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

Abstract

SummaryThe 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.

Differential Turnover of Myosin Chaperone UNC-45A Isoforms Increases in Metastatic Human Breast Cancer

“…9,31 The myosin levels were similar, in agreement with results in ovarian carcinoma cells, 21 and were distinct from the effects of transgenic UNC-45 overexpression leading to decreased myosin accumulation in C. elegans muscle cells. 13,38 In addition to regulation of UNC-45A gene transcription with distinct patterns of expression from the UNC-45B gene, 20 alternative splicing produced two major mRNAs that are expressed as two protein isoforms. These isoforms differed only § http://www.rcsb.org/pdb/explore/explore.do?…”

The UNC-45 Myosin Chaperone

“…These authors suggest that excess UNC-45 associates with myosin, making it more likely to remain in an unassembled state that is more susceptible to degradation. Ni et al (2011) used extra-chromosomal arrays expressing UNC-45 fragments to show that reductions in myosin levels, A-band assembly and motility are largely dependent on the presence of the UCS domain. Furthermore, the UCS domain is required to partially rescue lethality of an unc-45 null mutant and the motility and A-band assembly of a temperature-sensitive mutant (Ni et al, 2011).…”

“…Ni et al (2011) used extra-chromosomal arrays expressing UNC-45 fragments to show that reductions in myosin levels, A-band assembly and motility are largely dependent on the presence of the UCS domain. Furthermore, the UCS domain is required to partially rescue lethality of an unc-45 null mutant and the motility and A-band assembly of a temperature-sensitive mutant (Ni et al, 2011). Interestingly, the UCS domain alone appears more effective than full-length UNC-45 on a per molecule basis (Ni et al, 2011).…”

“…Furthermore, the UCS domain is required to partially rescue lethality of an unc-45 null mutant and the motility and A-band assembly of a temperature-sensitive mutant (Ni et al, 2011). Interestingly, the UCS domain alone appears more effective than full-length UNC-45 on a per molecule basis (Ni et al, 2011). This led to the conjecture that the TPR domain might actually inhibit UNC-45 function, perhaps through presentation of Hsp90.…”

Nematode Hsp90: highly conserved but functionally diverse

“…The details of how UNC-45 and DAF-21 work together are still unclear, although it has been reported that UNC-45 interacts with Hsp90 via a TPR domain (Russell et al 1999;Scheufler et al 2000;Barral et al 2002) and myosin motor domains through its COOH-terminal regions (Barral et al 1998(Barral et al , 2002. Elegant studies by Ni et al demonstrated that DAF-21 and UNC-45 interact in pull-down experiments using C. elegans lysates and proposed that this interaction might have an inhibitory effect on the myosin-chaperoning activity of UNC-45 (Ni et al 2011).…”