both relative to each other and also in comparison with previous estimates of Glut4 levels within these cells. To achieve this, we performed quantitative immunoblot analysis of these proteins in 3T3-L1 membranes compared to known amounts of purified recombinant proteins. Such analyses suggest that in 3T3-L1 adipocytes there are approximately 374,000 copies of syntaxin 4, 1.15 x 10(6) copies of SNAP23, 495,000 copies of VAMP2, 4.3 x 10(6) copies of cellubrevin and 452,000 copies of Munc18c per cell, compared to previous estimates of 280,000 copies of Glut4. Thus, the main SNARE proteins involved in insulin-stimulated Glut4 exocytosis (syntaxin 4 and VAMP2) are expressed in approximately equimolar amounts in adipocytes, whereas by contrast the endosomal v-SNARE cellubrevin is present at approximately 10-fold higher levels and the t-SNARE SNAP-23 is also present in an approximately 3-fold molar excess. The implications of this quantification for the mechanism of insulin-stimulated Glut4 translocation are discussed.

Mutational analysis of cysteine-string protein function in insulin exocytosis.

Cysteine-string proteins (Csps) are vesicle proteins involved in neurotransmission. They contain at least four domains: an N-terminal J-domain which can interact with the chaperone Hsc70, an adjacent linker region, the defining cysteine rich domain and a variable C terminus. As the relevance of these domains for the function of Csps in exocytosis is unknown, we have performed a mutational analysis of Csp domains using insulin release by large dense core vesicles (LDCVs) as a model of regulated exocytosis. All mutants were apparently palmitoylated and their subcellular distribution was similar to endogenous Csp. Point mutations within the highly conserved HPD motif of the J-domain abolished activation of Hsc70. However, these mutations altered the effect of Csp on exocytosis only after additional truncation of the extreme C terminus as found in the Csp splice variant Csp2. Furthermore, the strikingly conserved linker region adjacent to the J-domain was important for Csp function in exocytosis, but not for the activation of Hsc70 ATPase. The effects of Csp wild-type or mutants were preserved in permeabilized cells excluding an effect on transmembrane ion fluxes. These observations demonstrate a functional difference between the two isoforms and suggest a role for the J-domain co-chaperone function as well as for the newly defined linker region in LDCV exocytosis.

Cysteine-string proteins regulate exocytosis of insulin independent from transmembrane ion fluxes.

Cysteine-string proteins (Csps) are vesicle proteins involved in exocytosis of synaptic vesicles in Drosophila and modulation of presynaptic calcium influx. As both the contribution of calcium channel regulation to the role of Csp in exocytosis and a function of Csp outside the nervous system are unknown, we studied its function in endocrine exocytosis from large dense core vesicles (LDCVs) using insulin-secreting pancreatic beta-cells. Csps were expressed in primary and derived beta-cell lines on insulin-containing LDCVs. Suppression of Csp expression reduced not only depolarisation induced insulin release but also exocytosis in permeabilised cells directly stimulated by Ca2+. Thus, Csp is a secretory granule protein and is required for endocrine exocytosis independent of the modulation of transmembrane calcium fluxes.

The cysteine-string domain of the secretory vesicle cysteine-string protein is required for membrane targeting.

The post-translational addition of palmitic acid residues to cysteine-string protein (Csp) was originally thought to form the basis for membrane association of this secretory-vesicle protein. However, subsequent work showed that chemical depalmitoylation of Csp does not result in its release from membranes. We have confirmed these findings and employed [3H]palmitate labelling of PC12 cells to demonstrate that Csp1 remains associated with membranes following the complete removal of palmitic acid residues. Although palmitoylation is not essential for the stable membrane association of Csp, its role in membrane targeting has not been assessed. To examine this, we constructed a Csp mutant protein with seven cysteines replaced by serines in the cysteine-string domain. In contrast to wild-type Csps, this mutant protein was not targeted to membranes when expressed in PC12 or HeLa cells. We conclude that although a palmitoylated cysteine-string domain is not required for stable membrane association of Csp, it is essential for initial membrane targeting.

Cysteine string protein functions directly in regulated exocytosis.

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPgammaS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.

Activation of the ATPase activity of heat-shock proteins Hsc70/Hsp70 by cysteine-string protein.

DnaJ proteins are characterized by a 'J' domain which is homologous to a region of the Escherichia coli protein DnaJ. DnaJ has been shown to interact with the chaperone protein DnaK, and a number of eukaryotic DnaJ-like proteins have been found to interact with the 70 kDa heat-shock protein/70 kDa heat-shock cognate protein (Hsp70/Hsc70), the eukaryotic homologues of DnaK. Cysteine-string proteins (Csps) are believed to function in calcium-stimulated exocytosis and in this paper we describe a specific ATP-dependent interaction between a Csp (Csp1) and Hsc70/Hsp70. We also show that Csp1 can stimulate the ATPase activity of both Hsc70 and Hsp70 several-fold. Furthermore, we demonstrate that Csp2, a Csp variant found in adrenal chromaffin cells, can enhance the ATPase activity of Hsc70 to a similar extent as Csp1, whereas Csp(137-198), a truncated protein lacking the 'J' domain of Csp1 is unable to stimulate the ATPase activity of Hsc70. This suggests that the functions of Csp1 and Csp2 must differ in some aspect other than interaction with Hsc70. This study is also important from a general view of DnaJ/Hsc70 interactions, as Csps lack a G/F-rich region which has been suggested to be essential for activation of the ATPase activity of DnaK by DnaJ. Thus, this work would imply that a G/F-rich region is not an essential feature of DnaJ proteins for stimulation of the ATPase activity of Hsp70 proteins.

The molecular chaperone function of the secretory vesicle cysteine string proteins.

The "J" domains of eukaryotic DnaJ-like proteins specify interaction with various Hsp70s. The conserved tripeptide, HPD, present in all J domains has been shown to be important for the interaction between yeast and bacterial DnaJ/Hsp70 protein pairs. We have characterized mutations in the HPD motif of the synaptic vesicle protein cysteine-string protein (Csp). Mutation of the histidine (H43Q) or aspartic acid (D45A) residues of this motif reduced the ability of Csp to stimulate the ATPase activity of mammalian Hsc70. The H43Q and D45A mutant proteins were not able to stimulate the ATPase activity of Hsc70 to any significant extent. The mutant proteins were characterized by competition assays, tryptic digestion analysis, and direct binding analysis from which it was seen that these proteins were defective in binding to Hsc70. Thus, the HPD motif of Csp is required for binding to Hsc70. We also analyzed the interaction between Csp and a model substrate protein, denatured firefly luciferase. Both Csp1 and the C-terminally truncated isoform Csp2 were able to prevent aggregation of heat-denatured luciferase, and they also cooperated with Hsc70 to prevent aggregation. In addition, complexes of Csp1 or Csp2 with Hsc70 and luciferase were isolated, confirming that these proteins interact and that Csps can bind directly to denatured proteins. Csp1 and Csp2 isoforms must differ in some aspect other than interaction with Hsc70 and substrate protein. These results show that both Csp1 and Csp2 can bind a partially unfolded protein and act as chaperones. This suggests that Csps may have a general chaperone function in regulated exocytosis.

Cysteine string proteins are associated with chromaffin granules.

In this work, we have examined the subcellular distribution of cysteine string proteins (Csps) in bovine adrenal medullary chromaffin cells. Csps did not leak from digitonin-permeabilized chromaffin cells, suggesting that there is no cytosolic pool of the protein in these cells. Subcellular fractionation studies confirmed that there was essentially no Csp immunoreactivity in the cytosolic fraction. However, immunoreactivity was detected in the membrane fractions of these cells. Csp immunoreactivity codistributed with dopamine beta-hydroxylase, a granule marker protein, in sucrose gradient-separated granule fractions. Immunofluorescence studies showed that all chromaffin cells in culture were stained with a punctate appearance consistent with a granular localization. These results were confirmed by immunogold labeling, which demonstrated specific labeling of chromaffin granule membranes. In addition to its presence on synaptic vesicles, cysteine string protein is therefore a bona fide chromaffin granule membrane protein.

Identification of a novel cysteine string protein variant and expression of cysteine string proteins in non-neuronal cells.

Cysteine string proteins (Csps) are synaptic vesicle proteins thought to be involved in calcium-dependent neurotransmitter release at nerve endings. Here, we report the cloning of two Csp variants, termed Csp1 and Csp2, from bovine adrenal medullary chromaffin cells. The bovine Csp1 appears to be the homologue of rat brain Csp, sharing 95% identity at the amino acid level. The nucleotide sequence of csp2 is identical with that of csp1 except for a 72-base insert which introduces a stop codon into the coding sequence, which would be predicted to result in a truncated protein 3.3 kDa smaller than Csp1. Furthermore, polymerase chain reaction analysis detected homologues of Csp1 and Csp2 in rat kidney, liver, pancreas, spleen, lung, and adrenal gland. Expression of Csps in non-neuronal tissues was confirmed by Northern blotting and by immunoblotting with anti-Csp1 antiserum which also demonstrated expression of both full-length and truncated Csps in spleen. The widespread tissue distribution is inconsistent with a role of Csps as specific regulators of presynaptic calcium channels as previously proposed. We suggest that Csps may have a more general role in membrane traffic in non-neuronal as well as neuronal cells.

Distinct effects of alpha-SNAP, 14-3-3 proteins, and calmodulin on priming and triggering of regulated exocytosis.

We have used stage-specific assays for MgATP-dependent priming and for Ca(2+)-activated triggering in the absence of free MgATP to examine the effects of alpha-SNAP, 14-3-3 proteins and calmodulin on regulated exocytosis in permeabilized adrenal chromaffin cells. All three proteins lead to a Ca(2+)-dependent increase in catecholamine secretion. Both alpha-SNAP and 14-3-3 proteins stimulated in a priming but not in a triggering assay. In contrast, calmodulin was stimulatory in triggering but not priming. The effects of alpha-SNAP and 14-3-3 proteins were likely to be due to distinct mechanisms of action since they differed in Ca(2+)-dependency, time course and extent of stimulation and their effects were additive. alpha-SNAP and 14-3-3 proteins did not appear to exert their priming action through changes in synthesis of phosphatidylinositol (4,5) bisphosphate. The data show that these three proteins have distinct stage-specific actions on exocytosis and indicate that alpha-SNAP acts in an early MgATP-requiring stage and not in the late Ca(2+)-triggered steps immediately prior to membrane fusion as previously suggested.