(Received for publication, May 9, 1995; and in revised form, July 13, 1995)
From the
We used affinity chromatography to probe for a direct binding
interaction between cytoplasmic dynein and dynactin. Purified
cytoplasmic dynein was found to bind to an affinity column of
p150, the largest polypeptide in the dynactin
complex. To test the specificity of the interaction, we loaded rat
brain cytosol onto the p150
affinity column
and observed that cytoplasmic dynein from cytosol was specifically
retained on the column. Preincubation of the p150
affinity matrix with excess exogenous dynein intermediate
chain resulted in a significant reduction of dynein binding, suggesting
that p150
may be interacting with dynein via
this polypeptide. Therefore we constructed an affinity column of
recombinant dynein intermediate chain and observed that dynactin was
retained from rat brain cytosol. These results demonstrate that the
native dynein and dynactin complexes are capable of direct in vitro interaction mediated by a direct binding of the dynein
intermediate chain to the p150
component of
the dynactin complex. We have mapped the site of this interaction to
the amino-terminal region of p150
, which is
predicted to form an
-helical coiled-coil. Regulation of the
dynein-dynactin interaction may prove to be key in the control
mechanism for cytoplasmic dynein-mediated vesicular transport.
Coordinated trafficking of organelles along microtubules is central to the viability of cell and is powered by the mechanochemical ATPases kinesin and cytoplasmic dynein. While the mechanisms which govern the specificity and regulation of this transport remain to be determined, there is growing evidence for the role of accessory factors in the function of the molecular motors involved. Recently, an integral membrane protein, kinectin, was found to be the essential anchor for kinesin-driven vesicle motility(1, 2) . Although no membrane receptor for cytoplasmic dynein has been described yet, a distinct 20 S complex, dynactin, was shown to differentially co-purify with cytoplasmic dynein from a variety of sources(3, 4, 5) . Also, in an in vitro motility assay the dynactin complex was required to reconstitute dynein-mediated vesicular motility(6) . These observations suggest that dynactin may interact with cytoplasmic dynein transiently or in a regulatory manner. However, the mechanism of interaction is not clearly understood.
Dynactin is a macromolecular oligomeric complex
of at least 10 different
polypeptides(6, 7, 8) . The two best
characterized components of the dynactin complex are p150 and a 45-kDa protein, centractin. cDNA cloning and amino
acid sequence analysis revealed that rat p150
is 32% identical to the product of the Drosophila gene Glued(5, 9) . It has been shown
previously that the null mutation of Glued is embryonic
lethal(10) , thus suggesting that the p150
polypeptide has a role in an essential cell function such as
mitosis or vesicular transport. Centractin (also known as Arp1) (
)is a novel form of actin that shares
50% identity to
human
-actin(11, 12) . Analysis of the dynactin
complex by immunoelectron microscopy revealed short actin-like
filaments, most likely formed from the polymerization of
centractin(8) . Polypeptides of 135, 62, 50, 42, 37, 32, 27,
and 24 kDa are also thought to be components of the dynactin complex;
the 42-kDa polypeptide has been identified as conventional actin, and
the 37- and 32-kDa polypeptides are the
and
subunits of
CapZ (capping protein)(7, 8) .
A number of genetic
studies have provided indirect evidence for an in vivo interaction between cytoplasmic dynein and dynactin in that
mutations in components of either of the complexes give rise to similar
phenotypes. In Neurospora crassa, mutant alleles which result
in curled hyphal growth and altered nuclear distribution have been
shown to encode polypeptides with homology to the cytoplasmic dynein
heavy chain (DHC), p150 and centractin(13) . In S. cerevisiae, mutations in a putative centractin homologue
result in defects in spindle orientation and nuclear migration which
are similar to the phenotype observed in dynein heavy chain
mutants(14, 15, 16, 26) . In Drosophila, it has been reported that certain Dhc64C (cytoplasmic dynein heavy chain) mutations can act as dominant
suppressors or enhancers of the Glued phenotype(36) .
Taken together, these observations suggest that dynein and dynactin
interact in vivo or affect the same cellular processes.
In
order to investigate the hypothesis that there is a direct biochemical
interaction between cytoplasmic dynein and the dynactin complex, we
constructed affinity columns of p150 or dynein
intermediate chain (DIC), then passed over preparations of purified
dynein or of whole rat brain cytosol and looked for specific retention
of cytoplasmic dynein on a p150
affinity
column or of the dynactin complex on a DIC affinity column. The results
indicate that the cytoplasmic dynein complex interacts with the
dynactin complex via a direct binding of the dynein intermediate chain
to p150
component of the dynactin complex.
This direct binding between cytoplasmic dynein and dynactin provides
evidence in support of involvement of an accessory factor in dynein
function. These results also suggest that modulation of the
dynein-dynactin interaction in vivo may be a key step in the
mechanism of regulation of cytoplasmic dynein-mediated organelle
trafficking within the cell.
Figure 1:
Purified cytoplasmic
dynein binds to p150. The peak 20 S fraction
of cytoplasmic dynein purified from rat brain (lane 1) was
loaded on columns constructed of either bacterially expressed fragment
(amino acids 133-899) of p150
(lanes
2-5) or BSA (lanes 6-9) immobilized on
CH-Sepharose 4B beads. The columns were washed with 100 mM NaCl and eluted with 1 ml each 0.5 and 1.0 M NaCl. The
eluted fractions were analyzed by probing with antibodies to DIC. Lane 1, 20 S peak fraction loaded; lanes 2 and 6, flow-throughs; lanes 3 and 7, final wash; lanes 4 and 8, 0.5 M NaCl elution; lanes
5 and 9, 1.0 M NaCl
elution.
Figure 2:
Native cytoplasmic dynein binds to
p150 affinity column. Whole brain cytosol (lane 1) was loaded on a column of bacterially expressed
fragment (amino acids 133-899) of p150
(lanes 2-5) or BSA (lanes 6-9)
immobilized on CH-Sepharose 4B beads. The columns were washed with 25
mM NaCl and eluted with 1 ml each of 0.5 and 1.0 M NaCl. The eluates were subjected to 7% SDS-PAGE after methanol
precipitation and the resulting gel stained with Coomassie Brilliant
Blue. Lane 1, cytosol-loaded; lanes 2 and 6,
flow-throughs; lanes 3 and 7, final wash; lanes 4 and 8, 0.5 M NaCl elution; lanes 5 and 9, 1.0 M NaCl elution. The arrows indicate
polypeptides eluting from the column of >300, 75, and 50-58
kDa (bracket) which migrate on SDS-PAGE gels similar to the
DHC, DIC, and LIC polypeptides of purified cytoplasmic
dynein.
The identities of the polypeptides
specifically retained from cytosol by the p150 column
were verified by immunoblotting with antibodies specific for the dynein
intermediate chain (Fig. 3A) and heavy chain (Fig. 3B). Both the dynein heavy chain and intermediate
chain were retained specifically by the p150
column and
not by the control BSA column. Some cross-reactivity of the DIC
monoclonal antibody to native p150
present in the
cytosol is evident by the presence of the doublet at approximately
150/135 kDa in lanes 1 and 2 in Fig. 3A as was observed previously(5) .
Figure 3:
Cytosolic cytoplasmic dynein complex binds
to p150. Whole brain cytosol (lane 1)
was loaded on a column of bacterially expressed fragment (amino acids
133-899) of p150
(lanes
2-5) or BSA (lanes 6-9) immobilized on
CH-Sepharose 4B beads. The columns were washed with 100 mM NaCl and eluted with 1 ml each of 0.5 and 1.0 M NaCl. The
eluates were analyzed by probing with either anti-DIC antibodies (A) or anti-heavy chain antibodies (B). Lane
1, cytosol-loaded; lanes 2 and 6, flow-throughs; lanes 3 and 7, final wash; lanes 4 and 8, 0.5 M NaCl elution; lanes 5 and 9, 1.0 M NaCl elution. A and B are separate blots, but
have the same samples.
Comparison of the
immunoblots shown in Fig. 3, A and B, indicates that
comparatively more DIC than DHC was eluted from the p150 affinity column with 0.5 M NaCl. It is likely that this
is artifactual, due to the higher sensitivity of the anti-DIC antibody
as compared with the DHC antibody. Alternatively, it is possible that
there may be a limited pool of free dynein intermediate chain in the
cytosol, as was observed previously by Paschal et
al.(7) . This free DIC may bind to dynactin with a lower
affinity than does the intact dynein holoenzyme, thus eluting at a
lower ionic strength from the affinity column.
To test whether the dynein-dynactin interaction is mediated
by an interaction between p150 and DIC we prepared
p150
affinity beads as before but preincubated the
matrix with 2.5 M excess of bacterially expressed DIC before
loading the cytosol onto the column (Fig. 4). A control column
was pretreated with 25 M excess BSA. If DIC mediates the
dynein-dynactin interaction, then excess DIC should block the binding
sites on p150
, and therefore dynein should no longer
bind to the column. When the salt eluates from DIC-treated or
BSA-treated p150
affinity columns were probed for DHC,
we observed a significant reduction (>80%, as judged by comparative
densitometry) in the observed binding of DHC to the DIC-treated column,
whereas the BSA-treated p150
column showed high levels
of binding (compare lanes 4 and 5 with lanes 8 and 9). This result suggests that DIC mediates the
binding of cytoplasmic dynein to the p150
component of
the dynactin complex.
Figure 4:
The dynein intermediate chain blocks
dynein-dynactin binding. p150 affinity beads
were preincubated in 0.3 mg/ml bacterially expressed DIC (lanes
2-5) or 1 mg/ml BSA (lanes 6-9). After mild
washing (50 mM NaCl), whole brain cytosol was loaded and
processed as described in the legend to Fig. 3B. The
eluates were probed for DHC with anti-DHC antibodies. Results show that
excess DIC blocks dynein binding to p150
affinity column. Lane 1, cytosol-loaded; lanes
2 and 6, flow-throughs; lanes 3 and 7,
final wash; lanes 4 and 8, 0.5 M NaCl
elution; lanes 5 and 9, 1.0 M NaCl
elution.
Whole
brain cytosol was loaded onto an affinity column of bacterially
expressed DIC. The column was washed extensively and then eluted with
0.5 and 1.0 M NaCl. The resulting fractions were resolved by
SDS-PAGE and probed for the binding of dynactin complex to DIC by
Western blot using antibodies to p150 and centractin.
Because p150
and centractin are bona fide components of the dynactin complex, the presence of both
components would suggest that the intact dynactin complex is binding to
the DIC column. As shown in Fig. 5, both p150
(A) and centractin (B) were found to be
retained by the DIC affinity column (lanes 4 and 5).
In previous work we have determined that centractin binds directly to
p150
(27) . Thus the retention of centractin by
the DIC column is most likely mediated by its association with
p150
. Neither the p150
or centractin
polypeptides of the dynactin complex were found to be retained on a
similarly constructed BSA control column (lanes 8 and 9).
Figure 5:
The
dynactin complex binds to dynein intermediate chain. Whole brain
cytosol (lane 1) was loaded on a column of bacterially
expressed dynein intermediate chain (lanes 2-5) or BSA (lanes 6-9) immobilized on CH-Sepharose 4B beads. The
columns were washed with 50 mM NaCl and eluted with 1 ml each
of 0.5 and 1.0 M NaCl. The eluates were analyzed by probing
with either anti-p150 antibodies (A)
or anti-centractin antibodies (B). Lane 1,
cytosol-loaded; lanes 2 and 6, flow-throughs; lanes 3 and 7, final wash; lanes 4 and 8, 0.5 M NaCl elution; lanes 5 and 9, 1.0 M NaCl elution. A and B are
separate blots but have the same samples. CENT., centractin or
Arp1.
We have found that if we increase the stringency of our
affinity chromatography by increasing the ionic strength of the column
wash buffer to 200 mM NaCl, the 135-kDa band recognized by our
anti-p150 antibody is not retained by the DIC column.
The 135-kDa band corresponds to an alternatively spliced form of
p150
.
This observation indicates a weaker
interaction of 135-kDa species with DIC and suggests that isoform
diversity may modulate the affinity of cytoplasmic dynein and dynactin
within the cell.
Not all of the p150 or centractin
in the cytosol was observed to bind to the DIC affinity column. The
capacity of the column may have been exceeded. Nonetheless, a
significant fraction of p150
and centractin in brain
cytosol was retained by the DIC affinity column. This simultaneous
retention of both p150
and centractin by the DIC column
suggests that the dynactin complex as a whole binds directly to DIC.
Cytoplasmic dynein and dynactin have been suggested to interact in vitro and in vivo. Such an interaction is important, since this might provide the basis for the functional regulation of cytoplasmic dynein. Dynactin is a 20 S oligomeric complex that variably co-purifies with cytoplasmic dynein(5, 6, 7, 25) . However, the basis for this apparent co-purification has been unclear, since it might result from either a direct interaction or because the two complexes share similar physical properties such as microtubule affinity and size. A functional link between the two complexes has been suggested by in vitro assays in which the reconstitution of microtubule-based vesicular motility mediated by cytoplasmic dynein required a fraction that contained dynactin(6) . While it has been observed that antibodies to dynein failed to co-precipitate components of dynactin complex, and vice versa(7) , a direct in vivo interaction has been suggested by genetic evidence where mutations in components of either the dynein or the dynactin complex give rise to similar phenotypes(13, 14, 15, 16, 26) .
In our experiments we investigated the ability of column-bound
polypeptides to interact with native protein complexes. The results
presented in this paper clearly demonstrate that native cytoplasmic
dynein binds to column-immobilized p150 and that the
dynactin complex binds to column-immobilized DIC. We also show that
this interaction is mediated by the direct binding of the DIC to
p150
, since we could effectively block dynein binding
to the p150
column by excess exogenous DIC. A
biochemical interaction between polypeptides of cytoplasmic dynein and
the dynactin complex has also been observed by Vaughan and Vallee, (
)using the solid-phase blot overlay method.
We have
mapped the DIC binding domain to the amino-terminal half of
p150, between amino acids 133 and 899. This region is
predicted to form an extended
-helical coiled coil(5) .
Recent results from our laboratory demonstrate that the p150
component of the dynactin complex binds to both the microtubule
and to centractin(27) , an actin-related protein which is a
major stoichiometric component of the dynactin
complex(6, 7, 8, 11, 12) .
Taken together, these data suggest that p150
is a
multifunctional polypeptide with at least three interacting domains as
depicted in Fig. 6A.
Figure 6:
Map of
p150 and a model depicting dynein-dynactin
interaction. A, current results and our previous studies (27) have defined several domains of interaction on
p150
. There is a microtubule-binding domain at
the amino terminus of p150
(28) which
is homologous to the microtubule-binding domain of
CLIP-170(28) , and near the carboxyl terminus there is a highly
charged cluster of amino acids that mediates p150
binding to centractin. This study has identified an
additional domain in the amino-terminal half of p150
that binds DIC. B, a model for the biochemical
interaction among DIC, p150
, centractin, and
microtubule has been depicted. Immunoelectron microscopy analysis of
chick dynactin preparations (8) revealed a characteristic tilt
of p150
with respect to the centractin
filament as shown, as well as the immunolocalization of many of the
polypeptides within the dynactin complex. Dynein heavy chains are
depicted as two globular heads interacting with microtubule and contain
the catalytic ATPase domains(33) . Organelles that might
constitute minus end targeted cargo for cytoplasmic dynein have been
omitted, since their exact location in the context of dynein-dynactin
is not yet established. Note that this static model shows several
simultaneous biochemical interactions; however, in vivo these
interactions are likely to be dynamic and closely
regulated.
One perplexing observation is
that although dynein and the dynactin complex biochemically interact,
as we have demonstrated here, they do not co-precipitate when
antibodies to components of either complex are used(7) .
However, this apparent discrepancy may result from the blocking of the
sites of interaction by the immunoprecipitating antibodies. Recently
Waterman-Storer et al.()observed that two distinct
polyclonal antibodies to p150
blocked the interaction
between dynein and dynactin. This lends support to the idea that
antibodies used previously to co-precipitate dynein and dynactin
interfered with the dynein-dynactin interaction and hence led to the
failure to co-precipitate.
The observation that the binding of
cytoplasmic dynein to p150 is mediated by DIC is
interesting in view of the functional homology of this polypeptide to
the 70-kDa intermediate chain of flagellar outer arm dynein from Chlamydomonas. Studies on flagellar outer arm dynein suggest
that the 70-kDa intermediate chain is involved in the structural,
ATP-insensitive binding of axonemal dynein to the A subfiber
microtubule(22) . Paschal et al.(19) have
therefore predicted that the cytoplasmic DIC may function in an
analogous manner to attach cytoplasmic dynein to organelles or
kinetochores. Since p150
is localized to the membranous
structures in the cytoplasm, it is possible that the dynactin complex
on the surface of membranous structures targets the binding of the
dynein motor via the interaction between p150
and DIC.
It has been demonstrated recently that p150 binds to
microtubules independently of its association with cytoplasmic dynein (27) . This microtubule-binding motif shares homology with a
similar motif in the microtubule-organelle linking protein
CLIP-170(27, 28) . CLIP-170 has been proposed to act
as a docking protein for the binding of membranous vesicles to the
microtubule(28) . By analogy with CLIP-170, we speculate that
p150
may function to target organelles and vesicles to
the microtubule and subsequently allow cytoplasmic dynein to bind. Fig. 6B depicts a model in which p150
is simultaneously linked to the microtubule, the dynein
intermediate chain, and centractin. Alternatively, a continuous, but
weak, interaction with the microtubule (K
=
10 µM; (27) ) during vesicle translocation may
prevent diffusion of the vesicle during that stage of the dynein
cross-bridge cycle when both heads are predicted to be detached (see (35) for kinetics of axonemal dynein). In the model shown in Fig. 6B, centractin may be a structural link to the
organelle, potentially via the cortical cytoskeleton.
Now that our
results establish a direct binding between the cytoplasmic dynein and
dynactin complexes through DIC and p150, it will be
important to determine how the interactions among dynein, dynactin, the
microtubule, and the cellular cargo are regulated if we are to
understand the molecular mechanism of dynein-dynactin function. How
p150
that is bound to the microtubule in an
ATP-insensitive manner would facilitate dynein-based motility if it is
simultaneously bound to both dynein and microtubule is an important
issue and raises the possibility that interaction of dynactin to either
the microtubule or DIC is transient and regulatory. Phosphorylation of
p150
may regulate dynein function by altering the
affinity of the polypeptide for either the microtubule or the DIC.
Farshori and Holzbaur (
)have shown that p150
is differentially phosphorylated in response to cellular
effectors that have been reported to increase cellular vesicle
transport(29) . CLIP-170 has been shown to dissociate from
microtubules upon phosphorylation (30) and by analogy the
interaction of p150
to microtubules may also be
regulated in a similar manner to allow transport of organelles along
microtubules by cytoplasmic dynein. Alternatively, phosphorylation of
p150
may regulate its binding to dynein. In this
context it is interesting to note that Lin and Collins (31) and
Lin et al.(32) have demonstrated that okadaic acid
(an inhibitor of phosphatases 1 and 2a) causes redistribution of
cytoplasmic dynein from lysosomes to the cytosol. On the basis of our
current results, this redistribution could be due to phosphorylation of
p150
which induces the dissociation of DIC from
p150
and therefore the release of cytoplasmic dynein
from the organelles.
Only very recently have genetic and biochemical studies provided useful insight into the interaction between cytoplasmic dynein and its proposed activator, the dynactin complex, although we do not as yet understand the mechanism by which dynactin regulates dynein activity. In this paper we have described a system where the dynein-dynactin interaction can be observed in vitro and have identified the components involved in this interaction. These results now provide insight into the cellular basis for the lethal defect observed in the Glued mutation in Drosophila, as dynactin may be an essential component of the retrograde transport mechanism.