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. 2011 Sep 15;124(Pt 18):3164-73.
doi: 10.1242/jcs.087320.

The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans

Weiming Ni  1 , Alex H Hutagalung, Shumin Li, Henry F Epstein
Affiliations

The myosin-binding UCS domain but not the Hsp90-binding TPR domain of the UNC-45 chaperone is essential for function in Caenorhabditis elegans

Weiming Ni et al. J Cell Sci. .

Abstract

The UNC-45 family of molecular chaperones is expressed in metazoan organisms from Caenorhabditis elegans to humans. The UNC-45 protein is essential in C. elegans for early body-wall muscle cell development and A-band assembly. We show that the myosin-binding UCS domain of UNC-45 alone is sufficient to rescue lethal unc-45 null mutants arrested in embryonic muscle development and temperature-sensitive loss-of-function unc-45 mutants defective in worm A-band assembly. Removal of the Hsp90-binding TPR domain of UNC-45 does not affect rescue. Similar results were obtained with overexpression of the same fragments in wild-type nematodes when assayed for diminution of myosin accumulation and assembly. Titration experiments show that, on a per molecule basis, UCS has greater activity in C. elegans muscle in vivo than full-length UNC-45 protein, suggesting that UNC-45 is inhibited by either the TPR domain or its interaction with the general chaperone Hsp90. In vitro experiments with purified recombinant C. elegans Hsp90 and UNC-45 proteins show that they compete for binding to C. elegans myosin. Our in vivo genetic and in vitro biochemical experiments are consistent with a novel inhibitory role for Hsp90 with respect to UNC-45 action.

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Figures

Fig. 1.
Fig. 1.
UNC-45 is a multifunctional protein consisting of three identifiable regions. (A) Model of myosin assembly dependence on UNC-45 protein levels in C. elegans body-wall muscle cells. Myosin heavy chain accumulation and its consequent assembly are controlled by protein degradation on either side of the optimal UNC-45 concentration range (Hoppe et al., 2004; Landsverk et al., 2007). (B) Fragments cloned for injection under control of the body-wall muscle-specific unc-54 promoter with the FLAG tag on the C-terminus. (C) UNC-45 contains the N-terminal Hsp90-binding TPR domain, the central region and the C-terminal myosin-binding UCS domain (Barral et al., 1998; Barral et al., 2002). The arrows show the location of the embryonic lethal mutation st601 and the temperature-sensitive mutation e286. (D) The st601 mutation does not produce the predicted truncated protein fragment. st601 UNC-45 protein was examined in heterozygous st601, N2 and e286 worms by immunoblots with rabbit polyclonal anti-C. elegans UNC-45 antibody.
Fig. 2.
Fig. 2.
Reduction in wild-type nematode motility, thick filament assembly and body-wall muscle myosin accumulation by transgenic UCS-containing proteins. (A) The UCS domain and TPR(−) constructs significantly decrease wild-type nematode motility. Error bars indicate mean ± s.d. ***P<0.001. (B) The UCS domain and TPR(−) construct significantly diminish A-band assembly in wild-type nematode. (C) Quantification of A-band assembly diminution of transgenic wild type. Error bars indicate mean ± s.d. ***P<0.001. N.S., not significant. (D) The UCS domain is expressed at low levels compared with other transgenic proteins and the myosin-binding truncates show reduction in body-wall muscle myosin accumulation similar to FL UNC-45 in wild-type nematodes. Transgenic expression of UNC-45 fragments was detected by immunoblots with anti-FLAG antibody. UCS was almost invisible by anti-FLAG antibody. Body-wall muscle-specific myosin heavy chains A and B were detected and quantified by immunoblots with mAb 5–6 and mAb 28.2. Pharyngeal myosin heavy chain D, detected by mAb 5–17, was used as loading control. (E) UNC-45 truncate expression in wild type was examined by immunoblots with rabbit polyclonal anti-C. elegans UNC-45 antibody. UCS and other fragments were detectable on the blots. Endogenous UNC-45 and FLAG-tagged full-length UNC-45 overlapped on the top of the immunoblot. Pharyngeal myosin heavy chain D was used as loading control. (F) A titration experiment of transgenic UCS worms in wild type was performed to semi-quantitatively compare the level of UCS expression with that of FL in FL transgenic wild type by rabbit polyclonal anti-C. elegans UNC-45 antibody. Endogenous UNC-45 and FLAG-tagged full-length UNC-45 overlapped on the top of the immunoblot. UCS became detectable by polyclonal anti-C. elegans UNC-45 antibody. Pharyngeal myosin heavy chain D was used as loading control. (G) Table 1 shows the ratio of protein amounts. Table 2 shows ratios of the decreased body bends and the diminished A-bands between transgenic FL and UCS worms compared with those of wild type.
Fig. 3.
Fig. 3.
Rescue of st601 and e286 unc-45 mutants by transgenic UCS-containing proteins. (A) The UCS domain and TPR(−) construct are sufficient for significant rescue of st601 lethality. The percentage of dead eggs of the total progeny was measured after injection of the parental hermaphrodite with the specific constructs. Error bars indicate mean ± s.d. *P≤0.05; ***P<0.001. N. S., not significant. (B) The UCS domain and TPR(−) construct are sufficient to significantly rescue e286 motility at 25°C. Error bars indicate mean ± s.d. ***P<0.001. (C) The UCS domain and TPR(−) construct rescue A-band assembly in e286 at 25°C. The outline of worm muscle cell was faint in the UCS(−) or parental e286 mutant lines. (D) Quantification of A-band assembly rescue of transgenic e286 at 25°C. Error bars indicate mean ±s.d. ***P<0.001. (E) The UCS domain is expressed at lower levels compared with the other transgenic proteins, and the myosin-binding truncates rescue body-wall muscle myosin accumulation as does FL in e286. Transgenic expression of UNC-45 fragments was detected by immunoblots with anti-FLAG antibody. UCS band was quite weak with anti-FLAG antibody staining. Body-wall muscle-specific myosin heavy chains A and B were detected and quantified by immunoblots. Pharyngeal myosin heavy chain D was used as loading control. (F) The detection of UNC-45 truncates in e286 by rabbit polyclonal anti-C. elegans UNC-45 antibody. UCS and other fragments were detectable on the blots. Endogenous UNC-45 and FLAG-tagged full-length UNC-45 overlapped on the top of the immunoblot. Pharyngeal myosin heavy chain D was used as loading control. (G) A titration experiment of transgenic UCS worms in e286 was performed to semi-quantitatively compare the level of UCS expression with that of FL in FL transgenic e286 by reaction with rabbit polyclonal anti-C. elegans UNC-45 antibody. Endogenous UNC-45 and FLAG-tagged full-length UNC-45 overlapped on the top of the immunoblot. UCS was visible by polyclonal anti-C. elegans UNC-45 antibody. Pharyngeal myosin heavy chain D was used as loading control. (H) Table showing the ratios of protein amounts, body bends and A-bands between transgenic FL and UCS e286.
Fig. 4.
Fig. 4.
Hsp90 and UNC-45 compete for interaction with myosin. (A) UNC-45 interacts with Hsp90. FLAG beads were incubated with suIs2 worm lysate and myosin-containing complex eluted with FLAG peptide. Hsp90 and UNC-45 were determined by immunoblotting with anti-FLAG antibody and anti-Hsp90 antibody. (B) Titration of UNC-45 to a fixed amount of Hsp90 at a fixed concentration of myosin. The purified proteins were detected by Coomassie Blue staining.
Fig. 5.
Fig. 5.
Alternative models of UNC-45, Hsp90 and myosin motor interactions. In model I, UNC-45 and Hsp90 compete for myosin motor binding; binding of Hsp90 to the TPR domain and of myosin to the UCS domain would be sterically incompatible with one another. In this model, only binary complexes of the combinations of the three proteins would be significant. In model II, UNC-45 and Hsp90 cooperate for myosin motor binding in a ternary complex. Model II would require a major conformational change from the crystallographically based L-shaped structure (Lee et al., 2011; Shi and Blobel, 2010) to accommodate binding, because the Hsp90 dimer and myosin motor region have molecular weights of 182,000 and 115,000, respectively, significantly greater than the 107,000 of UNC-45 (Barral et al., 1998; Barral et al., 2002). Hsp90 assumes the open structure because the in vitro binding experiments in Figs 4B and supplementary material Fig. S1 were performed in the absence of ATP. Previous experiments (A. H. H. and H. F. E., unpublished results) showed that myosin did not bind UNC-45 in the presence of ATP.
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