Using a yeast two-hybrid system with the 70-kDa heat shock cognate protein (hsc70) or its C-terminal 30-kDa domain as baits, we isolated several proteins interacting with hsc70, including Hip/p48 and p60/Hop. Both are known to interact with hsc70. Except for Hip/p48, all of the proteins that we isolated interact with the 30-kDa domain. Moreover, the EEVD motif at the C terminus of the 30-kDa domain appears essential for this interaction. Sequence analysis of these hsc70-interacting proteins reveals that they all contain tetratricopeptide repeats. Using deletion mutants of these proteins, we demonstrated either by two-hybrid or in vitro binding assays that the tetratricopeptide repeat domains in these proteins are necessary and sufficient for mediating the interaction with hsc70.Members of the 70-kDa heat shock protein family (hsp70s) 1 and their cognates (hsc70s) have been implicated as ATP-dependent molecular chaperones (for reviews, see Refs. 1 and 2). Structurally, hsc70 is composed of two major domains. While the N-terminal 44-kDa domain is an ATPase (3-5), the Cterminal 30-kDa domain is capable of forming complexes with unfolded polypeptides (6, 7). Now, it is clear that hsp70s/hsc70s are acting in concert with other cellular proteins to exert the chaperoning functions such as folding of proteins, assembly, or disassembly of protein complexes or transport of proteins into organelles (for a review, see Ref. 1). For instance, hsc70 works together with auxilin to remove clathrin from coated vesicles (8). In Escherichia coli, DnaJ and GrpE are known to interact with DnaK (an hsc70 homolog) to assist protein folding (9). In yeast, interaction of SSA proteins (hsp70 homologs) with Ydj1p (a DnaJ homolog) was shown to play an important role in the translocation of some proteins across the endoplasmic reticulum membrane (10). Moreover, association of hsc70 with p60/ Hop and hsp90 appears to play an essential role in the assembly of the progesterone receptor (11,12). Using the yeast twohybrid approach with the conserved 44-kDa ATPase domain of hsc70 as a bait, Hohfeld et al. (13) have isolated an hsc70-interacting protein, Hip. Later, Hip was found to be identical to p48, a protein that interacts transiently with the steroid hormone receptor (14). More recently, BAG-1/Rap-46/Hap-46 was also shown to interact with the N-terminal domain of hsc70 (15-17) and inhibit the release of bound unfolded protein substrates (18). A number of other cellular proteins, including p16 (19) and HspBP1 (20), have been identified as hsp70/hsc70-interacting proteins. Nevertheless, the biological significance of these interactions is not well understood.During the last few years, the yeast two-hybrid system has been demonstrated as a powerful tool to identify interacting protein partners. However, it has not been investigated if intact hsc70 or its C-terminal 30-kDa domain can be effectively used as a bait in a two-hybrid screening, perhaps because the 30-kDa domain is known to bind short peptides or unfolded proteins (6, 7). In this study...
A group of tetratricopeptide repeat (TPR)-containing proteins has been shown to interact with the C-terminal domain of the 70 kDa heat-shock cognate protein (hsc70). In the present study, the effect of the TPR-containing proteins, including the C-terminus of hsc70-interacting protein (CHIP), TPR1 and human glutamine-rich TPR-containing protein (hSGT), on refolding of luciferase by DnaJ and hsc70 was investigated. These proteins inhibited the restoration of luciferase activity by the chaperones. The inhibitory effect exerted by TPR1 and hSGT depended upon their binding to hsc70. However, the interaction with hsc70 did not appear to be required for the inhibition of luciferase refolding by CHIP. We also demonstrate that the peptide, GPTIEEVD, corresponding to the C-terminal end of hsc70, abolished the association of [$H]hsc70 with CHIP, TPR1
A group of tetratricopeptide repeat (TPR)-containing proteins has been shown to interact with the C-terminal domain of the 70 kDa heat-shock cognate protein (hsc70). In the present study, the effect of the TPR-containing proteins, including the C-terminus of hsc70-interacting protein (CHIP), TPR1 and human glutamine-rich TPR-containing protein (hSGT), on refolding of luciferase by DnaJ and hsc70 was investigated. These proteins inhibited the restoration of luciferase activity by the chaperones. The inhibitory effect exerted by TPR1 and hSGT depended upon their binding to hsc70. However, the interaction with hsc70 did not appear to be required for the inhibition of luciferase refolding by CHIP. We also demonstrate that the peptide, GPTIEEVD, corresponding to the C-terminal end of hsc70, abolished the association of [(3)H]hsc70 with CHIP, TPR1 and hSGT. This implied that the GPTIEEVD motif of hsc70 was responsible for interacting with these TPR-containing proteins. However, the GGXP-repeats (where X is any aliphatic residue), another C-terminal conserved motif of vertebrate hsc70s, were not essential for interacting with the TPR-containing proteins. On the basis of mutagenesis studies, it was clear that a unique combination of the functional groups in the GPTIEEVD motif were utilized to interact with each TPR-containing protein, suggesting that inhibitors can be designed and used to elucidate the functional role of these interactions.
Seventy-kDa heat shock cognate protein (hsc70) and its homologs in bacteria, yeast and vertebrates are known to form complexes with S-carboxymethyl-a-lactalbumin (CMLA), an unfolded protein; and, this activity has been attributed to its C-terminal 30-kDa domain. Herein, we show that hsc70s isolated from the seeds of mung bean and peas, however, are not effective in complexing with CMLA, and that the 30-kDa domain of Arabidopsis hsc70 (At30) cannot form stable complexes with CMLA either. Moreover, chimeric 30-kDa domains, either composed of rat 18-kDa and Arabidopsis 10-kDa subdomains (R18At10) or with Arabidopsis 18-kDa and rat 10-kDa subdomains (At18R10), were prepared and tested for their ability to complex with CMLA or a heptapeptide FYQLALT. At18R10 cannot complex with both CMLA and FYQLALT. On the other hand, R18At10 is capable of forming complexes with FYQLALT at a level similar to that of the rat 30-kDa domain (R30). R18At10 also forms complexes with CMLA, but the amount of the R18At10/CMLA complexes is much less than that of R30/CMLA. The results imply that the 18-kDa subdomain dictates the binding specificity for heptapeptide, and that the C-terminal 10-kDa subdomain may also provide some selection or restriction for unfolded proteins to form complexes with hsc70.
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