Two distinct domains in hsc70 are essential for the interaction with the synaptic vesicle cysteine string protein

doi:10.1016/S0171-9335(99)80079-X

European Journal of Cell Biology

Volume 78, Issue 6, June 1999, Pages 375-381

https://doi.org/10.1016/S0171-9335(99)80079-X Get rights and content

The cysteine string protein (csp) is a synaptic vesicle protein found to be essential for normal neurotransmitter release. The precise function of csp in the synaptic vesicle cycle is still enigmatic. By interacting with the heat-shock cognate hsc70, a cochaperone-chaperone complex with an unknown function is formed. We report here that the formation of this complex is mediated by two distinct domains in hsc70. The ATPase domain and the substrate-binding domain must cooperate to create a binding site for csp. The C-terminal domain of hsc70 seems to function as a regulator for the formation of the cochaperone-chaperone complex. We also show that the interaction of csp with heat-shock proteins is confined to hsc70 and hsp70. Other heat-shock proteins, like hsp60 and hsp90, do not interact with csp.

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Cited by (33)

Comprehensive review on the HSC70 functions, interactions with related molecules and involvement in clinical diseases and therapeutic potential
2012, Pharmacology and Therapeutics
Citation Excerpt :
The interaction of Csp with HSC70 can stimulate the ATPase activity of HSC70 and cause a conformational change in HSC70. Interestingly, Csp can only interact with HSC70 and HSP70, but not with other heat shock proteins, such as HSP60 and HSP90 (Chamberlain & Burgoyne, 1997; Stahl et al., 1999). Small glutamine-rich TPR-containing protein (SGT) is a Csp partner which was originally identified because its interaction with envelope proteins of viruses.
Heat shock cognate protein 70 (HSC70) is a constitutively expressed molecular chaperone which belongs to the heat shock protein 70 (HSP70) family. HSC70 shares some of the structural and functional similarity with HSP70. HSC70 also has different properties compared with HSP70 and other heat shock family members.
HSC70 performs its full functions by the cooperation of co-chaperones. It interacts with many other molecules as well and regulates various cellular functions. It is also involved in various diseases and may become a biomarker for diagnosis and potential therapeutic targets for design, discovery, and development of novel drugs to treat various diseases.
In this article, we provide a comprehensive review on HSC70 from the literatures including the basic general information such as classification, structure and cellular location, genetics and function, as well as its protein association and interaction with other proteins. In addition, we also discussed the relationship of HSC70 and related clinical diseases such as cancer, cardiovascular, neurological, hepatic and many other diseases and possible therapeutic potential and highlight the progress and prospects of research in this field. Understanding the functions of HSC70 and its interaction with other molecules will help us to reveal other novel properties of this protein. Scientists may be able to utilize this protein as a biomarker and therapeutic target to make significant advancement in scientific research and clinical setting in the future.
The CSPα/G protein complex in PC12 cells
2007, Biochemical and Biophysical Research Communications
Cysteine string proteinα (CSPα) is a regulated vesicle protein and molecular chaperone that has been found to be critical for continuous synaptic transmission and is implicated in the defense against neurodegeneration. Previous work has revealed links between CSPα and heterotrimeric GTP binding protein (G protein) signal transduction pathways. We have shown that CSPα is a guanine nucleotide exchange factor (GEF) for G_αs. In vitro Hsc70 (70 kDa heat shock cognate protein) and SGT (small glutamine-rich tetratricopeptide repeat domain protein) switch CSPα from an inactive GEF to an active GEF. Here we have examined the cellular distribution of the CSPα system in the PC12 neuroendocrine cell line. CSPα, an established secretory vesicle protein, was found to concentrate in the processes of NGF-differentiated PC12 cells as expected. G_β subunits co-localized and G_αs subunits partially co-localized with CSPα. However, under the conditions examined, the GEF activity of CSPα is expected to be inactive, in that Hsc70 was not found in PC12 processes. These results indicate that CSPα activity is subject to regulation by factors that alter Hsc70 distribution and translocation within the cell.
Interaction between constitutively expressed heat shock protein, Hsc 70, and cysteine string protein is important for cortical granule exocytosis in Xenopus oocytes
2005, Journal of Biological Chemistry
Citation Excerpt :
First, by itself, overexpression of the J-domain of csp significantly blunts cortical granule exocytosis. Because the J-domain underlies the interaction of csp with members of the Hsp/Hsc 70 family of proteins (34–38), this result supports a role for csp-Hsp/Hsc 70 interaction in cortical granule exocytosis. Second, mutation of the J-domain of csp (to produce constructs that have been shown not to associate with Hsp/Hsc 70; see Refs. 35, 40, and 41) significantly reduces the inhibitory effect of these constructs on cortical granule exocytosis.
In many species, binding of sperm to the egg initiates cortical granule exocytosis, an event that contributes to a sustained block of polyspermy. Interestingly, cortical granule exocytosis can be elicited in immature Xenopus oocytes by the protein kinase C activator, phorbol-12-myristate-13-acetate. In this study, we investigated the role of cysteine string protein (csp) in phorbol-12-myristate-13-acetate-evoked cortical granule exocytosis. Prior work indicated that csp is associated with cortical granules of Xenopus oocytes. In oocytes exhibiting >20-fold overexpression of full-length Xenopus csp, cortical granule exocytosis was reduced by ∼80%. However, csp overexpression did not affect constitutive exocytosis. Subcellular fractionation and confocal fluorescence microscopy revealed that little or none of the overexpressed csp was associated with cortical granules. This accumulation of csp at sites other than cortical granules suggested that mislocalized csp might sequester a protein that is important for regulated exocytosis. Because the NH₂-terminal region of csp includes a J-domain, which interacts with constitutively expressed 70-kDa heat shock proteins (Hsc 70), we evaluated the effect of overexpressing the J-domain of csp. Although the native J-domain of csp inhibited cortical granule exocytosis, point mutations that interfere with J-domain binding to Hsc 70 eliminated this inhibition. These data indicate that csp interaction with Hsc 70 molecular chaperones is vital for regulated secretion in Xenopus oocytes.
Characterization of the Gα<inf>s</inf> regulator cysteine string protein
2005, Journal of Biological Chemistry
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 N-terminal J domain characteristic of the DnaJ/Hsp40 co-chaperone family. We have shown previously that CSP associates with heterotrimeric GTP-binding proteins (G proteins) and promotes G protein inhibition of N-type Ca²⁺ 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 Gα_s and increases steady-state GTP hydrolysis. CSP_1–198 modulation of Gα_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 Gα_s. Consistent with the stimulation of GTP hydrolysis, CSP_1–112 increased guanine nucleotide exchange of Gα_s. The interaction of native Gα_s and CSP was confirmed by coimmunoprecipitation and showed that Gα_s associates with CSP. Furthermore, transient expression of CSP in HEK cells increased cellular cAMP levels in the presence of the β₂ adrenergic agonist isoproterenol. Together, these results demonstrate that CSP modulates G protein function by preferentially targeting the inactive GDP-bound form of Gα_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.
Cysteine String Protein (CSP) Inhibition of N-type Calcium Channels Is Blocked by Mutant Huntingtin
2003, Journal of Biological Chemistry
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.
A brain-specific isoform of small glutamine-rich tetratricopeptide repeat-containing protein binds to Hsc70 and the cysteine string protein
2003, Journal of Biological Chemistry
Small glutamine-rich tetratricopeptide repeat-containing protein (SGT) is a ubiquitously expressed cochaperone of heat shock cognate protein of 70 kDa (Hsc70). SGT binds to the C terminus of Hsc70, a site used by several tetratricopeptide repeat-containing binding partners to recruit Hsc70 into complexes of diverse function. We describe here an isoform of SGT with 60% amino acid sequence identity that we name βSGT. In contrast to the previously published αSGT, βSGT is almost exclusively expressed in brain. Both isoforms of SGT possess similar binding properties toward Hsc70 and cysteine string protein, a synaptic vesicle-associated J-domain-containing protein. In addition, SGTs oligomerize without preferences among isoforms. The distribution of protein binding motifs on SGTs reveals a modular structure. The N-terminal domains mediate oligomerization. Binding to Hsc70 is impaired by mutations of basic residues within the central tetratricopeptide repeat domain of βSGT, indicating a two-carboxylate clamp as the binding mode. The tetratricopeptide repeats are also necessary for binding to the cysteine string protein. However, this binding mode is distinct from the two-carboxylate clamp that is involved in Hsc70 binding. The C-terminal regions of SGTs might constitute independent protein interaction domains. We conclude that βSGT is likely to cooperate with αSGT as co-chaperone of Hsc70 in the brain. The modular structure of SGTs allows them to recruit client proteins to Hsc70 and to direct the resulting complex toward downstream proteins that take over the respective client proteins.

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¹: Dr. Bernd Stahl, Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Str. 3, D-37075 Göttingen/Germany, Fax: ++5513899753.

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Two distinct domains in hsc70 are essential for the interaction with the synaptic vesicle cysteine string protein

Anal. Biochem.

Neuropharmacology

J. Biol. Chem.

Trends Neurosci.

J. Biol. Chem.

J. Biol. Chem.

Trends Biochem. Sci.

J. Biol. Chem.

Neuron

J. Biol. Chem.

Cell

J. Biol. Chem.

Cell