To elucidate the normal role of SGT/UBP in cells, we attempted to identify other cellular proteins that interact stably with SGT/UBP. To this end, HeLa cell lysates were subjected to Far-Western analysis. This entailed separation of the cell proteins on SDS gels and electrophoretic transfer to an Immobilon P membrane. The membrane was then incubated with GST-SGT/UBP, and stable association of the SGT/UBP with transferred protein was detected using anti-GST antibody as in conventional Western analysis. Data from this experiment indicated that a protein of about 70 kDa was the primary species that stably interacted with SGT/UBP (). Using a yeast 2-hybrid screen for proteins that interact with Hsp70, Liu et al (1999)
found that Hs70 interacts with SGT/UBP. Moreover, SGT/UBP contains TPR motifs in an array similar to that found in cochaperones such as Hip, Hop, and CHIP (Irmer and Hohfeld 1997
; Chen and Smith 1998
; Ballinger et al 1999
). Thus, we used purified Hsp70 and Hsc70 in Far-Western analysis in parallel with HeLa cell proteins to see whether the 70-kDa protein was Hs70. This indicated that SGT/UBP does associate stably with both Hsp70 and Hsc70 in this in vitro assay (). To examine the stability of this SGT/UBP-Hs70 interaction, we subjected the protein complexes to washes of increasing NaCl concentrations. This indicated that the protein was maintained even in the presence of relatively high salt concentrations (0.5 M NaCl) and is consistent with a robust interaction between SGT/UBP and Hs70 ().
Fig 1. Small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) binds directly to heat shock protein 70 (Hsp70) and its constitutive counterpart, Hsc70, with high affinity. (A) HeLa cell lysate (30 μg) was separated by (more ...)
Several cochaperones that mediate the activity of Hs70 contain TPR motifs and interact with Hs70 by way of their TPR motifs. TPRs are generally considered to be motifs that mediate intermolecular interaction by way of a signature alpha-helix (Das et al 1998
). Thus, we wanted to test the hypothesis that the TPRs in the N-terminal half of SGT/UBP are required for SGT/UBP-Hs70 interaction. Several deletion mutants of SGT/UBP were constructed that lack various segments of the protein, and these were tested for interaction with Hsc70 using Far-Western analysis () These data indicate that the TPRs of SGT/UBP are indeed necessary for interaction with Hs70. SGT/UBP mutants that maintained the 3 tandem TPRs (Δ1–93, Δ288–313, and TPR 2–4) were capable of stable interaction with Hsc70, whereas those that lacked the 3 TPRs (Δ95–195, N 1/2, and C 1/2) did not interact detectably with Hsc70. Further, the fact that N 1/2 and C 1/2, which contain a single intact TPR each, were unable to associate with Hs70 indicates that a single TPR is unlikely to be sufficient for association with Hsc70. TPR 2–4 is a mutant that expresses only the 3 tandem TPRs (a.a. 95–195). Because this mutant was able to interact stably with Hsc70, it appears that these 3 contiguous TPRs (a.a. 95–195) are sufficient for detectable SGT/UBP-Hs70 interaction. But in further experiments we found that this interaction was enhanced when a fragment contained TPR 2–4 as well as flanking charged residues (a.a. 81–209) such that the extent of binding was similar to that observed for SGT/UBP, Δ1–93, and Δ288–313 (data not shown).
Fig 2. Mapping of the small glutamine-rich protein (SGT)/viral protein U–binding protein (UBP)/Hs70 interaction, where Hs70 represents heat shock protein (Hsp) 70 and its constitutive counterpart, Hsc70. Seven identical sets of blots containing (more ...)
Hs70 contains an intrinsic ATPase activity that is important in the activity and function of the multiprotein chaperone complex. The ATP-bound form of Hs70 has relatively low affinity for protein substrates, whereas the ADP-bound form of Hs70 has relatively high affinity for protein substrates. Regulatory cochaperones that associate with Hs70, such as Hsp40, BAG-1, Hip, and CHIP, often exert their effect by affecting positively or negatively the ATPase activity of Hs70 (Irmer and Hohfeld 1997
; Bimston et al 1998
; Ballinger et al 1999
). To see whether SGT/UBP might similarly affect the ATPase activity of Hsc70, we carried out in vitro ATPase assays in the presence of SGT/UBP. This analysis indicated that SGT/UBP affected negatively the Hsc70-mediated hydrolysis of ATP (). The magnitude of this effect was similar to that of the cochaperone CHIP, a cochaperone that negatively affects the ATPase activity of Hs70. These data are consistent with the idea that SGT/UBP is also a cochaperone that affects the activity of Hs70.
Fig 3. Small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) inhibits the adenosine triphosphatase (ATPase) activity dependent on the constitutive counterpart, Hsc70, of heat shock protein (Hsp) 70. (A) Hsc70 (28 nM) was incubated (more ...)
Although Hs70 functions as part of a multiprotein complex, the protein is able to promote independently the refolding of denatured protein in vitro in the presence of ATP. To see whether SGT/UBP can affect this refolding activity of Hs70, we carried out an assay to detect the refolding of denatured luciferase in the presence and absence of SGT/UBP (). As expected, Hsc70 was able to catalyze the refolding of heat-denatured luciferase to functional form. SGT/UBP inhibited the Hs70-dependent refolding of luciferase by about 30%. This is consistent with the observed negative effect of SGT/UBP on the ATPase activity of Hsc70, similar in effect and magnitude to CHIP's affect on Hsc70-mediated protein refolding and again indicative of a likely role for SGT/UBP as a cochaperone.
Fig 4. Small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) inhibits refolding of denatured firefly luciferase dependent on the constitutive counterpart, Hsc70, of heat shock protein (Hsp) 70. Heat-denatured luciferase was (more ...)
Hsp90 is another key protein that is found in the chaperone complex and works in conjunction with other members of the complex to ensure correct substrate structure. Some TPR-containing cochaperones, such as Hip and Hop, are able to interact with Hs70 and Hsp90 by way of their TPRs; Hop interacts with both Hs70 and Hsp90 by way of different TPR-containing binding domains (Johnson et al 1998
). CHIP interacts with Hsc70 and can also interact with Hsp90 to redirect Hsp90 to function in the degradation of the glucocorticoid receptor (Connell et al 2001
). Additional Hsp90-associated proteins, such as the peptidylprolyl isomerases, Cyp40 and FKBP52, and the protein phosphatase PP5, also interact with Hsp90 by way of TPR motifs (Ratajczak and Carrello 1996
; Das et al 1998
). In our Far-Western analysis, we noticed that SGT/UBP was able to associate with a 90-kDa protein from HeLa cells (indicated with an asterisk in ), but the signal appeared to be markedly weaker compared with that for Hs70. To investigate this further, we analyzed purified Hsp90 using Far-Western analysis with SGT/UBP as a probe. On the basis of this experiment, it appeared that SGT/UBP could associate with Hsp90 in vitro (). We then evaluated the ability of the various SGT/UBP deletion mutants to interact stably with Hsp90. This analysis revealed that the 3 tandem TPRs of SGT/UBP were necessary for interaction between SGT/UBP and Hsp90. But in contrast to SGT/UBP-Hs70, the interaction of Hsp90 with the 3 TPRs was too weak to detect, suggesting that the specificity provided by the flanking charged amino acids was required for this interaction. Far-Western analysis is only semiquantitative. But the overall binding of SGT/UBP to Hsp90 appears weaker than that observed with Hs70. Moreover, in comparison with the known chaperones, SGT/UBP appears to resemble CHIP more closely than other cochaperones such as Hip or Hop; both SGT/UBP and CHIP interact with both Hs70 and Hsp90, and these interactions are mediated by the 3 tandem TPRs.
Fig 5. Mapping of the small glutamine-rich protein (SGT)/viral protein U–binding protein (UBP)/heat shock protein 90 (Hsp90) interaction. Identical blots containing 20 ng Hsp90 developed by SGT/UBP Far-Western analysis. The blots were probed (more ...)
SGT/UBP appears to be a highly conserved gene, and diverse organisms including Drosophila melanogaster
, Caenorhabditis elegans
, and Saccharomyces cerevisiae
contain apparent homologs to human SGT/UBP (Callahan et al 1998
; Cziepluch et al 1998
). To determine whether SGT/UBP may function as a cochaperone in yeast, we used homologous recombination to generate a knockout strain containing a deletion in the yeast SGT/UBP gene (y-SGT/UBP). This y-SGT/UBP mutant was indistinguishable from the parental strain when grown on a rich medium and at 37°C. Hsp70 and Hsp90 are members of a large class of “heat shock” proteins that were originally identified by increased expression after heat treatment. The increased expression of many of these heat shock proteins results in a corresponding increase in chaperone activity and concomitant refolding of heat-denatured proteins. Not surprisingly, many yeast strains that are deficient in appropriate cochaperone activity, and their regulation, are also poor in recovery from various heat shock treatments. We tested the ability of the y-SGT/UBP null mutant to recover from heat shock. The thermotolerance of the mutant was indistinguishable from that of the wild type when the cells were incubated at 18°C, 32°C, and 42°C (data not shown). But the y-SGT/UBP deletion mutant exhibited a marked reduction in viability after a high-temperature (55°C) heat shock (). The viability of the mutant was reduced by at least 50-fold relative to the wild type by this treatment. Such a phenotype is similar to that observed for some yeast strains containing lesions in genes encoding protein chaperone functions, such as the gene HSP104 (Sanchez and Lindquist 1990
). Thus, the phenotype of the y-SGT/UBP knockout mutant is consistent with a role for SGT/UBP as a functional component of the protein chaperone complex in yeast.
Fig 6. Deletion mutants of small glutamine-rich protein/viral protein U–binding protein (SGT/UBP) in Saccharomyces cerevisiae are defective for recovery from severe heat shock. Wild type (wt) or SGT/UBP deletion strains were subject to heat (more ...)