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INTRODUCTION |
Dynein is a microtubule-associated motor protein that is
responsible for several distinct motility processes in eukaryotic cells
(reviewed in Refs. 1-3). The dynein superfamily is divided into two
major classes based primarily on their cellular function. Cytoplasmic
dyneins act in positioning and transport of membrane-bound organelles
and help organize spindle assembly during cell division. The more
heterogeneous axonemal dyneins are largely responsible for the beating
motions of eukaryotic cilia and flagella. All dyneins characterized to
date are composed of multiple subunits. A complete dynein molecule
typically contains two or three heavy chains
(DHC)1 with molecular masses
of >400 kDa; intermediate chains (DIC) of 70-80 kDa, some of which
have been localized to the dynein tail (4, 5); and several light chains
of different molecular masses (1, 3, 6-8). Many cytoplasmic
forms also contain at least two light-intermediate chains (50-60 kDa)
(9, 10).
Comparisons between axonemal and cytoplasmic dyneins suggest a
functional division of the DHC into two regions. The carboxyl-terminal two-thirds is conserved and comprises the motor domain (11-14). The
more divergent amino-terminal third forms at least part of the stalk
and likely forms a scaffold upon which most of the other subunits
assemble to form the cargo-binding tail (1, 11, 15, 16). The molecular
interactions within the dynein tail are not well understood. They are
important, however, because variation in the subunit composition is
thought to specify and regulate cargo binding either directly or by
controlling the interaction of dynein with other proteins such as
dynactin (16-20).
Our work in Dictyostelium has shown that overexpression of
the motor domain in this organism dramatically alters the interphase microtubule array, but the cells are viable (13). In contrast, a
fragment containing the amino-terminal region can only be expressed at
low levels; its overexpression appears to be lethal (21). Because this
fragment can associate with the native DHC in vivo (21), its
overexpression may cause the formation of one-headed or headless
dyneins that can bind cargo but cannot function as motors (see also
Ref. 22).
To further investigate the toxic effect of this dynein fragment, and
characterize its domain structure, we have initiated a functional
dissection of the DHC amino terminus from Dictyostelium. We
identify here a highly conserved region that is important for DHC
homodimerization and for intermediate chain binding. Both these
interactive properties are confined to the same region of the DHC;
however, they have different sequence requirements and are
biochemically distinct in vitro. We further correlate these results with expression of similar regions of the DHC in
vivo. This is the first identification of a subunit-interaction
domain in a cytoplasmic dynein heavy chain, and it provides a target for investigations into the structure and assembly dynamics of the
dynein tail.
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EXPERIMENTAL PROCEDURES |
Construction of Vectors--
A fragment containing the
amino-terminal 4007 base pairs of the Dictyostelium DHC
cDNA was subcloned into the pET5 expression vector (Novagen) just
downstream of the T7 transcription initiation site. Smaller
amino-terminal fragments were generated by endo- and exonuclease
digestion and cloned into pET5 or pRSET (Invitrogen) expression vectors
(see Fig. 5 for summary).
A construct encoding the full-length Dictyostelium DIC
cDNA (652 aa, GenBankTM accession number U25116 (23)),
subcloned into pET 14 (Novagen), was used for the initial stages of
this work. However, we found that a large EcoRI fragment of
the DIC cDNA (aa 65-613) shows the same binding activity as the
full-length DIC but is produced in greater amounts by BL21(DE3) cells.
It is used almost exclusively in the data presented here. Plasmids were
purified according to the alkaline lysis/polyethylene glycol method
(24).
Construction of Affinity Resins--
Expression vectors encoding
the DHC amino-terminal fragment and the full-length and partial DIC
cDNAs were expressed in BL21(DE3) cells. Polypeptides were purified
from inclusion bodies following the methods of Lin and Cheng (25).
Inclusion bodies were solubilized in renaturing buffer (50 mM Tris, 500 mM NaCl, 0.05% Tween 80, 20%
glycerol, pH 7.6) containing 8 M urea. Soluble protein was clarified at 25,000 × g for 20 min and adjusted to a
concentration of 2 mg/ml. This solution was dialyzed stepwise at 2-h
intervals against 25 volumes of buffer with decreasing concentrations
of urea (4, 2, 0 M), then against CNBr coupling buffer (100 mM NaHCO3, 500 mM NaCl, pH 8.3)
overnight, and clarified at 25,000 × g for 20 min. The
concentration of soluble protein was determined by the Bradford assay,
and the protein was then coupled to one-fourth volume activated CNBr
resin (Amersham Pharmacia Biotech) overnight. This resulted in a final
protein concentration on the resin of ~3 mg/ml. The affinity resin
was blocked with 1 M glycine or 100 mM Tris, pH
8, washed, and stored in PHEM buffer (60 mM Pipes, 25 mM HEPES, 8 mM EGTA, 2 mM
MgCl2, pH 6.9 (26)) containing 0.02% sodium azide on ice.
Affinity Analysis of Reticulocyte Lysate-expressed
Proteins--
DHC constructs were expressed in vitro using
the TnTTM reticulocyte lysate system (Promega). 50 µl of the reaction
mixture was brought to 500 µl with PHEM + 250 mM NaCl
(PHEM 250). This concentration of NaCl reduces nonspecific interactions
between protein and resin but does not disrupt the subunit organization of native cytoplasmic dynein (not shown). The diluted lysate was clarified at 18,000 × g for 20 min and divided equally
into two or three aliquots. Each aliquot was brought to 1 ml with PHEM 250 and incubated with 50 µl of the appropriate affinity or control resin at 4 °C for 2-4 h. The resin was washed three times with 1-ml
volumes of PHEM 250, resuspended in an equal volume of 4× SDS-PAGE
sample buffer, and boiled. Bound polypeptides were resolved by
electrophoresis through a 7.5% SDS-polyacrylamide gel.
Reticulocyte-lysate expressed bands were detected by soaking the gel in
Amplify (Amersham Pharmacia Biotech), drying, and exposing to film. The
amount of [35S]methionine-labeled polypeptide was
determined using a Fujix phosphoimage BAS 2000 scanner (Fuji). Bands
were selected and analyzed with TINA analysis software (Raytest) using
the Autocontour feature.
Cross-linking of Reticulocyte Lysate-expressed Proteins--
DHC
amino-terminal fragments were expressed in vitro as
described above. The sample was divided into two or three aliquots and
brought to 50 µl by addition of buffer containing 4 µl of DSP stock
in N,N-dimethylformamide (final concentration 0.8 mg/ml), EDC stock in H2O (final concentration 0.16 mg/ml),
or addition of N,N-dimethylformamide or water as
control. Samples were incubated at room temperature for 30 min, and the
reaction was stopped with 5 µl of 1 M ethanolamine.
Samples were brought to 500 µl with PHEM 250, clarified at
18,000 × g for 20 min, and then incubated with
antibodies raised against the DHC amino terminus (27) bound to Protein
A-Sepharose (Amersham Pharmacia Biotech). The immunoprecipitate resin
was then washed three times, and bound polypeptides were resolved by
SDS-PAGE.
Statistical Methods--
The data from the DHC-DHC and DHC-DIC
affinity resin experiments were examined using a cluster analysis (28).
A set of 1000 distributions was generated by randomly assigning a
fragment to one of two groups, and a mean group affinity was
calculated. For each distribution, the mean squared error from the
group mean was calculated for every fragment. The randomly generated
distribution that had the lowest G statistic (defined as the sum of all
mean squared errors) was designated as "best" and was used as the
basis for designating fragments as either binding or nonbinding.
Expression of Polypeptides In Vivo--
Amino-terminal
constructs were tagged by oligonucleotide addition of the c-Myc epitope
(29) (details available upon request). These were subcloned downstream
of the native DHC promoter on a plasmid containing a G418 selection
marker (13, 21). Fresh AX-2 cell spores were plated onto 10-cm plastic
Petri dishes and were transformed with plasmid DNA (13, 21) just before
the cells reached confluency, at about 107 cells/dish.
Following an overnight recovery, the medium was changed to include 10 µg/ml G418. Colonies were detected after 3-5 days of drug selection.
Individual clones were picked with a pipette and transferred to a
24-well plate.
Biochemical Analysis of Dictyostelium Proteins--
Preparation
of high speed supernatants (HSS), electrophoresis, and immunoblotting
were performed as described in (30). c-Myc-tagged polypeptides were
purified from HSS by incubating 1 ml of supernatant with 25 µl of
anti-c-Myc monoclonal antibody from ascites for 1 h at 4 °C. 25 µl of Protein A-Sepharose (Amersham Pharmacia Biotech) was then added
to bind the antibodies. The resin was washed three times and
resuspended in 50 µl of PHEM buffer. Bound polypeptides were resolved
by SDS-PAGE.
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RESULTS |
Cross-linking of the DHC Amino Terminus--
Previous work has
suggested that the heavy chains of cytoplasmic dynein associate
directly in the absence of other dynein subunits (31). Because the two
heavy chains are believed to interact within the amino-terminal third
of the DHC, we tested whether this region can dimerize in
vitro. A reticulocyte lysate expressing the amino-terminal 1327 aa
(151 kDa) of the DHC was exposed to either DSP or EDC cross-linkers to
covalently link polypeptides in close contact with one another. DSP is
approximately 8 Å in length and cross-links the
-amine of lysine
residues; EDC is a zero-length cross-linker that forms a covalent bond
between aspartate or glutamate and lysine. As indicated in Fig.
1, both cross-linkers produced labeled
bands of MW ~300 kDa, a value close to the predicted dimer weight of
the amino-terminal fragment.

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Fig. 1.
Cross-linking of DHC amino terminus. A
reticulocyte lysate-expressed 1327-aa DHC amino-terminal fragment was
incubated with the cross-linkers DSP, EDC, or with
N,N-dimethylformamide (DMF) as a
control. Protein was immunoprecipitated with anti-DHC antibody bound to
Protein A-Sepharose. Both cross-linkers generate a band of
approximately 300 kDa, the predicted dimer size of the 151-kDa
fragment. No other high molecular weight bands are visible, even after
longer exposures.
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Purification of DHC Amino Terminus with DHC Affinity Resin--
To
further investigate DHC dimerization, a construct encoding the
amino-terminal 1327 aa of Dictyostelium DHC was expressed in
Escherichia coli (see Fig.
2A) and coupled to Sepharose,
producing a DHC affinity resin. Two types of control resins were
constructed to measure nonspecific interactions: non-dynein proteins
(actin or BSA) bound to Sepharose at the same concentration, and a
similarly-treated protein-free resin.

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Fig. 2.
Binding of the dynein amino terminus to
affinity resin. A, lane 1 shows a Coomassie
Blue-stained gel of the purified inclusion bodies that were used to
construct the amino-terminal 151-kDa DHC affinity resin. Lane
2 shows an immunoblot of the same sample probed with a polyclonal
DHC antibody. B, DHC amino terminus interaction with DHC
affinity resin. Equal amounts of reticulocyte-expressed polypeptide
(DHC aa 1-1327) were incubated with DHC affinity resin and with a
control resin, either BSA, actin, or protein-free-Sepharose
(Seph). Luciferase (luc), a control protein, was
similarly expressed and incubated with either DHC or BSA affinity
resin. This panel shows autoradiographs of material retained by the
resin after washing. DHC affinity resin retains significantly more DHC
than do any of the control resins (there were no statistical binding
differences between the control resins). In contrast, the luciferase
control bound with the same low affinity to the DHC as to control
resin. C, the amount of radiolabel in each gel band was
quantitated with a phosphoimager. This panel shows the mean ratio of
protein bound to the DHC affinity resin versus control resin
for the 1-1327 DHC and luciferase. DHC resin retains approximately
3-fold more DHC than do the control resins; luciferase binds equally
well to the DHC affinity and BSA resin.
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Incubation of the DHC affinity resin with the same, in vitro
expressed polypeptide showed an approximately 3-fold higher retention of the soluble fragment than on control resins (Fig. 2, B
and C). This suggests that the bound and soluble fragments
interact in the assay. The resin-bound DHC fragment does not retain
protein indiscriminately; luciferase does not bind to the affinity
resin any more strongly than to the control resins. Moreover, the
resins do not retain any of the more abundant protein components of the reticulocyte lysate at levels detectable by Coomassie Blue staining (not shown).
Dimerization Capabilities of DHC Subunits--
To determine a
minimal HC fragment that supports dimerization, we produced a series of
smaller fragments (summarized in Fig. 5) and tested their ability to
interact with the DHC resin and to cross-link. As expected, the
fragments showed different affinities for DHC resin (Fig.
3A). Specific retention was
measured as the ratio of band intensity on the affinity resin to its
relevant control. These ratios fell into two groups, suggesting a
defined binding site which some, but not all, constructs contain. One group comprises DHC fragments that bind more than twice as efficiently to affinity resin than to control resin; these were considered to
contain the binding site. In all cases, these polypeptides bound to DHC
resin at least as well as did the full-length DHC amino terminus. The
second group consists of those that exhibit a mean affinity
resin:control resin intensity ratio of less than 2; these were
considered noninteractors. Such low ratios are similar to the
nonspecific behavior exhibited by luciferase in this system (see Fig.
2B). Statistical analyses showed that, relative to the stated null distribution, these results are significant (P
value of 0.04). All of the constructs that showed binding activity
contain the region between aa 627 and 780. No fragment lacking this
region showed binding ability. However, this region alone is not
sufficient for dimer interaction in vitro.

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Fig. 3.
Fragments of DHC amino terminus exhibit
distinct DHC binding abilities. A, the autoradiograms
in the upper panel show representative pairs of several
amino-terminal fragments (encoded amino acids are listed
below each pair; see Fig. 5 for a summary) bound to the DHC
affinity resin and a control resin. The 1-1327 and luciferase samples
are the same as shown in the previous figure and are provided here for
reference. The lower panel provides the mean relative
affinity of the DHC resin versus control resin from multiple
independent experiments. The black bars represent fragments
statistically identified as interactors; the white bars
represent noninteractors. B, three examples of DSP
cross-linking experiments. The panels show autoradiograms
from reticulocyte lysate-expressed and immunoprecipitated samples
following SDS-PAGE. + or indicates whether DSP was added as a
cross-linker. Constructs 1-565 and 780-1327 show no evidence of
higher molecular weight bands as a result of adding cross-linker;
fragment 629-1337 (predicted mass of 81 kDa) shows a band of
approximately 160 kDa (arrow) in the presence of DSP. This
work is summarized in Fig. 5.
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These fragments also displayed differential cross-linking properties
(Fig. 3B). Two fragments, encoding aa 567-1327 and
629-1327, generated high molecular weight bands in the presence of
cross-linker. Both novel bands are approximately double the predicted
mass of the expressed fragments, suggesting dimerization rather than
nonspecific cross-linking to reticulocyte lysate proteins. DHC
fragments that only contain the central region produced some faint
higher molecular weight smearing in the presence of DSP, but no
distinct banding patterns could be detected. The fragments of the DHC
amino terminus that do not contain the central region do not form high
molecular weight species in the presence of cross-linker.
While the affinity resin and cross-linking assays do not generate
identical results (summarized in Fig. 5), this may reflect subtle
differences between the interactions being measured; the resin work
measures the ability of a fragment to interact with the full 1327-aa
amino-terminal polypeptide, whereas the cross-linking evaluates the
interaction between two identical polypeptides. Nonetheless, the two
assays agree that there is a crucial dimerization core region between
aa 629 and 780, whose interaction is supported by flanking sequence on
the carboxyl-terminal side. This additional sequence may play a role in
stabilizing the structure of the interaction region.
Differential Interaction of DHC Subunits with DIC--
Because DHC
amino terminus is also predicted to interact with the DIC, we
constructed an affinity resin with bacterially expressed DIC and tested
its ability to bind the DHC amino terminus.
Reticulocyte-lysate-produced DHC also binds to DIC resin (Fig.
4A), with a 4-fold mean
enhancement of binding over control. As in the DHC affinity resin,
luciferase does not show an increased affinity for the DIC resin. This
indicates that the DHC amino terminus also contains a binding site for
the DIC.

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Fig. 4.
DHC fragment binding identifies a DIC binding
site. A, lane 1 shows a Coomassie
Blue-stained gel of the purified inclusion bodies containing the 60-kDa
DIC fragment used to construct the DIC affinity resin. Lane
2 shows an immunoblot of the same sample probed with an
intermediate chain antibody (23). B, the autoradiogram shows
the relative binding of reticulocyte-expressed 1-1327-aa heavy chain
fragment and luciferase for the DIC affinity resin versus a
BSA control resin. Similar differences were obtained with actin or
protein-free control resins. The graph below shows the
quantified difference averages between the DIC and control resins for
several independent binding experiments. C, the
autoradiograms in the upper panel show representative pairs
of several DHC fragments (encoded amino acids are listed below each
pair; see Fig. 5 for a summary) bound to the DIC affinity resin and a
control resin. The 1-1327 and luciferase samples are the same as shown
in panel B. The lower panel provides the mean
relative affinity of the DIC resin versus control resin from
multiple independent experiments. The black bars represent
fragments statistically considered to interact, the white
bars represent noninteractors.
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To search for a DIC interaction site within the DHC amino terminus, we
tested the ability of the DHC fragment series to interact with DIC
resin (Fig. 4B). The mapping pattern is similar to the DHC
dimerization work but defines a smaller minimal region for this
activity. The best statistical grouping of fragments predicted to
contain the DIC-DHC interaction site is attained when fragments 1-1327, 567-1327, 629-1327, 1-730, 1-940, and 534-760 contain the
site (P value = 0.012)
(Fig. 5). This suggests that the DIC interaction site is located within aa 629-730, just upstream of the
DHC-DHC site.

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Fig. 5.
Summary diagram of the amino-terminal DHC
deletion series. The encoded DHC amino acids of each fragment are
listed on the left. The relative positions of the fragments
are shown in the middle. The right side of the
figure summarizes data from Figs. 1-4, indicating which of these
fragments did (+) or did not ( ) show evidence of chemical
cross-linking (X-LINK), binding to DHC affinity resin
(DHC), or DIC affinity resin (DIC).
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Heavy Chain and Intermediate Chain Binding Activities--
Despite
mapping to the same region of the DHC sequence, the DHC and the DIC
binding activities are not identical. We analyzed the resin assay
interaction under conditions of varying ionic strength. While the
homodimerization of the 1327-aa DHC fragments was salt-sensitive, the
DHC-DIC interaction was stable even in the presence of 2 M
NaCl (Fig. 6). This suggests that the
DHC-DHC and the DHC-DIC interact through different mechanisms. The
in vitro DHC-DIC interaction is partially disrupted in the
presence of 0.6 M KI (not shown), a result consistent with
the dissociation of the intermediate chain from native chicken
cytoplasmic dynein (9).

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Fig. 6.
DIC and DHC affinity resins bind expressed
DHC fragment by different interactions. Bound affinity resins were
washed with 4 volumes of PHEM buffer containing either 250 mM or 2 M NaCl. The upper panel
shows the representative autoradiogram while the bottom
panel provides a graphical quantitation of the number of bound
counts. The interaction between the 1-1327 polypeptide and the DIC
affinity resin was much more stable in high salt (2 M NaCl)
than with the DHC affinity resin.
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In Vivo Expression of DHC Amino-terminal Fragments--
Previous
attempts to overexpress the 1327-aa amino-terminal region of DHC in
Dictyostelium suggest that large amounts of this polypeptide
are toxic (21). A reasonable interpretation is that the fragment
interacts with native dynein to produce defective molecules that impact
cell viability. Because our in vitro work suggests that the
central amino-terminal region is crucial for dynein assembly, fragments
containing only this region should disrupt dynein assembly in
vivo and cause severe phenotypic effects. To test this hypothesis,
we attempted to express DHC constructs comprising aa 1-567 (amino),
567-817 (central), and 780-1327 (carboxy) in
Dictyostelium. The amino- and carboxy-fragment expression is shown in Fig. 7. In cells containing
either of these two constructs, a single polypeptide of the predicted
size is abundantly expressed and is readily immunoprecipitated by
anti-c-Myc monoclonal antibody. Consistent with the in vitro
work, none of the dominant dynein subunits (DHC, DIC, or light
intermediate chains) appear to co-purify here (Fig. 7) or on a c-Myc
immunoaffinity column (not shown). Moreover, cells expressing these
fragments do not have obvious phenotypic defects and grow at rates
similar to those of wild type cells.

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Fig. 7.
Expression of amino-terminal fragments
in vivo. Dictyostelium expression
constructs containing aa 1-567 and 780-1406 of the DHC amino terminus
were epitope-tagged with c-Myc and expressed in vivo. The
first two lanes show the Coomassie Blue-stained high speed
supernatant (HSS) and the c-Myc immunoprecipitate
(IP) from Dictyostelium cells expressing the
predicted 64-kDa polypeptide (aa 1-567). The second pair of
lanes show an immunoblot of the same samples probed with an
antibody to the amino terminus of the DHC (27). The high molecular mass
native heavy chain can be seen in the HSS, but it is not enriched in
the IP, suggesting that it does not interact with this amino-terminal
fragment in vivo. The expressed fragment is also labeled by
the antibody. The third pair of lanes show an identical
immunoblot probed with an antibody to the Dictyostelium
dynein intermediate chain (23). This polypeptide can also be seen in
the HSS but is not enriched by immunoprecipitation. The second
set of three panels follows the same order but show cells
expressing the predicted 61-kDa fragment encoded by aa 780-1406.
Again, there is no indication here that this fragment interacts with
either the DHC or the DIC in vivo.
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In contrast, attempts to express the central region (aa 561-817) of
the amino terminus have been unsuccessful. Despite several transformations, no viable clones have been identified that express detectable levels of the predicted 29 kDa fragment.
Conservation of Sequence Among Cytoplasmic Dyneins--
The
results described above suggest that at least two activities important
for dynein subunit assembly are localized to a relatively small region
of the DHC amino terminus. Sequences from nine species were compared
with the amino-terminal region of the DHC of Dictyostelium
(Fig. 8A). Homologous regions
were aligned and the degree of identity to the Dictyostelium
sequence was determined for intervals of 100 aa. While the
amino-terminal region is generally not as conserved as the rest of the
polypeptide (1, 11, 27), there are three striking areas of homology
evident among the ten cytoplasmic sequences. The most conserved lies
between aa 600 and 800 in the Dictyostelium sequence and
includes the sequence we experimentally identify as essential for
heavy-chain dimerization and heavy chain-intermediate chain binding.
This further suggests that the assembly activities localized within aa
629-780 in Dictyostelium DHC are conserved among the other
cytoplasmic dynein heavy chains.

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Fig. 8.
Sequence of the central region of the DHC
amino terminus is conserved among the cytoplasmic dyneins.
A, sequences homologous to Dictyostelium aa
1-1327 were determined for cytoplasmic DHCs of nine other species:
Aspergillus nidulans (GenBankTM accession number
P45444), Paramecium tetraurelia (Q27171),
Drosophila melanogaster (P37276), Rattus norvegicus
(P38650), Nectria hematococca
(P78716), Saccharomyces cerevisiae (P36022),
Schizosaccharomyces pombe (O13290), Neurospora
crassa (P45443), and Caenorhabditis elegans (Q19020)
(see Ref. 39). Sequence alignment was facilitated with the PileUp
utility of GCG, and identity was calculated over 100-aa intervals.
Three peaks of conservation are evident, and the most strongly
conserved (aa 600-800) contains the region of
Dictyostelium DHC essential for both DHC and DIC
interactions (shaded band). B, amino termini of
axonemal dynein heavy chains (Chlamydomonas reinhardtii (Q39565) and (Q39575), Anthocidaris crassispina (P39057), P. tetraurelia (Q94709) (39)) were compared
using C. reinhardtii DHC as the standard. Areas of high
homology are evident, but they do not align with the conserved regions
of the cytoplasmic DHC amino termini.
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Sequences for the axonemal Chlamydomonas, sea urchin, and
Paramecium
DHCs, and for Chlamydomonas
DHC, were also analyzed (Fig. 8B). The amino terminus of the
Chlamydomonas
DHC interacts with several other dynein
subunits (22), and the homologous regions of the sea urchin and
Paramecium
DHCs show regions of good conservation with
the Chlamydomonas
sequence. However, the overall pattern
of conservation within the
amino-terminal fragments is distinct
from the cytoplasmic dyneins.
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DISCUSSION |
The organization of the cytoplasmic dynein tail and the specific
interactions between its subunits at the amino acid level have not been
well characterized. Electron microscopy has been useful in localizing
some of the subunits (4, 5), and considerable progress has been made in
investigating interactions of these subunits with other
dynein-associated proteins (15, 18-20). Subunit organization of
axonemal dyneins has been primarily studied through isolation of dynein
gene mutations (reviewed in Ref. 2). These often produce organisms with
nonlethal motility defects, which are amenable to biochemical and
structural analyses. Similar analyses are becoming informative for
cytoplasmic dyneins (21, 23, 32-35), mapping out the heavy chain
domain structure and identifying the microtubule-binding motif.
However, the physical organization of the cytoplasmic dynein tail has
not been dissected in sufficient detail to identify heavy chain domains
that mediate interactions with other subunits. Because the tail region
links the motor to a cargo, understanding the interactions here is
important in determining how dynein is targeted and regulated.
Previous work in vivo suggested that the amino-terminal
third of the dynein heavy chain is responsible for interactions between the DHCs and with other dynein subunits (21, 22). We used both chemical
cross-linking and in vitro affinity methods here to identify
a region that promotes DHC dimerization. This supports an earlier
report suggesting recombinant cytoplasmic DHCs directly form multimers,
even in the absence of other subunits (31). We have further determined
that the sequences crucial for this interaction lie within an
approximately 150-aa region of the DHC. While this core region is
necessary for dimerization, it seems to function most efficiently in
the context of flanking sequences. The additional amino acids may be
important in stabilizing the structure of this central domain. The
sequence on the carboxyl-terminal side, which likely forms at least
part of the stalk connecting the tail to the globular head, is
especially effective in this role.
We also show that the central domain of the DHC amino terminus directly
interacts with the dynein intermediate chain in vitro. The
sequence does not appear to require an additional structural support to
bind the DIC, suggesting that the DHC and DIC interactions are distinct
and that their activities are not dependent on one another. The
different biochemical properties of the two interactions lend strength
to this hypothesis.
The in vitro work is supported by in vivo
expression of DHC fragments. The amino- and carboxy-regions of the DHC
amino terminus can be expressed at high levels in
Dictyostelium; selective immunoprecipitation of these
fragments shows no evidence that they bind the DHC or DIC. We cannot
rule out the possibility that the fragments are misfolded and their
activities do not reflect native behavior. However, previous expression
of the amino-terminal region indicated that larger fragments (158 kDa)
are able to dimerize with a native heavy chain, suggesting some degree
of proper folding (21). Attempts to overexpress the entire amino
terminus in Dictyostelium led to the idea that this fragment
contains a functional motif that can perturb native dynein activity,
resulting in cell death. This lethal activity appears to be preserved
within the central 29 kDa of the amino terminus, within the region we
indicate contains the dimerization and DIC binding sites. We do not
know whether the toxic effect is because of perturbation of DIC
binding, dissociation of the two heavy chains, or both.
Because most dyneins form multi-polypeptide assemblies through
interactions in their tail, it seems reasonable to expect some degree
of conservation within this region. While the amino-terminal tail
region of the DHC is less conserved than the carboxyl-terminal head
domain (1, 11, 12), it does contain small areas of strong conservation.
The highest degree of sequence identity corresponds to the area we
identify here to mediate DHC and DIC interactions. Among cytoplasmic
heavy chains, this region is generally 40-65% identical to the
Dictyostelium sequence. Even the most divergent yeast
sequences show some degree of conservation in this region. In contrast,
axonemal dynein heavy chains show very little sequence conservation
with cytoplasmic dyneins in this region (1, 2, 36-38, and this study).
Given their conserved structures seen by electron microscopy, this
seems surprising. It is certainly possible that tertiary structure is
preserved in this region and that similar interaction mechanisms occur.
However, axonemal dyneins more often form heterodimers or heterotrimers
of distinct DHC polypeptides, whereas cytoplasmic dyneins dimerize from
identical heavy chains. While the amino-terminal portion of the DHC is
important for assembly in both cytoplasmic and axonemal dyneins, the
mechanisms of interaction may be distinct.
The current work suggests a functional division of the DHC amino
terminus into three regions. The central core contains an interaction
domain that assembles two heavy chains and binds the intermediate
chain. Moreover, expression of this region in vivo appears
to generate a tool to disrupt native dynein assembly and activity.
Directed mutagenesis and in vivo expression should prove useful here in determining the exact location and mechanism of the DHC
dimerization and DIC interaction sites, thereby providing information
on the interaction dynamics of the cytoplasmic dynein tail.