©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Both the Amino and Carboxyl Termini of Dictyostelium Myosin Essential Light Chain Are Required for Binding to Myosin Heavy Chain (*)

(Received for publication, January 26, 1995; and in revised form, August 21, 1995)

Guyu Ho Tung-Ling L. Chen Rex L. Chisholm (§)

From the Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

Dictyostelium myosin deficient in the essential light chain (ELC) does not function normally either in vivo or in vitro (Pollenz, R. S., Chen, T. L., Trivinos-Lagos, L., and Chisholm, R. L.(1992) Cell 69, 951-962). Since normal [Medline] myosin function requires association of ELC, we investigated the domains of ELC that are necessary for binding to the myosin heavy chain (MHC). Deleting the NH(2)-terminal 11 or 28 amino acid residues (DeltaN11 or DeltaN28) or the COOH-terminal 15 amino acid residues (DeltaC15) abolished binding of the ELC to the MHC when the mutants were expressed in wild-type (WT) cells. In contrast, the ELC carrying deletion or insertion of four amino acid residues (D4 or I4) in the central linker segment bound the MHC in WT cells, although less efficient competition with WT ELC suggested that the affinity for the MHC is reduced. When these mutants were expressed in ELC-minus (mlcE) cells, where the binding to the heavy chain is not dependent on efficient competition with the endogenous ELC, DeltaN28 and DeltaN11 bound to the MHC at 15% of WT levels and DeltaC15 did not bind to a significant degree. I4 and D4, however, bound with normal stoichiometry. These data indicate that residues at both termini of the ELC are required for association with the MHC, while the central linker domain appears to be less critical for binding. When the mutants were analyzed for their ability to complement the cytokinesis defect displayed by mlcE cells, a correlation to the level of ELC carried by the MHC was observed, indicating that a stoichiometric ELC-MHC association is necessary for normal myosin function in vivo.


INTRODUCTION

Myosin is a mechanochemical enzyme that generates force during muscle contraction and has a fundamental role in a variety of cell movements (for review, see (2) ). Conventional myosin is a hexameric protein composed of a pair of heavy chains (MHC) (^1)and two pairs of light chains, called essential light chains (ELC) and regulatory light chains (RLC). Both light chains bind to the neck of myosin(3, 4) . We have shown that ELC-deficient Dictyostelium cell lines are defective in cytokinesis, and myosin purified from these cells does not show significant actin-activated ATPase activity(1) . Since normal myosin function requires association of the ELC, we set out to map the domains of the ELC that are critical for ELC-MHC interaction.

Sequence comparisons of ELCs from various sources show that the COOH-terminal half of the molecule is more conserved than the NH(2)-terminal half (5, 6) and that it may represent the site required for association with MHC. Skeletal muscle alkali light chain A1 in which the COOH-terminal 14 residues were chemically removed did not bind the S1 heavy chain, although the conformation remained largely unchanged(7) . Transfection studies also showed that a COOH-terminal truncation of the alkali light chain A1 failed to colocalize with acto-myosin structures in cultured myocytes (8) .

The ELC belongs to the calmodulin-troponin gene family, which contains four helix-loop-helix structures, also known as EF-hands (for review, see (9) ). The atomic structures of calmodulin (CaM) and troponin C show two terminal globular lobes linked by a central helix, with each lobe containing a pair of EF-hands(10, 11) . Although ELCs may no longer bind Ca due to amino acid deletions or substitutions in the Ca-coordinating sites(9) , several lines of evidence suggest that the overall structure has been preserved. The ELC has many physical and chemical properties similar to those of CaM and troponin C, including Stokes radius, molecular weight, sedimentation coefficient, and radius of gyration(4) . Secondary and tertiary structural modeling, based on factors thought to control protein folding, also predicted similar tertiary arrangements for ELC, troponin C, and CaM(12) . Recently, three-dimensional structures of myosin S1 (13) and the regulatory domain of scallop myosin (14) confirmed these predictions. Based on these structures, we divided the Dictyostelium ELC into three structural domains: an NH(2)-terminal globular domain, a COOH globular domain, and a central linker domain. The participation of these domains in ELC-MHC interaction was assessed. In vitro mutagenesis was used to modify the Dictyostelium ELC, and the altered ELCs were expressed in both WT and mlcE cells. The ability of mutant ELC to associate with the MHC in vivo and to rescue the phenotypic defects of mlcE cells was analyzed.


MATERIALS AND METHODS

Site-directed Mutagenesis

Mutagenesis employed a polymerase chain reaction (PCR)-based approach. To construct the myc-tagged and terminal-deleted ELCs, the conventional one-template PCR amplification was employed using the Dictyostelium ELC cDNA (15, 16) as a template. The primer used to introduce the myc epitope at the end of the ELC coding sequence was designed to match 17 nucleotides before the stop codon, followed by 30 nucleotides encoding the myc epitope and a stop codon. To create terminal deletions, the primers were made to match 17 nucleotides adjacent to the deletions, followed by either a start codon or a stop codon. A BamHI site and two extra bases were included at the 5` end of all primers to facilitate cloning(17) . To construct the I4, D4, and F15A ELCs, the overlap extension PCR technique was used(18, 19) . A pair of internal primers containing the desired mutations were complementary to each other for at least 17 nucleotides at their 5` ends. A pair of external primers, the forward and the reverse primers of the plasmid Bluescript pKS (Stratagene), flanked the entire ELC coding sequence since the ELC cDNA was cloned into the polylinker region of the plasmid pKS. The first stage PCR amplifications were performed in two separate reactions, with one internal and one external primers to produce the 5` and 3` fragments of the desired final PCR product. These two fragments were purified by agarose gel electrophoresis, treated with T4 DNA polymerase to remove overhanging 3` adenosine residues(20) , and used as template for the second stage PCR amplification with external primers to produce a full-length ELC mutant. All PCR products were sequenced. Mutated ELCs were then cloned into the pBORP expression vector at the BamHI site(21) .

Transformation and Growth Conditions

AX3 cells or mlcE cells were transformed by electroporation (22) . Transformants were selected and grown in HL-5 medium containing 10 µg/ml G418 and 100 µg/ml streptomycin.

Preparation of Protein Samples

Dictyostelium whole cell lysates were prepared as described by Pollenz et al.(1) , and cytoskeletons were prepared according to Giffard et al.(23) . To release myosin from cytoskeletons, cytoskeletal pellets were resuspended in a buffer containing 100 mM PIPES, pH 6.8, 2.5 mM EGTA, 1 mM MgCl(2), 200 mM KCl, and 2 mM ATP. The supernatant was collected after centrifugation and referred to as ATP-released myosin supernatant.

Immunoblot Analysis

Protein samples were electrophoresed on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and blotted with myosin anti-serum NU-48. Antibody binding was detected using a horseradish peroxidase-conjugated goat anti-rabbit IgG (Bio-Rad). For quantitation analysis, antibody-binding was detected with I-labeled protein A (gift of Dr. J. Bartles) or Western blot chemiluminescence reagent (DuPont NEN). Autoradiographs were scanned using laser densitometer (Pharmacia Biotech Inc.) to determine the relative levels of MHC, WT and mutant ELCs, and RLC.

Myosin Purification and Activity Assays

Myosin was purified as described previously (1) except that the ammonium sulfate precipitation was eliminated, and gel filtration chromatography employed a Superose 6 column (Pharmacia). Actin-activated ATPase was assayed in the 10 mM Tris, pH 7.6, 25 mM KCl, 5 mM MgCl(2), 0.1 mM CaCl(2), 1 mM ATP, 10 µM actin, and 50 µg/ml myosin. Reactions were incubated at room temperature for 5 min and quenched with acid, and the liberated P(i) was quantified following organic extraction(24, 25) . In vitro motility assays were performed as described by Uyeda et al.(26) . Myosin was diluted to 200 µg/ml in buffer AB (25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl(2), 1 mM EGTA, 1 mM dithiothreitol), and applied to a flow cell coated with nitrocellulose. After blocking with AB containing 0.5 mg/ml bovine serum albumin (AB/BSA), a solution of rhodamine-phalloidin-labeled F-actin in AB/BSA was introduced. Active movement was initiated at room temperature by introducing AB/BSA containing 1 mM ATP and the oxygen scavenger enzymes.

DAPI Staining

DAPI staining was performed as described previously(1) .

Gel Filtration

Wild-type and mlcE cells were harvested from plates and lysed in a buffer containing 25 mM HEPES, pH 7.4, 55 mM NaCl, 2 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100, and a mixture of proteinase inhibitors. The Triton-cytoskeletal pellet was collected by centrifugation at 20,000 times g for 15 min and resuspended in 0.1 volume/g of cells of a buffer containing 10 mM HEPES, pH 7.4, 250 mM NaCl, 3 mM MgCl(2), 2 mM ATP, 1 mM dithiothreitol. A 0.2-ml aliquot of the suspension was loaded on Sephacryl-500 column (0.5 cm times 60 cm), eluted at 0.5 ml/min with a buffer containing 10 mM triethanolamine, pH 7.5, 1 mM EDTA, 600 mM KCl, 0.2 mM ATP, 1 mM dithiothreitol. Fractions (0.2 ml) were collected, and the amount of myosin was measured by probing slot-blots with the MHC monoclonal 396 (provided by Dr. G. Gerisch, Max-Planck Institute). The void volume was 4 ml, and myosin eluted at 6 ml.


RESULTS

The myc Epitope-tagged WT ELC Associates with the MHC

To facilitate the detection of ELC mutants when expressed in WT cells, the ELCs were tagged at the carboxyl termini with a myc epitope (27) of 10 amino acid residues (EQKLISEEDL) and expressed in Dictyostelium AX3 cells using an expression vector pBORP (21) . Lane 1 in Fig. 1shows the expression of the tagged WT ELC (mycELC). Based on densitometry, the expression level of mycELC was about 10 times that of the endogenous ELC. This mycELC was present in both the Triton-insoluble cytoskeletons (lane 2) and the ATP-released myosin supernatant (lane 3), suggesting its association with the MHC. Myosin purified from the mycELC transformants showed that 98% of the MHC carried mycELC. Similar levels of binding were observed in the ATP-released myosin supernatant (Table 1). Because supernatants of ATP Triton cytoskeletons accurately reflected association of ELC with MHC, quantification of the mutant ELC-MHC association employed the ATP-released myosin supernatants. Myosin carrying 98% mycELC exhibited normal actin-activated ATPase activity (140 nmol/min/mg). Cytokinesis and development of cells expressing mycELC were indistinguishable from wild-type. These results establish that the myc-epitope tag does not affect either the binding of the ELC to the heavy chain or myosin function.


Figure 1: Expression and association of the mycELC in WT cells. Protein samples from a mycELC transformant were run on 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and probed with myosin polyclonal anti-serum NU 48. Lane 1, whole cell lysate of 1 times 10^6 cells; lane 2, Triton-insoluble cytoskeletons; lane 3, ATP-released myosin supernatant; lane 4, purified myosin. The arrowhead points to the mycELC.





Contribution of Terminal Residues of the Dictyostelium ELC to the MHC Association

To test the role of the NH(2)-terminal domain in MHC association, the first 28 amino acid residues of the ELC were removed. The deleted region contained the first helix (A helix) and loop of the ELC (Fig. 2). Fig. 3shows that the truncated molecule, DeltaN28, was expressed in WT cells (lane 1) but did not appear in the cytoskeletal fraction (lane 2), indicating that the deletion had abolished the ELC-MHC association. A similar deletion, which removed the first 11 amino acid residues corresponding to the A helix of the ELC (Fig. 2) gave the same result (Fig. 3, lanes 3 and 4). In the complementary analysis, an ELC, which lacked the COOH-terminal 15 residues corresponding to the H helix (Fig. 2), was constructed and expressed (Fig. 3, lane 5). DeltaC15 also failed to partition in the cytoskeletal fraction (Fig. 3, lane 6). These results suggest that residues at both the NH(2) and COOH termini of the ELC are necessary for the ELC-MHC association.


Figure 2: Structural representations of terminal deletion mutants. Positions of four helix-loop-helix structures of the Dictyostelium ELC (Dd ELC) are illustrated based on sequence alignment with the Dictyostelium CaM. Solid bars (denoted from A to H) represent helices, and curved lines represent loops. The myc-epitope tag is indicated.




Figure 3: Expressed terminal deletion mutants do not associate with MHC in WT cells. Transformants from DeltaN28, DeltaN11, and DeltaC15 were analyzed by Western blots as in Fig. 1. Lanes 1, 3, and 5, whole cell lysates; lanes 2, 4, and 6, Triton-insoluble cytoskeletons. Arrowheads point to the mutants.



The involvement of the COOH terminus of the ELC in heavy chain binding is consistent with previous observations(7, 8) , but participation of residues at NH(2) terminus of the ELC has not been previously demonstrated. We tested the possibility that the NH(2)-terminal residues are required for maintaining an overall conformation of the ELC rather than being in a direct association with the heavy chain. Amino acid residue 15, a phenylalanine residue that is conserved among ELCs(28) , was replaced by an alanine. This residue corresponds to Phe-19 of CaM, which has five contacts in the CaMbulletM13 peptide complex(29) . Fig. 4shows that the F15A ELC mutant was expressed to levels comparable with the mycELC (lane 1) and partitioned in the cytoskeletal fractions (lanes 2 and 3), suggesting that it is associated with the MHC. But unlike the mycELC transformants (Fig. 1), the endogenous ELC appeared in the cytoskeletal fractions of the F15A transformants (lanes 2 and 3), suggesting that the F15A ELC had reduced ability to compete with the endogenous ELC for the binding to the MHC. Quantification of MHC-bound ELC showed that 85% of the MHC carried the F15A ELC in WT cells (Table 1). The reduced binding affinity of this substitution mutant supports the idea that residues at the NH(2) terminus of ELC associate directly with the heavy chain.


Figure 4: Expression and association of the F15A mutant in WT cells. One of the F15A trasformants was analyzed by Western blot as in Fig. 1. Lane 1, whole cell lysate; lane 2, Triton-insoluble cytoskeletons; lane 3, ATP-released myosin supernatant.



While this work was in progress, mlcE cell lines produced by gene targeting became available. (^2)The ELC mutants were expressed in mlcE cells to assess their binding to MHC in the absence of competing endogenous ELC. While the levels of expression were comparable with those seen in WT cells, DeltaN28 and DeltaN11 bound to MHC at 15% of WT levels, whereas DeltaC15 did not bind to any significant extent. F15A showed binding comparable with WT ELC. Since F15A was at least 10-fold overexpressed, its reduced binding affinity was apparently masked by its overexpression in mlcE cells.

ELCs with Central Linker Domain Mutations Bind the MHC with Reduced Affinity

The central linker domain of the ELC, initially identified by sequence alignment with calmodulin and confirmed by the two available crystal structures(13, 14) , extends from residues 70 to 79. To study the role of this region in ELC-MHC association and to determine if the relative distance between the NH(2)-terminal and COOH-terminal globular domains was critical, insertion and deletion mutants of the central linker were produced. The deletion mutant, D4, was constructed by deleting residues Gln, Glu, Gln, and Gln from positions 74-77. The insertion mutant, I4, carried four additional residues with a helix forming potential Lys, Ser, Thr, and Asp (30) inserted between residues Glu-79 and Met-80. The sites for insertion and deletion were chosen because changes at these locations were predicted by the Garnier method for secondary structure predication (31) to have little effect on the conformation of neighboring structures. Theoretically each amino acid residue in an alpha-helix causes 100° twist and a 1.5-Å rise(32) . Insertion or deletion of four residues in the linker domain would cause an increase or decrease of about one helical turn in relative distance of the two terminal lobes, while the relative orientation is approximately maintained.

Despite the fact that both I4 and D4 were expressed to a level comparable with that of mycELC in WT cells, they did not compete as efficiently for the MHC binding as the mycELC (Fig. 5). These two mutants bound to MHC at 40% of the mycELC level in WT cells (Table 1), indicating that the ELC carrying deletion or insertion of residues in the central domain bind MHC with reduced affinity.


Figure 5: Association of I4 and D4 with MHC in WT cells. The ATP-released myosin supernatants from I4 and D4 transformants were analyzed by Western blot as in Fig. 1. The ATP-released myosin supernatant from mycELC transformants is used as control. Arrowheads indicate mycELC, I4, and D4, respectively.



When the I4 and D4 mutants were overexpressed in mlcE cells, they associated with MHC stoichiometrically, a situation similar to the F15A ELC. To test the importance of the linker domain in myosin enzymatic function, myosin containing I4 and D4 was purified and assayed for ATPase activity and the ability to support movement of actin filaments in an in vitro motility assay. As shown in Table 2, both myosins had WT activities.



To determine the biochemical properties of a ``weak binding'' mutant, myosin was purified from cells expressing DeltaN11. In purified myosin preparations, DeltaN11 bound to MHC at 5% of WT levels. Similar to ELC-minus myosin, DeltaN11 myosin did not exhibit significant actin-ATPase and failed to move actin filaments in vitro (Table 2).

Abilities of Mutant ELCs to Rescue the Cytokinesis Defect of mlcE Cells

The most prominent phenotype of the mlcE cells is the formation of large multinucleate cells when cultured in suspension.^2 The ELC mutants were analyzed for their ability to rescue the defect in suspension growth. ELC-minus cells carrying the mutants had normal levels of MHC expression and normal stoichiometric association between the RLC and MHC (data not shown). Fig. 6shows that cells expressing DeltaC15 had a phenotype similar to mlcE cells, with only 13% of the cells mono- or dinucleated. Cells expressing DeltaN28 and DeltaN11 had intermediate phenotypes with 35-45% of the cells carrying one or two nuclei, while those expressing F15A, I4, and D4 were indistinguishable from the wild-type. The results show a strong correlation between the level of ELC association with the MHC and the ability to perform myosin-dependent function in vivo.


Figure 6: The abilities of ELC mutants to rescue the cytokinesis defect of mlcE cells. Cells transformed with ELC mutants were grown in suspension for 3 days and taken for nuclei staining by DAPI. More than 100 cells were scored for each strain. The numbers are the average of two or three independent experiments. Solid bars represent the efficiency of cytokinesis, and stippled bars represent the levels of mutant ELCs associated with the heavy chain.



It has been reported that myosin lacking the RLCs has a tendency to aggregate(33, 34, 35) , presumably mediated by the exposed, hydrophobic alpha-helix that serves as binding sites for myosin light chains(13, 14) . An important question is whether the poor cellular myosin function observed with the weak binding mutants DeltaN28, DeltaN11, DeltaC15 is due to a defect in enzymatic activity or aggregation of a significant amount of the myosin lacking bound ELC. To address this question, crude cellular extracts of mlcE and WT cells were subjected to gel filtration to compare the association state of the myosin. As shown in Fig. 7, the ELC-minus myosin fractionated with the same pattern as wild-type, suggesting there are no gross differences in the physical state of myosin in the two cells. Thus the multinucleate phenotype associated with the weak binding mutants seems unlikely to result from abnormal myosin aggregation.


Figure 7: Gel filtration profiles of myosin cytoskeletons from mlcE and WT cells. High salt cytoskeletons obtained from mlcE and WT cells were fractionated on Sephacryl-500 column. Elution of myosin was monitored by measuring the densities of MHC in eluted fractions.




DISCUSSION

The structure of CaMbulletM13 peptide complex shows that residues in both lobes of calmodulin are in contact with the M13 peptide (a 26-amino acid peptide containing the CaM binding domain of myosin light chain kinase); residues in the helices account for the vast majority of those contacts(29, 36) . By analogy to this structure, the two terminal helices A and H of the Dictyostelium ELC were deleted. The deletions resulted in a loss of ELC binding to the MHC when the mutants were expressed in WT cells. Since the atomic structure of CaM shows independent folding of the two lobes and no interactions between them, it is unlikely that mutations in one lobe affect the folding or tertiary structure of the other. If this is true with ELC, then the deletion results suggest that residues in both the COOH-terminal lobe and NH(2)-terminal lobe are important for association with the heavy chain. The participation of the NH(2) terminus is further supported by the substitution mutant F15A, in which a phenylalanine to alanine substitution resulted in reduced binding of the ELC to the MHC. It is conceivable, therefore, that ELC may bind MHC with both lobes in a manner similar to that of CaM binding its substrates. The recently published atomic structures of myosin S1 (13) and the regulatory domain of scallop myosin (14) are consistent with this idea. Our results further indicate that neither lobe is sufficient and that both are necessary for the ELC-MHC association. When expressed in mlcE cells, DeltaC15 did not show significant binding to the MHC, whereas DeltaN11 and DeltaN28 retained low levels of association. It is possible that the COOH terminus of the ELC may contain a major binding site for the heavy chain, and the binding is strengthened by the association with the NH(2) terminus. A similar binding mode has been observed for smooth muscle regulatory light chain(37) .

Similar to the CaMbulletM13 peptide complex, the major contacts between ELC and MHC are hydrophobic(13, 14) . Phenylalanine 15, a conserved hydrophobic residue, is shown in this study to take part in the process. There are in total 204 contacts observed between the ELC and MHC(14) , implying that many residues of the ELC participate in the ELC-MHC association. A single amino acid substitution, therefore, might not be expected to produce a dramatic effect on binding, as observed for the F15A mutant, which caused a modest reduction in the binding affinity for the heavy chain.

The ELC has a dumbbell-like fold similar to that of CaM(13, 14) . Like the bound form of CaM, the helix in the linker region of ELC is unwound to facilitate binding of the two lobes on the heavy chain. It is likely, therefore, that deletion or insertion of residues in the linker region will lead to suboptimal contacts of the terminal domains with respect to the binding site on the heavy chain. This seems likely to provide a structural basis for the reduced binding affinity seen with the I4 and D4 mutations. The D4 mutant, in which only six out of 10 residues in the linker region are retained, still bound the MHC, albeit with a reduced affinity, suggesting that the linker segment must be flexible enough to tolerate significant shortening. In addition, the deletion or insertion of four residues in the linker domain does not seem to affect myosin enzymatic function as assayed by ATPase activity measurements and in vitro motility assays. Similar results were observed in mutagenesis studies of CaM, where it has been proposed that the linker domain functions as a flexible structure to accommodate binding of the two lobes to a range of substrates(31, 38, 39) . It is likely that the linker region of ELC may also function primarily as a structural element to place the two globular domains in an optimal contact with the heavy chain.

It has been suggested that one function of the myosin light chains is to provide structural support for the region of single alpha-helix of the heavy chain that exists between the head and the coiled-coil tail domain. One notion is that by stiffening the myosin neck, the light chains contribute to an extension of the effective power stroke resulting from conformational changes in the head(13, 33, 40) . If this is the case, then the I4 mutant, which extends the central linker domain but apparently has normal myosin function, must still provide adequate structural support. It would be interesting to determine how large a central linker region could become before it would no longer provide sufficient structural support.

The light chain binding region of the heavy chain, a long alpha-helix, is hydrophobic in nature and shielded from solvent by association with light chains(13, 14) . Thus, one apparent function of light chains is the stabilization of this structure. Consistent with this structural analysis, myosins lacking the RLCs have a tendency to aggregate through the neck region(33, 34, 35) . Our results show that the ability of ELC mutants to rescue the cytokinesis defect of mlcE cells correlates with their ability to bind to heavy chain, establishing that a stoichiometric binding of ELC is necessary in vivo for normal myosin function. On the other hand, the results from mutants F15A, I4, and D4 seem to suggest that as long as the stoichiometric association between ELC and MHC is maintained, myosin would be sufficiently functional to rescue the defects seen in the ELC null mutant. In addition, the similarity of gel filtration profiles of WT and mlcE cells suggests that the defects observed in mlcE myosin are unlikely to be due to abnormal aggregation of mlcE myosin. This raises the question of whether the ELC merely serves a structural support role for the neck of myosin, or might it have other functions? ELC binds to the heavy chain between the active site and the regulatory light chain(13) . It is possible that it may also play a role in modulating myosin enzymatic function and/or in transmitting regulatory signals from the regulatory light chain to the active center. With the atomic structures now in hand, further mutagenesis to explore other functions of ELC are in progress.

In summary, we have shown that residues at both termini of the ELC are necessary for the ELC-MHC association. These data provide direct experimental support for the structural models presented recently(13, 14) . Residues in the central linker domain do not appear to be essential for either ELC's binding or function, but they do seem to be required for the optimal association of the two terminal domains on the alpha-helix of heavy chain. Furthermore, a stoichiometric ELC-MHC association is necessary for normal myosin-dependent function in vivo.


FOOTNOTES

*
This work was supported in part by Grant GM-39264 from the National Institutes of Health (to R. L. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 312-503-4151; Fax: 312-503-5994.

(^1)
The abbreviations used are: MHC, myosin heavy chain; ELC, essential light chain; RLC, regulatory light chain; CaM, calmodulin; WT, wild-type; PCR, polymerase chain reaction; PIPES, 1,4-piperazinediethanesulfonic acid; BSA, bovine serum albumin; DAPI, 4`,6-diamino-2-phenylindole-2HCl.

(^2)
Chen, T.-L. L., Kowalczyk, P. A., Ho, G., and Chisholm, R. L., J. Cell Sci., in press.


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