The UNC-45 chaperone mediates sarcomere assembly through myosin degradation in Caenorhabditis elegans

Abstract

Myosin motors are central to diverse cellular processes in eukaryotes. Homologues of the myosin chaperone UNC-45 have been implicated in the assembly and function of myosin-containing structures in organisms from fungi to humans. In muscle, the assembly of sarcomeric myosin is regulated to produce stable, uniform thick filaments. Loss-of-function mutations in Caenorhabditis elegans UNC-45 lead to decreased muscle myosin accumulation and defective thick filament assembly, resulting in paralyzed animals. We report that transgenic worms overexpressing UNC-45 also display defects in myosin assembly, with decreased myosin content and a mild paralysis phenotype. We find that the reduced myosin accumulation is the result of degradation through the ubiquitin/proteasome system. Partial proteasome inhibition is able to restore myosin protein and worm motility to nearly wild-type levels. These findings suggest a mechanism in which UNC-45-related proteins may contribute to the degradation of myosin in conditions such as heart failure and muscle wasting.

Figure 1.

Overexpression of UNC-45^FLAG results in decreased thick filament assembly and a mild paralysis phenotype. (A) Expression levels of UNC-45^FLAG protein by two different integrated lines. Two integrated lines overexpressing UNC-45, suIs1, and suIs2 were analyzed for UNC-45^FLAG levels by Western blots using a mAb to the C-terminal FLAG tag on transgenic UNC-45. Each lane contains 10 worms total. (B) UNC-45^FLAG overexpression results in a mild paralysis phenotype. Motility in liquid was measured at 20°C for N2 and suIs2 grown at 20°C, unc-45(e286) grown at 15 and 20°C, and suIs2;unc-45(e286) grown at 25°C . N2 and suIs2 worms showed a mean of 33 and 21 body bends, respectively, over 15 s. The UNC-45^FLAG transgene expressed in suIs2 was able to rescue the motility defect of unc-45(e286) at 25°C from a mean of 2 to 16 bends in 15 s. *, P < 0.001 versus N2 wild type. (C) suIs2 worms have decreased number of MHC-containing A bands in body wall muscle cells. Ya worms were labeled with an anti-MHC A mAb to reveal the presence of A bands. (D) Quantitation of the myosin assembly defect of suIs2 worms. The number of A bands per cell in regions I and II, posterior to the pharynx (Mackenzie et al., 1978b) of N2 and suIs2 worms, was counted. In region II, N2 worms have a mean of 7.8 A bands per cell, whereas susI2 worms have a mean of 5.8. In region III, N2 and suIs2 worms have 8.7 and 6.3 A bands per cell, respectively. *, P < 0.0001 versus N2 wild type. Error bars indicate SD.

Figure 2.

Overexpression of UNC-45^FLAG results in diminished accumulation of body wall myosins. (A) C. elegans worms overexpressing UNC-45^FLAG or harboring an UNC-45 Lof mutation have decreased levels of body wall muscle MHC. Individual worms from N2, suIs2, and unc-45(e286) strains at either the permissive (15°C) or restrictive (25°C) temperatures were isolated at the Ya stage. Western blots against MHC A, B, and D show that only body wall muscle MHC A and B are affected in transgenic UNC-45^FLAG worms, whereas MHC D is also decreased in e286 mutant worms. (B) Quantification of MHC levels in worms overexpressing UNC-45^FLAG and in UNC-45 Lof mutants. Analysis of Western blots performed in triplicate show that suIs2 worms have 30% less MHC A and B than N2 wild-type worms. Note that this decrease is not as great as that of e286 mutants, which have 70% less body wall MHC than wild type. *, P < 0.5; **, P < 0.01, versus N2 wild type. (C) MHC mRNA levels between N2 and suIs2 remain unchanged. The threshold cycle (Ct) was set as the fractional cycle number at which the real-time fluorescence signal of the reaction could be measured above background. The cycle at which N2 wild type and suIs2 crossed the threshold was within one cycle each for unc-54, myo-3, and GAPDH. Error bars indicate SD.

Figure 3.

The UPS is responsible for degrading myosin in worms overexpressing UNC-45^FLAG. (A) C. elegans myosin is ubiquitinated in vivo. Pull downs from N2 wild-type worms using anti-MHC and antiubiquitin antibodies subsequently subjected to anti-MHC immunoblotting. Bands of slower mobility can be observed in the lane corresponding to the antiubiquitin pull down, which may correspond to multiubiquitinated myosin species. Bands of faster mobility observed in lanes of both pull downs may represent proteolytic fragments of myosin. (B) Degradation of myosin in C. elegans is dependent on ATP and is inhibited by the proteasome inhibitor MG132. Supernatants of worm lysates were incubated with no supplementation, ATP, MG132, or ATP/MG132 at room temperature. Samples were removed at the indicated time points. Western blots to detect MHC B were performed using mAb 28.2. (C) Partial inhibition of proteasomal function through RPT2 RNAi leads to restoration of both MHC isoforms in suIs2 worms. Western blots for MHC A and B were performed on suIs2 worms grown on Escherichia coli containing an empty vector or an RPT2 RNAi vector, as indicated. (D) Quantification of MHC levels in suIs2 worms after partial inhibition of the proteasome. Analysis of Western blots performed in triplicate shows that inhibition of the proteasome induces an increase in myosin levels in suIs2 worms. *, P < 0.5, versus N2 wild type; **, P < 0.5 suIs2 RNAi versus suIs2 empty vector. (E) Partial inhibition of proteasomal function results in limited rescue of mobility defects associated with UNC-45^FLAG overexpression. Inhibiting the proteasome in suIs2 worms increases the number of body bends in 15 s. suIs2-empty vector– and suIs2-RPT2-RNAi–treated worms displayed a mean of 21 and 27 body bends, respectively. *, P < 0.001 versus N2; **, P < 0.001 susI2 RNAi versus susI2 empty vector. Error bars indicate SD.

Figure 4.

Mechanism of UNC-45 chaperone–mediated proteasomal degradation of myosin. (A) Myosin assembly as an idealized function of UNC-45 chaperone levels. The two are linked by control of myosin accumulation through its degradation. ts, temperature sensitive. (B) Proposed mechanism of UNC-45 chaperone–mediated myosin assembly, disassembly, and consequent degradation. All reactions represented by opposing arrows are reversible; myosin degradation represented by a single arrow is irreversible. By mass action, chaperone deficiency leads to a buildup of free, nonnative myosin, a substrate for the proteasome. Similarly, excess chaperone leads to a buildup of its complexes with myosin molecules and filaments. These complexes lead to increased levels of free, nonnative myosin and its proteasomal degradation.