The predicted UNC-45 sequence contains three distinct regions: an NH2-terminal TPR domain, a central region with no significant similarity to any known protein, and a COOH-terminal CRO1/She4p-like domain. This last region is the target of three of the four ts mutations analyzed, which are caused by base substitutions leading to missense mutations in codons for conserved residues (Fig. ). The two lethal alleles studied are the result of base substitutions in the central region, which lead to premature chain termination, so that the CRO1/She4p-like domain cannot be expressed.
This work has also provided discrete evidence of abnormal structure in unc-45 mutant thick filaments. Comparison of thick filaments from the CB286 strain grown at the restrictive versus the permissive temperature has demonstrated that this ts mutation, located within the CRO1/ She4p-like domain, affects the accumulation of assembled thick filaments, as well as their myosin isoform distribution and in vitro stability.
The results presented here suggest that UNC-45 functions as a thick filament assemblase through its CRO1/ She4p-like domain. Assemblase activity may be defined as a catalyst or chaperone that mediates the incorporation of myosin and related proteins into thick filaments (Liu et al., 1997
). Several mechanisms may be involved, including catalysis of myosin folding at the ribosome, intracellular myosin transport, posttranslational modification of myosin and associated proteins, catalysis of myosin polymerization, sorting of myosin isoforms, and scaffolding of myosin, paramyosin and the filagenins during the actual formation of the filament. An assemblase molecule may participate in one or more of these processes as a catalyst and may be subject to regulation by the interaction of additional proteins.
The scrambling of myosins A and B in the central region of 25°C CB286 filaments indicates that both isoforms contribute to the anti-parallel interactions of the central region of the filament, as has been demonstrated to be the case in filaments isolated from paramyosin loss-of-function mutant nematodes (Epstein et al., 1986
). Therefore, additional factors are necessary to recognize these isoforms and place them differentially along the length of the filament. One probable function of UNC-45 may be to sort each myosin isoform during the assembly process. This model of directed assembly of muscle thick filaments proposes that additional processes and specific proteins are required to explain the behavior of myosin besides its simple self-assembly as discussed by McLachlan and Karn (1982)
and Hoppe and Waterston (1996)
Several independent lines of evidence support the requirement for additional functions during the construction of thick filaments. In Drosophila melanogaster
, a single mhc gene is alternatively spliced to produce different isoforms that assemble into thick filaments of different characteristics, depending on the tissue and developmental stage of the organism (Bernstein et al., 1983
). Transgenic flies that express only a single mhc isoform, however, are still able to assemble different types of thick filaments with characteristics appropriate to the tissue and developmental stage (Wells et al., 1996
). Additional factors must then enable this single myosin species to assemble into distinct structures. In C
body wall muscle cells, thick filament length varies according to the developmental stage of the organism. Embryonic filaments cannot be longer than the greatest dimension of the embryonic muscle cell (<5 μm; Epstein et al., 1993
), whereas adult thick filaments are 9.7 μm long (Mackenzie and Epstein, 1980
). There must be factors that regulate filament elongation according to the developmental stage of the muscle cell other than myosins A and B and paramyosin, which are present continously. Moreover, myosins from different species that assemble in vivo into filaments of different characteristics (Epstein, 1989
) form very similar filamentous structures under the same conditions in vitro (Harris and Epstein, 1977
). Also, mutant CB675 myosin, which disrupts thick filament assembly in vivo, forms indistinguishable filamentous structures from those formed by wild-type molecules under the same conditions in vitro (Harris and Epstein, 1977
). This indicates that myosin molecules must be interacting with additional factors in vivo that regulate their proper assembly into thick filaments. Three-dimensional structural work on nematode thick filament cores has shown that these consist of paramyosin subfilaments held together in a tubular arrangement by additional protein structures (Epstein et al., 1995
). Several of these proteins have now been identified, including β-filagenin (Liu et al., 1998
), and their role as cross-linking proteins demonstrates the presence of additional proteins involved in the assembly of myosin filaments.
One possible role for the TPR domain of UNC-45 may be to bring interacting proteins to the site of thick filament assembly. These may be proteins that are additional components of the proposed assemblase, which contribute their particular functions during the assembly process, including protein chaperones, prolyl isomerases, and phosphoprotein phosphatases. Other partner proteins brought to the site of filament assembly through TPR binding may provide regulatory properties on the assemblase, appropriate to tissue and developmental stage of thick filament construction. We currently have no information as to the functional role the central region. It may be involved in an assemblase activity because one of the ts mutations is localized within its boundaries. The multi-domain nature of UNC-45 (Fig. ) suggests that each region may specifically interact with different kinds of protein molecules and perhaps act at distinct steps during thick filament assembly.
Several observations suggest that the actual process of thick filament assembly rather than the stability of the UNC-45 protein or the filaments themselves may be sensitive to elevated temperature in the unc-45 ts
alleles. A significant loss of UNC-45 function leads to lethality (Venolia and Waterston, 1990
). Therefore, the ts
mutations cannot cause complete misfolding of UNC-45 at the restrictive temperature; a partial function (or set of functions) must be present to allow survival of the organism. Also, our finding that three ts
alleles are caused by mutations in residues conserved in UNC-45, CRO1, and She4p suggests that these residues may represent key interactions in the UNC-45 active site and that their substitution causes specific defects rather than destabilization of the protein. Furthermore, there is genetic evidence that the protein product of the ts
alleles is not wild-type at the permissive temperature (Venolia and Waterston, 1990
); the mr/mr
progeny of a mr/ts
hermaphrodite are F1
lethals at either 15 or 25°C, whereas the mr/mr
progeny of a mr/
+ hermaphrodite are viable and produce mostly F2
lethals. A plausible interpretation of these interallelic interactions is that the UNC-45 protein may work as an oligomer, and the presence of a ts
protein, even at 15°C, hinders UNC-45 activity. It is unlikely that the thick filaments are sensitive to the elevated temperature because adult nematodes from any of the ts
strains grown at the permissive temperature do not show phenotypic reversal when switched to 25°C. This indicates that the myofibrils in these worms are stable structures not susceptible to depolymerization due to increased temperature.
Our results on the accumulation of mhc isoforms in the CB286 strain grown at 15 versus 25°C suggest that this mutation, localized within the CRO1/She4p-like domain, may affect the dynamics of myosin B polymerization. Work by others (Bejsovec and Anderson, 1990) has demonstrated that dominant lethal mutations in the myosin B head lead to impaired filament assembly and decreased accumulation of myosin B. A plausible interpretation of these results is that the mutations cause misfolding of the myosin molecule, which in turn hinders the thick filament assembly process. The presumably misfolded myosin B is then degraded. A similar situation may be occurring in CB286 nematodes at 25°C. A defect of UNC-45 function may lead to an abnormal myosin B, which then causes an impairment of filament assembly reflected by reduced numbers of filaments assembled in vivo, scrambling of myosin isoforms, and in vitro filament depolymerization. The decreased accumulation of myosin B may be explained by increased degradation of unincorporated molecules. Alternatively, the CB286 defect may cause a reduced number of functional myosin B molecules to be available for assembly. This could explain the similarity with the mhc B null phenotype observed in cross-sections by electron microscopy, and the decreased accumulation of mhc B. The yeast protein She4p may play a similar role to UNC-45 during the assembly of Myo4p (She1p) into structures capable of transporting the repressor Ahs1p. A common substrate of both molecules may be the myosin head, since this domain is conserved in both unconventional and sarcomeric myosins (Cheney and Mooseker, 1992
An alternative, but unlikely, explanation for the observed myosin isoform scrambling in the 25°C CB286 strain could be myosin repolymerization in vitro. Our data indicated that there was substantial filament depolymerization during the isolation procedure. Some of the myosin molecules could have dissociated from the filament and reassembled in a disorganized fashion in vitro to produce the abnormal structures observed by immunofluorescence and immunoelectron microscopy. If these structures originally had a myosin isoform distribution similar to wild-type in vivo, they would contain a central myosin A region. It has been shown that this myosin isoform remains tightly associated with the filament until the paramyosin core itself depolymerizes (Deitiker and Epstein, 1993
). Because the 25°C filaments contained paramyosin cores, which always retain myosin A in vitro, it appears unlikely that myosin A could have dissociated and then repolymerized with myosin B along nearly the entire length of the thick filament, including the central zone.
Biochemical investigation of the potential physical interactions between UNC-45 and the myosin isoforms is required in order to understand the mechanisms involved in its wild-type function(s). UNC-45 may associate with thick filament components in a stable fashion. In this case, it may act as part of a protein scaffold in the positioning of specific myosin isoforms and related proteins along the filament. As a scaffolding protein, UNC-45 may have additional nonstructural activities, in a manner analogous to twitchin, which is both a structural component of the sarcomere and a myosin light chain kinase (Hu et al., 1994
; Lei et al., 1994
) or the sarcomeric myosin itself, which is an ATPase, a protein motor and a structural protein (Harris and Epstein, 1977
; Warrick and Spudich, 1987
). In this case, the UNC-45 protein may be a structural component of the filament. On the other hand, involvement of UNC-45 in any of the other possible roles of a thick filament assemblase would require only transient association with filament components and not stable association with a particular sarcomeric structure.
The information related to myosin assembly presented here for UNC-45 suggests that UCS proteins (UNC-45/ CRO1/She4p) may serve as components of protein machines that recognize specific myosin isoforms and localize them to subcellular sites, where they can be assembled into structures appropriate for their particular cellular function.