(Received for publication, February 29, 1996, and in revised form, October 22, 1996)
From the Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, Illinois 60611
Myosin essential light chain (ELC) wraps around
an -helix that extends from the myosin head, where it is believed to
play a structural support role. To identify other role(s) of the ELC in
myosin function, we have used an alanine scanning mutagenesis approach
to convert charged residues in loops I, II, III, and helix G of the
Dictyostelium ELC into uncharged alanines.
Dictyostelium was used as a host system to study the
phenotypic and biochemical consequences associated with the mutations.
The ELC carrying loop mutations bound with normal stoichiometry to the
myosin heavy chain when expressed in ELC-minus cells. When expressed in
wild type cells these mutants competed efficiently with the endogenous ELC for binding, suggesting that the affinity of their interaction with
the heavy chain is comparable to that of wild type. However, despite
apparently normal association of ELC the cells still exhibited a
reduced efficiency to undergo cytokinesis in suspension. Myosin purified from these cells exhibited 4-5-fold reduction in
actin-activated ATPase activity and a decrease in motor function as
assessed by an in vitro motility assay. These results
suggest that the ELC contributes to myosin's enzymatic activity in
addition to providing structural support for the
-helical neck
region of myosin heavy chain.
Myosin is a mechanochemical enzyme that converts the energy of ATP hydrolysis into diverse actin-based cellular movements such as muscle contraction and cytokinesis (1, 2). Conventional myosin is composed of two heavy chains (~200 kDa) and two pairs of light chains (16-20 kDa), the regulatory light chain (RLC)1 and the essential light chain (ELC). Observed under electron microscopy, conventional myosin consists of two globular heads joined to a long rod-like tail. The globular heads contain the nucleotide binding sites and actin binding regions and are sufficient to generate movement (3, 4). One copy of each of the light chains associates with myosin head near the head-rod junction, also known as myosin neck (5, 6).
One striking feature of the recently reported crystal structures of
myosin is the neck domain, where the light chains arranged in tandem
with the ELC more proximal to the head wrap around a 10-nm long,
hydrophobic -helix that extends from the COOH terminus of the myosin
head (7, 8). An apparent function of light chains is the stabilization
of this
-helix. Consistent with this structural analysis, studies
have shown that myosins from which the RLCs are removed display
collapsed heads (9) and tend to form head-to-head aggregates (10-13),
presumably mediated by the exposed hydrophobic neck region. Myosin
aggregates also form in vivo when the RLC is eliminated
through a targeted gene disruption (14). Although no abnormal
aggregation is observed in ELC-deficient Dictyostelium
cells, the loss of RLC during myosin purification suggests that the
RLC-MHC interaction has a reduced affinity in the absence of ELC (15,
16).
In addition to protecting the -helix the light chains are postulated
to provide rigidity to the neck which is hypothesized to function as a
"lever arm" for generating an effective power stroke (7). This
lever arm concept is supported by the fact that weakening of the lever
arm by removal of one or both light chains causes 60-90% reduction in
the velocity with which myosin translocates actin filament in
vitro without significantly altering the actin-activated ATPase
(13). Shortening the lever arm 50% by deleting the RLC binding site
produces myosin that moves actin at one-half the wild type velocity in
the in vitro motility assays without a corresponding drop in
the actin-activated ATPase (17). Mutant myosin bearing light chain
binding domains of varying lengths translocate actin filaments in
in vitro assays at rates directly proportional to the length
of the postulated lever arm (18).
Although the contribution of the RLC to myosin's enzymatic activity has been well studied (19), the role of ELC in this respect is less understood. Early experiments showed that skeletal muscle myosin stripped of the ELC does not appear to affect the actin-activated ATPase (20, 21). In contrast, generation of ELC-deficient Dictyostelium cell lines by molecular genetic techniques has demonstrated the functional importance of the non-muscle ELC in vivo (15, 16). Biochemical analysis of myosin isolated from these ELC-deficient cells does not exhibit significant actin-activated ATPase activity. These data suggest that the ELC is required for actin-activated ATPase activity, but the interpretation is complicated by the apparently decreased stability of the myosin in the absence of the ELC and by a possible effect of ELC on RLC-MHC association (15, 16).
In an attempt to distinguish other possible role(s) of ELC from its structural function, we set out to screen for mutants that were able to stabilize the myosin neck but failed to exhibit normal myosin function. The clustered charged-to-alanine scanning mutagenesis technique was employed, for it has proven to be an efficient method to survey functional surfaces of a protein (22-25). The rationale for the method is that clusters of charged amino acid residues (i.e. two or more charged residues present in a stretch of five) are likely to occupy exposed positions, and substitution of these residues with alanines is structurally conservative at any residues that are not buried deep in the hydrophobic core and hence unlikely to disrupt the overall conformation of the molecule.
The ELC belongs to calmodulin and troponin C gene family and is composed of four helix-loop-helix motifs (26). Of the 14 charged clusters identified in the Dictyostelium ELC sequence, three fall in loop structures, and the others are in helices. To obtain the mutants that are able to stabilize the myosin neck, the surface residues of ELC which do not participate in the binding to the heavy chain were targeted for mutagenesis. The structure of myosin S1 (7) and scallop regulatory domain (8) shows the loops of the ELC to be solvent exposed. Thus the three charged clusters in loops (I, II, III) of the ELC were targeted for mutagenesis. In addition, a charged cluster in the G helix which is adjacent to the fourth loop was mutagenized.
When expressed in Dictyostelium ELC-minus cells, the ELC
carrying mutations in loops I, II, III bind to the heavy chain with normal stoichiometry. However, myosin carrying the ELC mutants exhibited decreased actin-activated ATPase activity and decreased motor
function as assessed by an in vitro motility assay. Cells expressing the mutants exhibited reduced efficiency in performing cytokinesis in suspension. These results provide biochemical and biological evidence that the ELC is important for normal enzymatic activity in addition to providing a structural support for the -helix of the myosin neck.
A full-length
Dictyostelium ELC cDNA (Ref. 27, accession no. M19337[GenBank];
Ref. 28, accession no. X54161[GenBank]) in plasmid BlueScript pKS (Stratagene,
La Jolla, CA) was used as a template for the polymerase chain
reaction-based mutagenesis (29). The pairs of primers (5, 3
) used to
introduce mutations include (all sequences are shown 5
to 3
; mutated
sequences are underlined): L1,
TT
AAT
GGTAAGGTCTCAGTTG and
ATT
AAAGATACTGAAACAT; L2,
TTT
ATCAATACATTAAAGAG and
AAA
AGCATTAATTTCAGTCT; L3,
GGCCATGGTACCATTCA and
CC
GAGGGCTTTGAATGCAT; L4,
CCGCT
GTT
TTATTTAAGGAAATCTC and
AAC
AGCGGTTGATAAGTAA. The primer
used to introduce the epitope tag (underlined) to the carboxyl terminus
of the L1, L2, and L3 cDNAs is
5
-CCGGATCCTTA
GAAACCGCCTAATGGAT-3
with a
BamHI site and 2 extra bases included at the 5
end for cloning purposes. All polymerase chain reaction products were sequenced
to confirm the presence of the desired mutations and the absence of
random polymerase chain reaction-induced mutations; they were
subsequently cloned into the integrating expression vector pBORP (30)
at the BamHI site.
Expression constructs were introduced into Dictyostelium AX3 cells or ELC-minus cells by electroporation (31). Transformants were selected and grown in HL-5 medium containing 10 µg/ml G418 and 100 µg/ml streptomycin.
Preparation of Cell ExtractsDictyostelium whole cell lysates were prepared as descried by Pollenz et al. (15). Myosin extracted from cytoskeletons was prepared according to Giffard et al. (32). Briefly, 1 × 107 cells were collected, washed in wash buffer (100 mM PIPES, pH 6.8, 2.5 mM EGTA, 1 mM MgCl2, 10 mM N-p-tosyl-arginine-methyl ester, 20 mM benzamidine), and lysed in wash buffer containing 0.5% Triton X-100. The Triton-insoluble pellet (cytoskeletons) was collected and resuspended in wash buffer containing 200 mM KCl and 2 mM ATP. Following centrifugation in a microcentrifuge, the supernatant was recovered, and one-fifth of it was used for analysis.
Western Blot AnalysisProtein samples were electrophoresed on a 15% SDS-polyacrylamide gel, transferred to nitrocellulose, and blotted with myosin polyclonal antiserum NU48. Antibody binding was detected using the Western blot Chemiluminescence Reagent (Du Pont NEN). For quantification of stoichiometry of myosin subunits, Western blots of myosin extracts from approximately 2 × 106 cells were scanned using a laser densitometer (LKB).
Protein PurificationWild type (JH10) and mutant myosins were purified according to the method described by Uyeda and Spudich (17) and Ruppel et al. (33), except cells expressing mutants were seeded from 40 confluent 24-cm plastic dishes into suspension (10 liters) and grown for approximately 40 h before harvesting for purification. Purified myosin was treated with a recombinant Dictyostelium myosin light chain kinase (a gift of Drs. J. Smith and J. A. Spudich, Stanford University) as described by Uyeda and Spudich (17). Myosin activity was assayed within 2 days of purification. Stoichiometry of myosin subunits was also determined by separating the heavy and light chains on 5-20% SDS-polyacrylamide gradient gels and staining the protein with Coomassie Brilliant Blue. Gels were scanned using a Bio-Rad densitometer and the Molecular Analyst software (Bio-Rad).
ATPase AssaysActin-activated ATPase was assayed in 10 mM Tris-HCl, pH 7.6, 25 mM KCl, 5 mM MgCl2, 0.1 mM CaCl2, 1 mM ATP, varying concentrations of actin (0-10 µM), and 50 µg/ml myosin. Ca2+-activated ATPase was assayed in 20 mM Tris-HCl, pH 8.0, 500 mM KCl, 10 mM CaCl2, 1 mM ATP, and 50 µg/ml myosin. Reactions were incubated at room temperature for 5 min, quenched with acid, and the liberated Pi was quantified following organic extraction (34, 35).
Measurement of Myosin Active HeadsThe number of myosin
active heads was measured using a protocol suggested by Dr. J. R.
Sellers (National Institutes of Health). 5 µg of purified myosin
(containing 20 pmol of heads) was incubated with 40 pmol of
[-32P]ATP in 100 µl of actin-ATPase assay buffer (no
actin) as above. Following a 20-s incubation at room temperature, 1 mM unlabeled ATP was added to chase the hydrolysis of the
labeled ATP. The liberated Pi was extracted after 10 min by
the organic partition method as above.
In vitro motility assays were performed as described previously (3, 36-38). Myosin was diluted to 200 µg/ml in buffer AB (25 mM imidazole, pH 7.4, 25 mM KCl, 4 mM MgCl2, 1 mM EGTA, 10 mM dithiothreitol), applied to a flow cell coated with nitrocellulose, and blocked with AB containing 0.5 mg/ml BSA (AB/BSA). To remove myosin heads that bound actin in a rigor fashion, a solution of phalloidin-labeled actin was perfused followed by 1 mM ATP in AB/BSA. After washing with AB/BSA to remove the excess nonfluorescent actin, a solution of rhodamine-phalloidin-labeled actin in AB/BSA was introduced. Active movement was initiated at room temperature by introducing AB/BSA containing 1 mM ATP and oxygen scavenger enzymes.
DAPI StainingDAPI staining was performed as described previously (15).
Charged residues in
loops I, II, III, and helix G of Dictyostelium ELC were
mutated by the polymerase chain reaction-based mutagenesis approach.
Three or four charged amino acid residues in each mutant were replaced
with alanines (Fig. 1). The approximate locations of
these charged residues are illustrated on the three-dimensional structure of chicken skeletal muscle myosin S1 head (Fig. 1). When
expressed in Dictyostelium ELC-minus cells using the
expression vector pBORP (30), the mutant ELCs accumulated to levels
similar to the endogenous ELC in wild type cells (Fig.
2A). All mutants bound to the heavy chain as
indicated by their ability to copartition with the MHC in ATP-extracted
cytoskeletal preparations (Fig. 2A). Based on densitometric
analysis of Western blots of these preparations, as well as direct
staining of purified protein with Coomassie Blue (Fig. 2B),
the loop mutants L1, L2, and L3 associated with the heavy chain with
1:1 molar ratio, whereas the helix mutant L4 bound at 0.2 mol/mol heavy
chain (Table I).
|
To assess the binding affinity of mutants L1, L2, and L3,
they were tagged at the carboxyl terminus by the addition of five amino
acid residues (EQKLI) of a myc epitope, which does not
affect the ELC-MHC association or myosin function (29). The tagged mutants were expressed in wild type cells using the pBORP vector. A
Western blot of the respective cell lysates shows the expression of the
mutants that have a larger size than the endogenous ELC (Fig.
3A). Analysis of supernatants from
ATP-extracted cytoskeletons indicates that the endogenous and the
tagged ELCs copartitioned (Fig. 3B), indicating that both
species associated with the MHC. The relative expression levels of the
tagged versus endogenous ELCs were 1:1 in cell lysates,
and similar ratios were obtained from purified myosin, suggesting that
the mutant ELCs have an affinity for the heavy chain similar to that of
wild type.
ELC Charge Mutants Have Defective Actin-activated ATPase Activities
Myosin was purified from both wild type and ELC mutant cell lines by a rapid purification method developed by Uyeda and Spudich (17) and Ruppel et al. (33). This method involves three rounds of salt-dependent assembly and disassembly of myosin filaments. Although we could not purify myosin from ELC-minus cells by this method, myosins carrying the ELC charge mutations were recovered as efficiently as wild type, indicating that they possessed assembly and disassembly properties typical of wild type myosin. The normal 1:1:1 stoichiometry of the three myosin subunits was maintained after purification (Fig. 2B).
When assayed for actin-activated ATPase, myosins with L1, L2, and L3 ELCs exhibited a 4-5-fold reduction in the activity compared with wild type (Table II). In contrast, the high salt Ca2+-ATPase, a property that assesses the integrity of the active site, was within the normal range for all three mutants (Table II), suggesting that each of the mutant myosins was capable of nucleotide hydrolysis. Because the actin-activated ATPase was greatly reduced in mutants L1, L2, and L3, it was important to know whether this was due to a significantly increased number of inactive molecules in the mutant myosin preparations. A crude assay was employed to determine the relative number of active heads in each myosin preparation. This assay was performed by incubating myosin with labeled ATP at 1:2 molar concentrations for a time duration short enough to allow each head the chance to hydrolyze a single labeled ATP. The amount of radioactive phosphate liberated therefore provides a crude assessment of the relative number of active heads. As shown in Table II, myosin preparation containing ELC mutants L1, L2, and L3 showed 81, 66, and 118% of the number of active heads of wild type preparations. Thus it is unlikely that the reduced actin-activated ATPase is due to a decreased stability of the mutant myosins but rather is the result of a decreased turnover rate of the enzyme.
|
Like other non-muscle myosins, the actin-activated ATPase of
Dictyostelium myosin is regulated by RLC phosphorylation
(39). Myosin purified by the rapid method has a low level of the
phosphorylated RLC (33). When these myosins were treated with a
recombinant myosin light chain kinase (40), the actin-activated ATPase
activity of wild type was increased to 163 nmol/min/mg, representing an approximately 3-fold enhancement (Table II and Fig. 4).
The activity of the mutants also increased 3-4-fold after
phosphorylation (Table I), but the Vmax was
reduced greatly relative to wild type (Fig. 4). The kinase treatment
resulted in uniformly high levels of RLC phosphorylation in both wild
type and mutant myosins (data not shown). Analysis of the
Km for actin on kinase-treated myosins yielded
similar values between the mutants and wild type (Table II), suggesting
that the reduced actin-activated ATPase activity is not due to a
decreased affinity for actin. When examined for low salt
Mg2+-ATPase, the wild type and mutant myosins exhibited
similar basal activities of 6-8 nmol/mg/min.
There is a good correlation between the actin-activated ATPase activity
and the motor function of myosin as assayed by in vitro
motility assays (1). But uncoupling of the two events has been observed
in several recent studies (13, 17, 41). When analyzed by an in
vitro motility assay, myosins containing ELC charge mutations
moved actin filaments at rates of 0.58-0.84 µm/s compared with 2.1 µm/s for wild type (Fig. 5). The mutant myosins did
not generate significant actin movement without prior treatment with
myosin light chain kinase.
Dictyostelium Cells Expressing the Mutant ELCs Perform Cytokinesis with Reduced Efficiency
Myosin is essential for cytokinesis of
Dictyostelium when grown in suspension (14-16, 42-44).
When cells expressing the mutants L1, L2, and L3 were analyzed for
their ability to perform cytokinesis in suspension, many cells with
multiple nuclei were observed (Fig. 6). The cytokinesis
defect was quantified further by scoring the percentage of
multinucleate cells (with three or more nuclei) in each cell
population. As shown in Fig. 7, cells carrying L1, L2,
and L3 mutants contained 30, 40, and 28% multinucleated cells, in
contrast to 5-6% observed in ELC-minus cells expressing a wild type
copy ELC or parental JH10 cells. Furthermore, the phenotype worsened
progressively, and by day 6 the multinucleate population rose to
50-60% the majority of which contained six or more nuclei. Those big
cells eventually lysed. Thus it is clear that the L1, L2, and L3
mutants show decreased myosin function in vivo.
It has been hypothesized that myosin essential light chains play a
structural support role for the neck of myosin, perhaps by providing
added rigidity for the proposed lever arm function of the neck (7, 8).
This concept is reinforced by the atomic resolution structures showing
that the ELC envelopes a naked -helix that forms the backbone of the
neck domain (7, 8), as well as biochemical studies that show a linear
relationship between the length of the light chain binding domain and
the rate with which actin filaments are translocated using in
vitro motility assays (18). In an attempt to explore other
possible role(s) of ELC, a site-directed mutagenesis approach was taken
to identify the mutants that bind to heavy chain, and therefore
maintain the stability of the neck, but which otherwise impair myosin
function. The results showed that substitution mutations of the charged residues in loops I, II, and III of Dictyostelium ELC did
not affect the ability of the ELC to bind the heavy chain. However, myosin carrying these mutations exhibited a 4-5-fold reduction in
actin-activated ATPase activity which was coupled to a 2.5-3.6-fold reduction in the velocity of actin filament translocation
in vitro. These defects in myosin produced
Dictyostelium cells with reduced cytokinesis efficiency.
Assessment of the affinity of mutant ELCs for the heavy chain using
in vivo competition showed that mutant ELCs compete
efficiently with the endogenous wild type ELC, suggesting that the MHC
binding affinities are similar. The depressed actin-activated ATPase
activity therefore seems unlikely to be caused by gross structural
changes or inappropriate binding of the ELC to the -helix of MHC.
Instead the defects observed most likely result from local changes in charge distribution or side chain orientation in the ELC mutants. Because myosin carrying the mutant light chains could be purified through rounds of filament assembly and disassembly, the
charge-to-alanine substitutions do not affect myosin filament formation
significantly. Furthermore, the stability of purified mutant myosins,
measured as the number of active heads relative to the wild type,
showed levels of 81, 66, and 118%. The slightly reduced number of
active heads observed in the L1 and L2 myosin preparations is not
sufficient to produce the 4-5-fold reduction observed in the
actin-activated ATPase. Similar to wild type, the loop mutants showed a
3-4-fold enhancement of activity following myosin light chain kinase
treatment, indicating that the mutations did not alter the regulation
associated with the RLC phosphorylation but instead lowered the basal
rate of actin-activated ATP hydrolysis. Because the mutant myosins had
a Km for actin similar to that of wild type, the reduced basal rate of ATP hydrolysis was not due to a change in the
affinity for actin.
What mechanism might account for the depressed actin-activated ATPase seen with the ELC charge mutations? The atomic structure of myosin S1 head suggests that the ELC does not contribute directly to formation of the active site. However, based on the chicken S1 structure, the ELC has a substantial protein-protein interface with amino acid residues 720-730 of the heavy chain (7, 45). In addition, the recently reported structures of the Dictyostelium motor domain (46, 47) suggest that the COOH terminus exists in dramatically different positions in MgADP·BeFx and MgADP·AlF4 structures thought to mimic the ATP bound and transition state for hydrolysis, respectively. This arrangement would allow movements of this domain to be transduced to the ELC which may in turn modulate or amplify the conformational changes in the active site during the binding and release of nucleotides (7, 48), thereby facilitating the ATPase cycle. Thus finding that mutations in the ELC reduce the basal rate of actin-stimulated ATP hydrolysis supports the idea that the ELC could in some way modulate the communication between the active site and other domains of the head such as the actin binding face. Since the ELC has not been shown to contain known regulatory elements, it is plausible that the MHC head together with the ELC determine the basal activity of the actin ATPase. Consistent with this idea, removal of the neck domain (which binds ELC) produced a motor domain that exhibits higher actin-activated ATPase activity than does intact myosin (49, 50).
Fisher et al. (46) have suggested that the ELC may be
important for stabilizing the COOH-terminal regions of the motor domain and perhaps in transmitting the conformational changes induced by ATP
hydrolysis. The results presented here provide direct experimental support for this idea. It is interesting to note that the mutations in
L3 are located along an interface between the ELC which has also been
shown to contain several -MHC mutations that have been implicated in
familial hypertrophic cardiomyopathy (51). This result raises the
possibility that mutations in the ventricular ELC (vMLC1) might also
lead to cardiac defects. Mutations of both the ELC and RLC have
recently been shown to produce hypertrophic cardiomyopathy (52). One of
the ELC mutations identified in this study is located at a position
very near the L3 mutation described here.
Based on the S1 structure, the L3 mutation, which is in the COOH-terminal lobe of the ELC, directly interfaces with the NH2-terminal domain of the heavy chain; whereas the L1 and L2 mutations, which are in the NH2-terminal lobe of the ELC, do not seem to contact that domain (Fig. 1 and Ref. 7). Nevertheless, a similar degree of reduction in the actin-activated ATPase activity in all three mutations was observed, suggesting that either there is a significant conformational change to allow the NH2-terminal lobe of the ELC to interact with the motor domain of the HC, or there is a cooperativity between the two lobes of the ELC during the ATP hydrolysis. It is difficult to distinguish between these two possibilities with biophysical methods currently available. However, a gross S1 shape transition at its distal region, where the ELC resides, has been suggested to occur during the ATP hydrolysis based on modeling of small angle synchrotron x-ray scattering data (48). A major domain-domain rearrangement such as would occur in this scenario has been observed in the crystal structures of elongation factor Tu during the binding and release of nucleotide (53).
Why the polar loops of the ELC have the observed effect on the actin-ATPase is not clear. One possibility is that because of their flexible nature, the polar loops could represent surfaces for domain-domain interactions without altering the folding of the main chain of the individual domains, which otherwise would not be possible. Such interdomain interactions at polar interfaces are evident in the domain rearrangements of elongation factor Tu (53).
Transient kinetic studies of the ATP hydrolysis of myosin establishes that ATP binding and hydrolysis occur at fast rates to produce ADP and Pi at the active site and that slow product release limits the steady-state rate in the absence of actin (54, 55). Although myosin carrying the ELC charge mutations does not affect the affinity for actin, this does not preclude the possibility that the ELC acts to facilitate the product release at the active site induced by actin binding. Detailed kinetic studies are necessary to dissect the role of ELC in the process.
The data presented here suggest that the ELC in a non-muscle system may play a significant role in determining the rate of actin-activated ATP hydrolysis. These are in contrast to its less significant role observed in skeletal muscle myosin (20, 21). It is interesting that the RLC regulates the actin-activated ATPase of both smooth muscle and non-muscle myosins but not the skeletal myosin (19), a feature that seems to correlate with functional significance of their counterpart ELC in these systems. Regulation of skeletal muscle myosin is governed by the thin filament-associated troponin-tropomyosin system. It is possible that in smooth muscle and non-muscle systems where myosin is regulated by its RLC subunit, the ELC may participate in more aspects of myosin function than merely as a structural support, especially considering the fact that it is located between the RLC and the active site (7, 8).