Small glutamine-rich protein/viral protein U-binding protein is a novel cochaperone that affects heat shock protein 70 activity

Abstract

Molecular chaperone complexes containing heat shock protein (Hsp) 70 and Hsp90 are regulated by cochaperones, including a subclass of regulators, such as Hsp70 interacting protein (Hip), C-terminus of Hsp70 interacting protein (CHIP), and Hsp70-Hsp90 organizing factor (Hop), that contain tetratricopeptide repeats (TPRs), where Hsp70 refers to Hsp70 and its nearly identical constitutive counterpart, Hsc70, together. These proteins interact with the Hsp70 to regulate adenosine triphosphatase (ATPase) and folding activities or to generate the chaperone complex. Here we provide evidence that small glutamine-rich protein/viral protein U-binding protein (SGT/UBP) is a cochaperone that negatively regulates Hsp70. By "Far-Western" and pull-down assays, SGT/UBP was shown to interact directly with Hsp70 and weakly with Hsp90. The interaction of SGT/UBP with both these protein chaperones was mapped to 3 TPRs in SGT/UBP (amino acids 95-195) that are flanked by charged residues. Moreover, SGT/UBP caused an approximately 30% reduction in both the intrinsic ATPase activity of Hsc70 and the ability of Hsc70 to refold denatured luciferase in vitro. This negative effect of SGT/UBP on Hsc70 is similar in magnitude to that observed for the cochaperone CHIP. A role for SGT/UBP in protein folding is also supported by evidence that a yeast strain containing a deletion in the yeast homolog to SGT/UBP (delta SGT/UBP) displays a 50-fold reduction in recovery from heat shock compared with the wild type parent. Together, these results are consistent with a regulatory role for SGT/UBP in the chaperone complex.

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 sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 3 identical lanes blotted to nitrocellulose and probed with no probe (none), GST alone, or GST-SGT/UBP. The Far-Western blot was then developed with anti-GST antibody to reveal bands that interact with SGT/UBP. The arrow to the right indicates a 70-kDa band detected by the GST-SGT/UBP fusion. The numbers to the left of each blot represent molecular weights in kilodaltons. (B) Identical blots containing 30 μg of HeLa lysate, 50 ng pure Hsc70, and 50 ng pure Hsp70, as indicated, were subjected to SGT/UBP Far-Western (SGT/UBP FW) or anti-Hsp70 Western analysis (αHSP70). The asterisk indicates the position of Hsp90, also detected in HeLa lysate by SGT/UBP Far-Western interaction. (C) Samples from the GST-SGT/UBP pull-down assay were analyzed by Western blot for the presence of Hsc70. At the bottom of the gel, the contents of each reaction are indicated by the plus symbols. The NaCl concentration of the washing buffer (100, 200, 300, and 500 mM) is indicated at the top of each lane. The arrow to the right of the blot indicates the position of the Hsc70 band

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 Hsc70 and Hsp70 were subjected to SGT/UBP Far-Western analysis. The blots were probed with GST fusions as indicated: full-length SGT/UBP, Δ1–93, Δ95–195, Δ288–313, N1/2 (amino acids [a.a.] 1–145), C1/2 (a.a. 145–313), and tetratricopeptide repeats 2–4 (a.a. 95–195). The numbers to the left of the blots indicate the molecular weights in kilodaltons. The diagram below the blots indicates regions of SGT/UBP contained in each fusion construct. The column to the right summarizes the results; a plus symbol indicates binding and a minus symbol indicates no binding

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 with 0, 12, 58, 118, or 176 nM SGT/UBP or bovine serum albumin (BSA) and 1 μCi of [α-³²P]ATP (13 nM) for 1 hour at 30°C. Adenosine diphosphate was separated from ATP by thin-layer chromatography and quantified using a Phosphorimager. Data are represented as the percent total ATPase activity, with the Hsc70 ATPase activity in the absence of BSA or SGT/UBP set to 100%. The Hsc70 ATPase activity in the presence of BSA (diamonds) or SGT/UBP (squares) is graphed as shown. (B) Hsc70 (28 nM) was incubated with 1 μCi of [α-³²P]ATP with or without SGT/UBP at a final concentration of 28 nM. Hydrolysis of ATP was monitored in the presence of Hsc70 (triangle), Hsc70 and SGT/UBP (circle), SGT/UBP alone (squares), or with no protein (diamond). The data are represented as the percent ATP hydrolysis and are plotted as a function of time from 0 minutes to 75 minutes

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 incubated with Hsc70 (square), Hsc70 and SGT/UBP, or no Hsc70 (circle). Luciferase activity was quantified using a luminometer. Data are plotted as the relative light units as a function of time from 0 minutes to 100 minutes

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 with GST or GST fusions having full-length SGT/UBP, Δ1–93, Δ95–195, Δ288–313, N1/2 (amino acids [a.a.] 1–145), C1/2 (a.a. 145–313), and tetratricopeptide repeats 2–4 (a.a. 95–195). The numbers to the left of the blots indicate the molecular weights in kilodaltons. The regions of SGT/UBP contained in each fusion construct are shown below the blots. The right columns summarize the results; a plus symbol indicates binding and a minus symbol indicates no binding

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 shock at 55°C for 1 hour and then plated at appropriate dilutions to rich media. The plates at the top of the bar graph show representative surviving colonies from wild type or SGT/UBP-deletion strains. The number of surviving colonies was counted, and the data were expressed as the percent total survivor, with wild type SGT/UBP set to 100%

Fig 7.

Comparison of several tetratricopeptide repeat–containing cochaperones