(Received for publication, September 18, 1995; and in revised form, January 17, 1996)
From the
The complete sequence of the Dictyostelium myo J heavy
chain gene has been determined from overlapping genomic clones. The
gene spans 7400 base pairs, is split by two small introns, and
encodes a 2241-residue, 258-kDa heavy chain polypeptide that that is
composed of an N-terminal 944-residue myosin head domain, a central
863-residue domain that is predicted to form an
helical
coiled-coil containing six hinges, and a C-terminal 434-residue
globular domain. The head domain is notable in that it contains a
30 residue insert near the nucleotide binding pocket, and five
potential calmodulin/myosin light chain binding sites at the head/tail
junction. The existence within the Myo J tail domain of both an
extensive coiled-coil structure and a large globular domain suggests
that this myosin is dimeric and incapable of self-assembly into
filaments. While these properties, as well as the overall predicted
structure of the Myo J protein, are reminiscent of class V myosins, the
sequence of the 434-residue globular tail piece of Myo J shows no
similarity to that of either yeast or vertebrate myosins V. Consistent
with this, phylogenetic analyses based on myosin head sequence
comparisons do not classify Myo J as a type V myosin. These and other
sequence comparisons indicate that Myo J and two as-yet-unclassified
unconventional myosins from Arabidopsis represent members of
the newest class within the myosin superfamily (class XI). Northern
blots analyses suggest that Myo J may function predominantly in
vegetative Dictyostelium cells. Finally, Southern blot
analyses suggest that Dictyostelium possesses another myosin
that is very closely related to Myo J.
To date, 10 classes of myosin have been identified, nine of
which are considered to be
unconventional(1, 2, 3, 4, 5) .
Like the myosins I, which were the first unconventional myosins
identified(6) , all of these unconventional myosins contain
within their heavy chains the 80-kDa mechanochemical domain that
corresponds in sequence to the globular head of conventional,
filament-forming myosins (myosin II). The sequences that comprise the
remainder of their heavy chains are, on the other hand, widely
divergent. While these sequences, which always extend C-terminal of the
motor domain and in some cases N-terminal as well, are similar between
members of the same class of unconventional myosin, they are unique
relative to all other classes. These class-specific sequences are
thought to confer functional specificity on a more or less generic
motor domain by mediating specific interactions with different cellular
proteins, structures, or membranes (for review, see (7, 8, 9, 10, 11) ).
The cellular slime mold Dictyostelium discoideum represents a reasonable system for the analysis of unconventional myosin structure and function because this ameboid cell demonstrates many different types of cellular and intracellular motility(12) , and because it exhibits reasonable frequencies of homologous recombination, allowing null mutations to be created by targeted gene disruptions(13) . This approach, when coupled with the identification of the behavioral abnormalities exhibited by these myosin-deficient cell lines, should allow conclusions to be drawn regarding myosin function in vivo. To date, a single myosin II heavy chain gene (mhc a) and five myosin I heavy chain genes (myo A-E) have been cloned from Dictyostelium and completely sequenced(14, 15, 16, 17, 18, 19) . While analyses of myosin II-deficient cells have yielded a wealth of information about the function of this class of myosin in vivo (for reviews, see (20) and (21) ), the identification of myosin I-dependent functions has been complicated by the fact that at least some of the many myosin I isoforms expressed by this organism probably have partially overlapping functions(18, 22, 23, 24) . The analysis of cell lines in which multiple, closely related myosin I isoforms have been eliminated from the cell is yielding more useful results, however(25, 26) .
Southern blot analyses(18) , together with the recent analysis of a Dictyostelium YAC library(27) , indicate that this organism contains a total of 10-13 actin-based motor proteins (mhc a and myo A-L). While these additional four to seven uncharacterized myosin genes may simply represent additional myosin I heavy chain isoforms, it seems reasonable to suspect that at least some of them represent members of other classes of unconventional myosin, especially since several of these classes (e.g. myosin V) have been identified in both lower and higher eukaryotes(28, 29, 30, 31) . We set out, therefore, to identify within this group of four to seven putative myosin genes any that encode heavy chains other than for a type I myosin. Here we present the complete sequence of the Dictyostelium myo J heavy chain gene. The deduced amino acid sequence of the Myo J heavy chain polypeptide predicts a high molecular weight, dimeric myosin that is probably incapable of self-association into filaments and that may be regulated by calcium ions through multiple, heavy chain-associated calmodulin ``light chains.'' Myo J represents, therefore, the first unconventional myosin identified in this model system that is not a type I myosin. While the overall predicted structure of the Myo J protein is similar to that of class V myosins(32) , sequence comparisons indicate that Myo J is not a type V myosin. Moreover, Myo J does not fall neatly into any of the other nine classes of myosin identified to date. Therefore, by the criteria used to group previously identified myosins into 10 classes, Myo J represents the newest class within the myosin superfamily (class XI). Other sequence comparisons suggest that MYA1 and MYA2, two recently identified unconventional myosins from Arabidopsis(33, 34) , may also be class XI myosins. Finally Southern blot analyses indicate that Dictyostelium probably possesses a second type XI myosin gene. The analysis of cells in which both genes have been disrupted will hopefully provide definitive information regarding the function of class XI myosins in vivo.
Figure 6: Dot matrix comparisons between the globular tail domain sequences of the vertebrate and yeast myosins V, Myo J, and Arabidopsis MYA1 and MYA2. Only the globular tail domain portions of these myosins are compared here. The window size was 11 residues, and the minimum acceptable score was 7 matches out of 11. Conservative substitutions, as defined by the PAM matrix, were counted. Chicken p190 refers to the myosin V homologue from chicken (29) .
Figure 1:
Southern blot analyses. Lanes 1 and 2, whole Dictyostelium genomic DNA was
digested with EcoRI and probed at moderate stringency with
restriction enzyme fragments containing portions of the coding sequence
surrounding the ATP binding site of Dictyostelium myosin II (lane 1) and Myo E (lane 2). The EcoRI
fragments that correspond to myo A through myo E are
indicated. The fragment assignments for myo C, myo E,
and the putative myosin gene corresponding to the 13.5-kb band
were confirmed by pulse field gel electrophoresis (data not shown). The
strong band at
0.56 kb in lane 1 is from the myosin II
gene. Lanes 3-6, whole Dictyostelium genomic
DNA was digested with EcoRI, SstI, ClaI, and XbaI, respectively, and probed at moderate stringency with DNA
fragments that together span most of the coding sequence for the
coiled-coil tail domain of Myo J (Probe A in Fig. 2). Lanes 7-10, whole genomic DNA was digested with EcoRI, BclI, SstI, and XbaI,
respectively, and probed at moderate stringency with a DNA fragment
that spans most of the coding sequence for the globular tailpiece of
Myo J (Probe B in Fig. 2). The size standards for lanes 1 and 2, and for lanes 3-10, are
included.
Figure 2: Schematic depicting the strategy used to obtain overlapping genomic clones that span the myo J heavy chain gene (A) and the predicted domain structure of the Myo J heavy chain polypeptide (B). In A, 3.2 RI, 2.7 RI, 1.9 XBA, and 4.5 RI are the four overlapping genomic clones that span the myo J heavy chain gene. PCR 1.1 was the PCR-generated fragment used to identify the 2.7 RI genomic clone. Probe A and Probe B were the probes used in the Southern blots in Fig. 1(lanes 3-10; see ``Materials and Methods'' for details). Probe A was also used for the Northern blots in Fig. 8.
Figure 8: Northern blot analyses. Lane 1, total RNA from vegetative Dictyostelium cells was probed at high stringency with probe A, which is a gene-specific probe that contains most of the coding sequence for the coiled-coil domain of Myo J (see Fig. 2). The migration of the RNA standards is indicated. Lanes 2-6, 5 µg of total RNAs from cells starved for 0, 3, 6, 9, and 12 h were hybridized with probe A.
In order to clone the gene (or
portion of the gene) that corresponded to the 3.2-kb EcoRI
band, an EcoRI genomic sublibrary was created. Specifically,
genomic DNA was cut preparatively with EcoRI, fractionated by
agarose gel electrophoresis, and DNA fragments ranging in size from
3.0 to
3.4 kb were obtained by electroelution. These
fragments were then cloned into EcoRI-cut
ZAP, and the
subsequent sublibrary was probed with the 560-bp myosin II probe. As
expected, this approach yielded a large number of genomic clones that
contained the 3.2-kb EcoRI insert of interest (Fig. 2).
After partial DNA sequence analysis indicated that this 3.2-kb EcoRI fragment contained the coding sequence for the
N-terminal two-thirds of a myosin head domain (see below), efforts were
directed at obtaining the remainder of the gene. Because of the extreme
AT bias of Dictyostelium DNA(12) , large fragments of
cloned Dictyostelium genomic DNA are very unstable. This fact
precludes the use of cosmid libraries or standard phage libraries
(containing 20-kb inserts) and usually forces one to engineer
chromosome walks in small steps of several kilobase pairs each. In the
case of Myo J, the size of the Myo J transcript (see below) suggested
that at least
5 kb of 3` DNA would be required to complete the
coding sequence. As a first step, PCR was used to create a probe that
would identify the genomic EcoRI fragment that lies
immediately 3` of the 3.2-kb EcoRI fragment. This probe was
generated using an exact primer corresponding to sequences lying
200 bp from 3` end of the 3.2-kb EcoRI fragment and a
degenerate primer corresponding to a highly conserved myosin sequence
found near the C-terminal end of the head domain (and predicted to be
900-bp 3` of the 3` end of the 3.2-kb EcoRI band). The
1.1-kb PCR fragment that was obtained with these primers and whole
genomic DNA as a template (Fig. 2, 1.1 PCR) was found
to recognize, in EcoRI genomic Southern blots, a 2.7-kb EcoRI band, as well as the 3.2-kb EcoRI band
identified first (data not shown). This 2.7-kb EcoRI band was
cloned by again creating a genomic sublibrary, this time using EcoRI genomic fragments ranging in size from
2.5 to
2.9 kb. Restriction enzyme analysis of the 2.7-kb EcoRI
genomic clone obtained revealed an XbaI site
450 bp from
the 3` end of the 2.7-kb EcoRI fragment (Fig. 2). This
450-bp XbaI/EcoRI fragment recognized a 1.9-kb XbaI band in genomic Southern blots (data not shown). The
1.9-kb XbaI fragment was obtained from a genomic sublibrary
created using XbaI-cut genomic DNA fragments ranging in size
from
1.7 to
2.1 kb and cloned into XbaI-cut
ZAP
II. The final 3` genomic clone was identified using as a probe a
950-bp EcoRI/XbaI fragment from the 3` end of
the 1.9 XbaI clone (Fig. 2). This probe recognized a
4.5-kb EcoRI band in genomic Southern blots (data not shown),
which was obtained from a genomic sublibrary created using EcoRI fragments ranging in size from
4.3 to
4.7 kb.
Northern blot analyses performed with various restriction enzyme
fragments obtained from this 4.5-kb EcoRI clone revealed that
the Myo J heavy chain coding sequence ended near the middle of the
4.5-kb insert (data not shown).
Figure 3:
The complete deduced amino acid sequence
of the Myo J heavy chain polypeptide. The DNA sequence, which includes
two small introns that interrupt the coding sequence near the 5` end
(the positions where they interrupt the coding sequence are marked
here; ), has been submitted to the GenBank data base. The
30-residue insertion near the ATP binding pocket (residues
208-237) and the five IQ motifs in the neck domain (residues
821-939) are underlined. The sixth putative IQ motif,
which is not underlined, begins at residue 940 and overlaps
the first 18 residues predicted to be coiled-coil. The seven discrete
segments of
helical coiled-coil (CC
1-CC
7 in Fig. 2) are indicated by a double underline. The
reactive thiols are marked with asterisks.
With regard to the myosin head domain, pairwise alignments with other myosin head sequences show that Myo J contains all of the highly conserved regions of sequence common to myosin motor domains ((7) ; data not shown). The Myo J head sequence does contain several interesting features, however. First, the flexible ``25 kDa/50 kDa'' loop that occurs just C-terminal of the conserved GESGAKT sequence contains considerably more residues in Myo J than in most other myosins (Fig. 3). For example, Myo J contains 64 residues between the T of GESGAKT and the beginning of the next highly conserved sequence below the ``25 kDa/50 kDa'' loop (LEAFGNAK), while Dictyostelium Myo A, B, C, D, and E and myosin II contain 33, 33, 39, 33, 36, and 34 residues, respectively, over this same distance. Thus, while different myosins do vary considerably in both the number of residues that comprise the ``25 kDa/50 kDa'' loop, and in the actual sequences of the loop, Myo J appears to be near the extreme end of the spectrum in terms of the size of this loop. The functional significance, if any, of differences in the size and sequence of this loop is not known, however (7, 44) . Second, the sequence of the Myo J head domain begins at a position analogous to that of conventional myosins(7) . In this regard, therefore, Myo J is more similar to the head domain of type II myosins than type I myosins, which begin at a position corresponding approximately to residue 75 in conventional myosin head sequences(7) . Third, Myo J has cysteine residues in both of the positions (residues 725 and 735) that correspond to the reactive thiols in muscle myosins (Fig. 3). This fact contrasts with many unconventional myosins that lack one or both cysteines residues in these positions(7, 8) .
With regard to
the neck domain, between residues 821 and 939 there are five imperfect
25-residue repeats that occur in tandem and that correspond to the
``IQ motif'' described by Cheney and Mooseker (9) (Fig. 4A). This motif, which is
characterized by a conserved IQ (or LQ) pair and basic residues at two
conserved positions C-terminal of the IQ pair, has been implicated as
the heavy chain binding site for authentic calmodulin and for myosin
light chains (for review, see (9) and (45) ). In
addition to these five IQ repeats, there is a potential sixth repeat
(residues 940-962) that immediately follows the fifth repeat.
Whether or not this sixth repeat is a bona fide calmodulin/myosin light
chain binding site is in question, however, since coiled-coil
prediction programs (see below) indicate that the coiled-coil begins at
residue 945. In summary, therefore, Myo J may bind per heavy chain a
total of five to six light chains, which could be either authentic
calmodulin, myosin light chains (which are members of the calmodulin
superfamily), or a combination of both.
Figure 4: The sequences of the six IQ motifs in the Myo J heavy chain (A) and the major region of sequence similarity identified by the BLAST program between the globular tailpiece sequences of Myo J and those of Arabidopsis MYA1 and MYA2 (B). In A, the IQ pairs and the conserved basic residues C-terminal of them are in bold and italics. The consensus IQ motif is from Cheney and Mooseker (9) . In B, exact matches are connected by bars, while conservative substitutions are connected by colons. The residue positions that these sequences correspond to in their respective heavy chains are indicated.
With regard to the central
coiled-coil domain, the program of Lupas et al.(39) ,
which predicts the probability of sequences forming helical
coiled-coils, indicates that most (but not all) of the Myo J sequence
between residues 945 and 1807 would form this supersecondary structure
(a probability of >90% was used as a cutoff for defining segments of
coiled-coil structure) (Fig. 5). Those areas that should not
form coiled-coil exist as six short stretches of sequence that break up
the coiled-coil into seven discrete regions (CC
1 through
CC
7; see Fig. 2B and 3). These six hinges range
in size from 16 to 25 residues. If all seven coiled-coil regions were
actually to dimerize, and if one uses a rise of 0.1485 nm per residue
in the coiled-coil structure(46) , then the central domain of
Myo J should fold into a rod that is
110 nm in length and contains
six discrete bend sites. Furthermore, if the central domain of Myo J
does form an extensive coiled-coil, which is likely, then the Myo J
motor protein should be dimeric, i.e. two-headed.
Figure 5:
Presence of helical coiled-coil
domains within the Myo J heavy chain polypeptide. The program of Lupas et al.(39) was used to estimate the probability that
the Myo J heavy chain sequence forms
helical coiled-coil. The
plots indicate the probability from 0% to 100% (see scale). For
comparisons sake, the complete heavy chain amino acid sequences of Dictyostelium myosin II ((14) ; MYOSIN II), Dictyostelium Myo B ((16) ; MYOSIN I), and
chicken myosin V ((29) , MYOSIN V) were also analyzed.
The tail of myosin II forms an almost uninterrupted
helical
coiled-coil, the tail of myosin I is devoid of coiled-coil, and the
tail of myosin V contains a central coiled-coil domain that is
interrupted in numerous places.
With
regard to the remaining 434 residues of the Myo J heavy chain (1808
through 2241), the program of Lupas et al.(39) reveals that this region exhibits no tendency to form
coiled-coil. This 48-kDa C-terminal domain may, therefore, form a
globular structure, as do the last
410 residues of type V
myosins(32) . The apparent existence of this large globular
tailpiece, which in the folded Myo J molecule would exist as a pair of
globular domains C-terminal of the central rod, would appear to
preclude the ability of Myo J to form filaments. We predict, therefore,
that Myo J is not only two-headed, but is also incapable of
self-assembly into filaments. Both of these are demonstrated properties
of type V myosins(29, 32) .
To search for other sequences in the data base (both myosin and non myosin) that might exhibit significant similarity to the globular tailpiece sequence of Myo J, the program BLAST(37) , which looks for local regions of homology, was used. This program did not identify any regions of significant sequence similarity between Myo J and any myosin that has previously been placed within the 10 current classes. The Myo J globular tailpiece also showed no regions of significant local sequence similarity with any non myosin sequence in the data base. The BLAST program did yield two sequences with reasonable scores, however, both of which are recently sequenced (and as-yet-unclassified) unconventional myosins from the plant Arabidopsis thaliana. MYA1 and MYA2 are closely related Arabidopsis myosin heavy chains whose overall deduced structure is again reminiscent of type V myosins, but whose globular tail domain sequences show only very limited similarity to that of vertebrate and yeast type V myosins(33, 34) . Furthermore, while Kinekema et al.(34) did not formally classify MYA1 and MYA2, phylogenetic analyses based on myosin head sequence comparisons do not group the Arabidopsis myosins with the myosins V ((34) ; see below). The local regions of sequence similarity between Myo J and MYA1/MYA2 that were identified by the BLAST program are concentrated in the C-terminal half of the tailpiece and are evident in the dot matrixes (Fig. 6, E and F), where an identity line containing many interruptions is present in the second half of both matrixes. Fig. 4B shows the pairwise alignment of Myo J with MYA1 and MYA2 across the largest region of sequence similarity within the globular tailpiece. These alignments suggest that Myo J and MYA1/MYA2 are related.
Figure 7: Rooted phylogenetic tree of myosin head domain sequences. The tree was generated using the neighbor joining program Clustal V(40) , with branch lengths corrected by the method of Kimura(41) . The sequence divergence between any pair of sequences is equal to the sum of the horizontal branch lengths connecting the two sequences. The tree was rooted using the longest branch (Nina C). The bootstrapping values at each node indicate the number of times out of 1000 data resamplings that the sequences below a given node clustered together. Bootstrapping values that are greater than 950 indicate a significant relationship. The different classes of myosins (I-XI) are indicated to the right. The extra bracket that encompasses both class V and class XI myosins (labeled Class V?) is meant to indicate the possibility that, as more sequences are added to the tree and/or other factors are considered in classification (e.g. function), class XI myosins may be reassigned at a later date as class V myosins (albeit divergent ones). To obtain the accession numbers for these sequences, see (47) .
With regard to type V myosins, a bootstrapping value of 996 at the node that joins the vertebrate myosins V (mouse dilute and chicken myosin V) and the yeast myosins V (Myo 2 and Myo 4) strongly supports the idea that these four myosins are all members of the same myosin class (Fig. 7) (for details, see Refs. 1, 2, and 29). Myo J, on the other hand, clearly does not cluster with the type V myosins. This result, together with the globular tail domain sequence comparisons described above, indicates that Myo J is not a type V myosin. Fig. 7also shows that Myo J does not fall into any of the other nine classes of myosin already identified. Myo J appears, therefore, to represent a new class (class XI) within the myosin superfamily. Fig. 7also shows that Myo J does cluster in 810 out of 1000 random reshufflings with a branch containing the Arabidopsis myosins MYA1, MYA2, and MYA3 (note that while MYA3 groups with MYA1 and MYA2 in head sequence comparisons, its tail sequence is very different from that of MYA1 and MYA2; see (34) ). While the pairing of Myo J with MYA1 and MYA2 in the tree is below the standard level for significance, it is consistent with the globular tailpiece sequence comparisons between Myo J and both MYA1 and MYA2 (Fig. 4) and suggests that Dictyostelium Myo J and Arabidopsis MYA1 and MYA2 are all type XI myosins.
Myo J represents the first myosin heavy chain gene identified in Dictyostelium that is neither a class I or class II myosin. While the domain structure of the Myo J heavy chain and the predicted properties of the folded protein resemble those of type V myosins(32) , the phylogenetic program that is currently being used to classify myosins according to their head sequences (Clustal V; (40) ) indicates that Myo J is not a type V myosin. Consistent with this, the sequence of the Myo J globular tailpiece shows no significant similarity to the globular tailpiece sequences of the bona fide vertebrate and yeast myosins V. Myo J also does not cluster with any of the other nine classes of myosin identified to date. Therefore, by the criteria used to define the previous 10 classes of myosin, Myo J represents a member of a new myosin class, class XI. We also found that some regions of the Myo J tailpiece show limited similarity to the globular tailpiece sequences of MYA1 and MYA2, two unconventional myosins from Arabidopsis that have not yet been formally classified, but clearly do not group with type V myosins in phylogenetic analyses(33, 34) . Furthermore, head sequence comparisons performed using Clustal V place Myo J closest to MYA1 and MYA2. For the moment, therefore, we are classifying MYA1 and MYA2 along with Myo J as type XI myosins (see also (47) ).
While it is true that these class XI myosins probably have the physical properties of class V myosins (i.e. two-headed and nonfilamentous), these are also the predicted properties of class VI (48, 49) and class VIII (3) unconventional myosins, and possibly class VII myosins (50) as well. These properties appear, therefore, to be a common theme among distinct classes within the myosin superfamily. Having said this, however, we cannot completely exclude the possibility that Myo J, MYA1, and MYA2 are actually class V myosins (albeit with very divergent tail domains). The area of the phylogenetic tree that contains the four bona fide myosins V, Arabidopsis MYA1 and MYA2, and Dictyostelium Myo J is one that at the moment is difficult to interpret unequivocally. The addition of more sequences to the tree should allow a more confident assignment of Myo J in the future. In addition, future studies of the physiological roles of myosin in vivo may force a partial redefinition of the myosin superfamily that takes into account similarities in intracellular function. For example, two-headed myosins that do not self-assemble may be required features of actin-based motors that serve as intracellular vesicle motors.
When Dictyostelium amoebae are starved, they undergo a
24-h-long process that involves chemotactic aggregation and
differentiation of the aggregate into a fruiting body composed of a
stalk supporting a spore-filled sac(12) . Many cytoskeletal
protein genes are strongly up-regulated during the early stages of
development (5-10 h) when cells become highly motile and are
chemotaxing toward cAMP(12) . For example, the steady state
level of the Myo B heavy chain transcript at 10 h of development is
7-fold higher than in vegetative cells(16) . This enhanced
gene expression is mirrored in elevated levels of Myo B heavy chain
protein (25) and is consistent with the demonstrated role of
Myo B in the locomotion of aggregation-stage
cells(22, 23, 25) . In contrast to Myo B, Myo
J mRNA levels fall fairly dramatically from vegetative cell levels as
cells progress through chemotactic aggregation and early development.
This result suggests that Myo J's primary function may be in
actively growing cells.
A combination of approaches will probably be required to elucidate the function(s) of Myo J in vivo. For example, the localization of the protein at the light and electron microscopic levels, and the biochemical identification of cellular proteins/structures that bind to its globular tail domain, should provide important clues as to the function of Myo J. Finally, the generation of Dictyostelium cell lines that lack Myo J, using either gene targeting techniques or antisense RNA approaches, and the identification of the behavioral abnormalities exhibited by these mutant cells, will hopefully allow firm conclusions to be drawn regarding Myo J function in vivo. Along these lines, however, we found strong evidence that Dictyostelium contains another myosin heavy chain that is closely related to Myo J and that may overlap functionally with it. In this vein, while single mutants of some myosin I isoforms have given reasonable phenotypes(22, 23, 24) , single mutants of other isoforms have not(17, 18) , presumably because of functional redundancy. Furthermore, while some of the defects exhibited by myosin I single mutants are clearly scorable(22, 23, 24) , they are often not extremely striking. This is again probably due to functional overlap between-closely related myosin I isoforms. To clearly define the functions of class XI myosins in Dictyostelium it may be necessary, therefore, to create single cells that lack both myosin XI isoforms. DNA fragments that encode the globular tailpiece of Myo J will be useful in cloning the putative second myosin XI isoform.
The presence of the five to six IQ repeats within the sequence of Myo J, together with recent studies of vertebrate myosins I and the myosin V homologue from chicken (see (45) for review), suggest that Myo J may bind numerous calmodulin molecules (up to 12/folded dimeric molecule) within the ``neck'' domain of the molecule. These calmodulins could play a critical role in regulating the ATPase and/or mechanochemical properties of Myo J. Additionally, Myo J may influence to a considerable extent the intracellular localization of calmodulin, much as do a yeast myosin V homologue (Myo 2) (51) and the 174-kDa Nina C myosin isoform in Drosphila photoreceptor cells(52) . In this regard, it will be interesting to see if Myo J plays a role in contractile vacuole function, since calmodulin is highly concentrated on the contractile vacuole membrane in Dictyostelium(53, 54) and since myosins have been implicated in contractile vacuole function(55) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) 442409.