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...

This study was aimed at evaluating the cloning and expression of three rumen microbial fibrolytic enzyme genes in a strain of Lactobacillus reuteri and investigating the probiotic characteristics of these genetically modified lactobacilli. The Neocallimastix patriciarum xylanase gene xynCDBFV, the Fibrobacter succinogenes ␤-glucanase (1,3-1,4-␤-D-glucan 4-glucanohydrolase [EC 3.2.1.73]) gene, and the Piromyces rhizinflata cellulase gene eglA were cloned in a strain of L. reuteri isolated from the gastrointestinal tract of broilers. The enzymes were expressed and secreted under the control of the Lactococcus lactis lacA promoter and its secretion signal. The L. reuteri transformed strains not only acquired the capacity to break down soluble carboxymethyl cellulose, ␤-glucan, or xylan but also showed high adhesion efficiency to mucin and mucus and resistance to bile salt and acid.

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.

Differential viral gene expression during both productive and persistent infections of Hz-1 virus in insect cells was elucidated. Despite more than 100 viral transcripts being expressed during productive viral infection, massive viral gene shutoff was observed during viral persistency, leaving the 2.9-kb persistence-associated transcript 1 (PAT1) as the only detectable viral RNA. Persistence-associated gene 1 (pag1), which encodes PAT1, was cloned and found to contain no significant open reading frames. PAT1 is not associated with the cellular translation machinery and is located exclusively in the nucleus. Further experiments showed that PAT1 is functional in the establishment of persistent Hz-1 viral infection in the cells. All the evidence collectively indicates that PAT1 is a novel nuclear transcript of viral origin. Our results showed that although PAT1 and XIST RNA, a mammalian X-inactive specific transcript, are transcribed by different genes, they have interesting similarities.