The two members of the Staufen family of RNA-binding proteins, Stau1 and Stau2, are present in distinct ribonucleoprotein complexes and associate with different mRNAs. Stau1 is required for protein synthesis -dependent long-term potentiation (L-LTP) in hippocampal pyramidal cells. However, the role of Stau2 in synaptic plasticity remains unexplored. We found that unlike Stau1, Stau2 is not required for L-LTP. In contrast, Stau2, but not Stau1, is necessary for DHPG-induced protein synthesis-dependent long-term depression (mGluR-LTD). While Stau2 is involved in early development of spines, its down-regulation does not alter spine morphology or spontaneous miniature synaptic activity in older cultures where LTD occurs. In addition, Stau2, but not Stau1, knockdown reduces the dendritic localization of Map1b mRNA, a specific transcript involved in mGluR-LTD. Moreover, mGluR stimulation with DHPG induces Map1b, but not Map2, mRNA dissociation from mRNA granules containing Stau2 and the ribosomal protein P0. This dissociation was not observed in cells in which Stau2 was depleted. Finally, Stau2 knockdown reduces basal Map1b protein expression in dendrites and prevents DHPG-induced increases in dendritic Map1b protein level. We suggest a role for Stau2 in the generation and regulation of Map1b mRNA containing granules that are required for mGluR-LTD.

Cysteine string protein (CSP) is an abundant regulated secretory vesicle protein that is composed of a string of cysteine residues, a linker domain, and an Nterminal J domain characteristic of the DnaJ/Hsp40 cochaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca 2؉ channels. To elucidate the mechanisms by which CSP modulates G protein signaling, we examined the effects of CSP 1-198 (full-length), CSP 1-112 , and CSP 1-82 on the kinetics of guanine nucleotide exchange and GTP hydrolysis. In this report, we demonstrate that CSP selectively interacts with G␣ s and increases steady-state GTP hydrolysis. CSP 1-198 modulation of G␣ s was dependent on Hsc70 (70-kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein), whereas modulation by CSP 1-112 was Hsc70-SGT-independent. CSP 1-112 preferentially associated with the inactive GDP-bound conformation of G␣ s . Consistent with the stimulation of GTP hydrolysis, CSP 1-112 increased guanine nucleotide exchange of G␣ s. The interaction of native G␣ s and CSP was confirmed by coimmunoprecipitation and showed that G␣ s associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the ␤ 2 adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of G␣ s and promoting GDP/GTP exchange. Our results show that the guanine nucleotide exchange activity of full-length CSP is, in turn, regulated by Hsc70-SGT.G proteins constitute a family of heterotrimeric GTP-binding proteins that act as transducers in a variety of transmembrane signaling systems. G proteins are composed of ␣, ␤, and ␥ subunits that dissociate into G␣ and G␤␥ upon activation. Activation of G proteins involves an exchange of GDP for GTP on G␣ subunits and the release of GTP-bound G␣ and G␤␥ to interact with effector molecules. Effector molecules include adenylate cyclase, phospholipases, phosphodiesterases, and ion channels. Several modulators of G proteins have been identified and are thought to play crucial roles in the kinetics of G protein signaling (e.g. guanine nucleotide exchange factors (GEFs), 1 guanine nucleotide dissociation inhibitors, and GTPase-activating proteins). Our previous work has identified cysteine string protein (CSP) as a novel modulator of G proteins (1-3). Although the functional parallels between CSP and established G protein modulators are evident, the molecular mechanism by which CSP regulates G proteins is not yet known.CSPs are secretory vesicle proteins of 34 kDa that contain three conserved domains: a J domain, a linker domain, and a cysteine string region (Fig. 1A). The J domain is a 70-amino acid region of homology shared by DnaJ (a well characterized bacterial co-chaperone) and many otherwise unrelated eukaryotic proteins. The linker domain is a 30-amino acid ...

IntroductionCSPs (cysteine string proteins) are secretory vesicle chaperone proteins that are evolutionarily conserved. Deletion of CSP in Drosophila is semi-lethal; only 4% of the flies develop into adulthood . Adult survivors exhibit uncoordinated motor behavior that progresses to paralysis. Recordings from mutant neuromuscular junctions reveal that neurotransmission is reduced by 50% at 22°C and completely abolished above 29°C, indicating that the function of CSP is critical. Spontaneous vesicle release in these CSP-null mutants is not temperature sensitive Saitoe et al., 2001). CSPs have three domains: an N-terminal J domain, a linker-domain and a cysteine string domain. The J domain of CSP is a 70 amino-acid region homologous to the well-characterized bacterial chaperone protein DnaJ and many otherwise unrelated eukaryotic proteins. Although CSP is thought to be important in synaptic transmission, the exact details regarding the role of this synaptic chaperone in neurotransmission are not yet defined. Conflicting reports support either (i) a role for CSP in exocytosis or (ii) a role for CSP in the regulation of transmembrane Ca 2+ fluxes (reviewed by Zinsmaier and Bronk, 2001) (Chamberlain and Burgoyne, 2000).We have recently shown that CSP is capable of binding to both the N-type calcium channel and to Gβγ in vitro and that the interaction between CSP and the N-type calcium channel results in a robust tonic inhibition of channel activity by G protein βγ subunits . The CSP/G protein interaction was confirmed by co-immunoprecipitation, GST pull-down assays, crosslinking in intact brain slices as well as evaluation of functional proteins in HEK cells and has recently been confirmed by others (Evans et al., 2001). Numerous synaptic proteins were absent from the CSP immunoprecipitations and GST pull-down assays, demonstrating the specificity of the CSP/G protein interaction. Interestingly, CSP and G proteins have been shown to coenrich in detergent-insoluble lipid raft fractions from rat hippocampus (Magga et al., 2002). Binding of G proteins appears to involve two separate regions of CSP, such that Gα interacts with the J domain of CSP in an ATP-dependent manner, whereas Gβγ associates with full-length CSP but not the J domain of CSP in an ATP-independent fashion. Although CSP interacts with G proteins, it is not clear exactly how CSP affects G protein function. In particular, it is unclear exactly which regions of CSP associate with G protein subunits. Furthermore, it is unknown whether CSP's interaction with Gα proteins is direct or requires an additional component. Understanding the nature of the interaction between CSP, G proteins and calcium channels is crucial in understanding the molecular role of CSP. In the present study we have therefore analyzed the association of CSP with G proteins and N-type calcium channels. Materials and MethodsPreparation of rat hippocampal homogenate Rat hippocampi were hand homogenized with a teflon-coated

Cysteine string protein (CSP), a 34-kDa molecular chaperone, is expressed on synaptic vesicles in neurons and on secretory vesicles in endocrine, neuroendocrine, and exocrine cells. CSP can be found in a complex with two other chaperones, the heat shock cognate protein Hsc70, and small glutamine-rich tetratricopeptide repeat domain protein (SGT). CSP function is vital in synaptic transmission; however, the precise nature of its role remains controversial. We have previously reported interactions of CSP with both heterotrimeric GTP-binding proteins (G proteins) and N-type calcium channels. These associations give rise to a tonic G protein inhibition of the channels. Here we have examined the effects of huntingtin fragments (exon 1) with (huntingtin exon1/exp ) and without (huntingtin exon1/nonexp ) expanded polyglutamine (polyQ) tracts on the CSP chaperone system. In vitro huntingtin exon1/exp sequestered CSP and blocked the association of CSP with G proteins. In contrast, huntingtin exon1/nonexp did not interact with CSP and did not alter the CSP/G protein association. Similarly, co-expression of huntingtin exon1/exp with CSP and N-type calcium channels eliminated CSP's tonic G protein inhibition of the channels, while coexpression of huntingtin exon1/nonexp did not alter the robust inhibition promoted by CSP. These results indicate that CSP's modulation of G protein inhibition of calcium channel activity is blocked in the presence of a huntingtin fragment with expanded polyglutamine tracts.

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Organellar proteomics is a useful tool for gaining biological insights about structures in the cell. Here, we discuss the tools used in organellar proteomics and the impact of this technique in understanding nervous system function. We will review insights gained from the proteomes of nervous system-specific organelles such as synaptic vesicles and the postsynaptic density. Moreover, we will show how comparison of proteomes between organelles isolated from the nervous system and from other tissues highlight nervous system-specific functions using the examples of clathrin-coated vesicles and RNA granules.