From the Department of Biological Chemistry, the
Jonsson Comprehensive Cancer Center, and the ** Molecular Biology
Institute, University of California,
Los Angeles, California 90095-1737
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ABSTRACT |
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Dynamin is a 100-kDa GTPase that assembles
into multimeric spirals at the necks of budding clathrin-coated
vesicles. We describe three different intramolecular binding
interactions that may account for the process of dynamin self-assembly.
The first binding interaction is the dimerization of a 100-amino acid
segment in the C-terminal half of dynamin. We call this segment the
assembly domain, because it appears to be critical for multimerization.
The second binding interaction occurs between the assembly domain and
the N-terminal GTPase domain. The strength of this interaction is
controlled by the nucleotide-bound state of the GTPase domain, as shown
with mutations in GTP binding motifs and in vitro binding
experiments. The third binding interaction occurs between the assembly
domain and a segment that we call the middle domain. This is the
segment between the N-terminal GTPase domain and the pleckstrin
homology domain. The three different binding interactions suggest a
model in which dynamin molecules first dimerize. The dimers are then linked into a chain by a second binding reaction. The third binding interaction might connect adjacent rungs of the spiral.
The discovery that the Drosophila shibire gene encodes
the homologue of dynamin led to the realization that these proteins are
important for an early stage in endocytosis (1, 2). Functional assays
with transfected mammalian cells demonstrated that dynamin is also
required for clathrin-mediated endocytosis in mammals (3). A possible
mechanism was suggested by the discovery that dynamin forms a spiral at
the neck of budding clathrin-coated vesicles when nerve endings are
incubated with GTP Dynamin is a member of a family of structurally related but
functionally diverse GTP-binding proteins with large C-terminal extensions (9). Three large blocks of sequences are highly conserved
within the family of dynamin-related proteins. The N-terminal segment
contains a GTPase domain, which is highly conserved between dynamin-related proteins, comprising a distinct subgroup within the
GTPase superfamily. The second domain, which we call the middle domain,
has no known function. The third domain is a putative coiled-coil,
which we call the assembly domain, because it may play an important
role in forming dynamin multimers. Most family members have divergent
segments inserted between the middle and the assembly domains. The
inserted segment in dynamin is a pleckstrin homology (PH) domain, which
binds to phosphatidylinositol 4,5-bisphosphate and, therefore, may be
important for interactions between dynamin and the plasma membrane (10,
11). The inserted segments of the other family members are completely
divergent from each other and from other proteins. This diversity in
sequence may reflect the different functions performed by dynamin
family members.
Dynamin is the only member of the dynamin family with an additional
C-terminal extension, which was called the proline-rich domain (PRD)
because it has more than 30% proline residues. The PRD interacts with
microtubules or SH3 domains in vitro, thereby stimulating
the dynamin GTPase (12, 13). However, all the intermolecular binding
interactions that are necessary for spiral formation are specified by
dynamin sequences, because no other proteins are required for in
vitro assembly of a dynamin spiral (5). C-terminal fragments help
activate the N-terminal GTPases of dynamin (14), but little else is
known about the internal organization of the dynamin spiral. There may
be as many as three steps in the formation of a dynamin spiral. 1)
Cytosolic dynamin forms a tetramer, possibly by joining two dynamin
dimers (14). 2) Dynamin oligomers are then linked into longer chains at
the plasma membrane. (3) Once the dynamin spiral has made a full circle, the rungs of the spiral might contact each other in a third
binding interaction that would help facilitate constriction. Here we
describe the discovery of a series of binding interactions between
three domains of dynamin that are conserved within the dynamin family:
the GTPase domain, the middle domain, and the assembly domain. These
binding interactions suggest how dynamin might form a spiral and then constrict.
Yeast Two Hybrid Analyses--
DNA fragments encoding different
parts of dynamin were generated by polymerase chain reaction with
Pfu polymerase (Stratagene, La Jolla, CA) with
oligonucleotides that have restriction sites at their 5'-ends.
The GTPase domain was cloned into NdeI and SalI sites of the bait plasmid (pAS1-CYH2) and into BamHI and
XhoI sites of the target plasmid (pAct-II) (15). Other
fragments were cloned into NcoI and SalI sites of
pAS1-CYH2 or into NcoI and XhoI sites of pAct.
The positions of the fragments used in yeast 2-hybrid and in
vitro binding studies are shown in Fig. 1. Where indicated,
mutations were introduced by fusion polymerase chain reaction with
complementary mutagenic oligonucleotides as described (3). Polymerase
chain reaction constructs were sequenced to rule out replication
errors. The two hybrid constructs were transformed into the yeast
strain Y190 (15) with polyethylene glycol/lithium acetate, as described
(16). A yeast 2-hybrid library made from human brain cDNA
(CLONTECH Laboratories, Palo Alto, CA) was screened
by growing the transformed yeast on selective plates without leucine,
tryptophan, or histidine but with 25 mM 3-amino-1,2,4-triazole (15). Surviving colonies were tested with a
filter assay for Expression and Purification of Proteins--
Protein fragments
were expressed with baculovirus, which is known to give high yields of
active dynamin (5, 17). To this end, DNA fragments were cloned into
pBlueBac4 (Invitrogen, Carlsbad, CA) with six histidines or GST-coding
sequences added by polymerase chain reaction. Recombinant virus was
produced in Sf9 cells as recommended by Invitrogen. Infected
cells were harvested after 48 h, resuspended in lysis buffer (20 mM Hepes, pH 7.2, 160 mM NaCl, 2 mM
MgCl2, 0.5 mM EDTA, 0.5 mM
dithiothreitol) with 0.1% Triton and protease inhibitors and lysed
with a French pressure cell (American Instrument Co, Inc., Silver
Spring, MD) adjusted to 20,000 psi. Recombinant proteins were bound to
His-Bind resin (Novagen Inc., Madison, WI) or glutathione-Sepharose
(Amersham Pharmacia Biotech) and eluted with 250 mM
imidazole in lysis buffer without Triton or with 10 mM
glutathione in 50 mM Tris-HCl, pH 8.0. The purified
proteins were then dialyzed into lysis buffer and stored at
To make radiolabeled protein for in vitro binding studies,
cDNA fragments were recloned into pET vectors (Novagen). Proteins were synthesized with the TNT®T7 Coupled
Transcription/Translation System (Promega Corp., Madison, WI) using
[35S]methionine (Amersham Pharmacia Biotech). Where
indicated, unlabeled methionine was added to 100 µM in
the transcription/translation reactions, thereby increasing the amount
of synthesized protein but decreasing the specific activity
approximately 100-fold.
Protein Binding and Cross-linking--
In vitro
binding assays were conducted with 1 µg of unlabeled GST fusion
protein and [35S]methionine-labeled target protein, or
both proteins were labeled with [35S]methionine. The
proteins were incubated in 250 µl of binding buffer
(phosphate-buffered saline with 10 mM EDTA or
phosphate-buffered saline with 5 mM MgCl2 and
100 µM GTP) and 20 µl glutathione-Sepharose beads and
gently mixed at 4 °C for 1 h (18). The beads were then pelleted
by centrifugation and washed in binding buffer. The bound proteins were
eluted with 10 mM glutathione in 50 mM Tris-HCl, pH 8.0, and fractionated by SDS-PAGE. The gels were dried and
subjected to autoradiography or quantitated with a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).
Cross-linking was essentially as described (19). Briefly, 1 µg of
His-tagged protein was incubated for 5 min in 20 µl of phosphate-buffered saline with 2 mM nickel acetate on ice.
Where indicated, 3 µg of carbonic anhydrase (Sigma) was included as a
control. Then magnesium monoperoxyphtalic acid was added to a final
concentration of 1 mM, and the mixture was incubated for another 15 min on ice. The reactions were stopped with SDS sample buffer. SDS-PAGE gels were stained with Coomassie Blue and scanned with
a Molecular Dynamics densitometer.
Binding between Dynamin Domains Discovered with the Yeast 2-Hybrid
System--
We conducted a yeast 2-hybrid screen to identify human
brain proteins that interact with a 377-amino acid fragment of dynamin containing the N-terminal GTPase domain. In a screen of 600,000 colonies transformed with human brain cDNA, we isolated 300 colonies that could grow without histidine, because they expressed the his-3 reporter gene (15). Upon further analysis, only one of those transformants was found to require both bait and target plasmids
to express a second reporter (
The sequences within the C-terminal half that bind to the N-terminal
GTPase domain were identified by testing short segments in the yeast
2-hybrid system. We found that those sequences were contained within a
100-amino acid segment, between the PH domain and the PRD (Fig.
2). This segment had previously been
suggested to form a coiled coil (20), although the coiled coil-forming probability predicted by programs such as Coils is relatively weak
(21). Support for a role in dimerization comes from a comparison with
the homologous segment in Mx proteins, which contains leucine zippers.
To test whether the dynamin fragment dimerizes, we expressed this
fragment both as bait and as target in the yeast 2-hybrid system.
Dimerization was readily detectable with a 100-amino acid segment (Fig.
2) but not with shorter fragments (data not shown). We call this
segment the assembly domain, because it binds to itself and to the
GTPase domain, which suggests that it plays a central role in the
assembly process.
To characterize the newly discovered interactions between the GTPase
domain and the assembly domain, we introduced mutations that are
predicted to alter the GTP binding properties of the GTPase domain and
tested their effects on binding between the GTPase domain and the
assembly domain. Unfortunately, we were unable to utilize well
characterized Ras-activating mutations, as was done previously with
other GTP-binding proteins, because the dynamin GTPase domain is too
dissimilar from Ras. We could, however, introduce mutations that are
predicted to block guanine nucleotide binding (K44A and S45N). These
two mutations cause the GTPase to bind much more strongly to the
assembly domain in the yeast 2-hybrid system (Fig. 2). The effect of
the K44A and S45N mutations indicates that the strength of the binding
interaction is coupled to the cycling of GTP.
Binding between the Middle Domain and the Assembly
Domain--
Further experiments with the yeast 2 hybrid system showed
that the assembly domain is capable of a third binding interaction besides binding to itself and to the GTPase domain. We found that the
assembly domain also binds strongly to the sequences between the GTPase
domain and the PH domain, which we call the middle domain (Fig.
3). Our results therefore suggest that
the assembly domain has three binding interactions. It can dimerize
(1), bind to the GTPase domain (2), and bind to the middle domain (3).
These three binding interactions might be needed for dynamin to form a
multimeric spiral. However, the number of binding interactions also
raises the question of whether these interactions are specific or
instead are because of nonspecific "stickiness" of the assembly domain. With the GTPase domain, this issue was addressed by testing point mutations known to affect GTP binding (Fig. 2). With the middle
domain, it was not obvious which mutations to test, because nothing
else is known about the middle domain.
To investigate the specificity of binding between the middle domain and
the assembly domain, we determined whether homologous domains from
another dynamin family member could replace them. As a counterpart, we
chose Mx1, which is one of the mammalian Mx proteins known to inhibit
viral replication. In yeast 2-hybrid experiments, we found that the
dynamin middle domain did not bind to the Mx1 assembly domain, nor did
the Mx1 middle domain bind to the dynamin assembly domain (Fig. 3).
However, the Mx1 middle domain did bind to the Mx1 assembly domain,
similar to the dynamin-dynamin interactions described above. This
result demonstrates that the middle domains bind specifically to their
cognate assembly domains but not to the assembly domains of
heterologous proteins. Such specificity could be beneficial by
preventing the incorporation of different dynamin family members into
heterologous complexes. Curiously, we could not detect dimerization of
the Mx1 assembly domain in the yeast 2-hybrid system, although the
presence of leucine zippers suggests that this is likely to occur
in vivo (Fig. 3). Dimerization of the Mx assembly domain
might not be detectable, because the Gal4 fragment, encoded by the
yeast 2-hybrid bait plasmid, also dimerizes, thus favoring a homodimer
of bait chimeras over binding between bait and target chimeras. The
dynamin assembly domain might be less affected by Gal4 dimerization,
because the dynamin coiled coil is much weaker than that of Mx proteins.
The dynamin GTPase domain and middle domain fragments used in the
experiments described above overlap by 63 amino acids. To rule out the
possibility that both binding activities are contained within the
overlapping segment, we delineated the boundary between the GTPase and
the middle domains with a deletion series (Fig. 1) that we tested with
the yeast 2-hybrid system. A boundary at position 320 allowed both the
GTPase domain and the middle domain to bind to the assembly domain,
thereby demonstrating that these two interactions are independent (Fig.
4). Further deletions at the C terminus
of the GTPase domain or at the N terminus of the middle domain
completely abolishes binding, suggesting that those domains must be
intact to bind to the assembly domain. Interestingly, the C-terminal 20 amino acids of the GTPase domain are predicted to form a coiled-coil
with the Coils program (21). However, the interaction between the
GTPase and the assembly domain is strongly influenced by mutations
affecting GTP binding (Fig. 2), which suggests that an intact GTPase
domain is required to achieve optimal binding to the assembly domain.
Taken together, our yeast 2-hybrid results indicate that dynamin has
three independent binding activities. These interactions may provide
the building blocks with which dynamin assembles into a multimeric
spiral.
Dimerization of the Assembly Domain Tested with Cross-linking
Reagents--
We used cross-linking to test whether dimerization of
the assembly domain can be replicated in vitro. Initial
trials with cross-linkers such as BS3 (3) were
unsuccessful, suggesting that the few lysine residues in the assembly
domain were not oriented properly for cross-linking. Instead, we used a
novel cross-linking strategy, which takes advantage of the histidine
tag that was included for protein purification. Nickel, chelated by the
histidine tag, can be activated by magnesium monoperoxyphtalic acid
to cross-link nearby proteins (19).
The results of in vitro cross-linking of the assembly
domain, which migrates as a 15-kDa fragment, are shown in Fig.
5. A reaction product with an apparent
molecular mass of 30 kDa is formed upon the addition of the
cross-linking reagents nickel acetate and magnesium monoperoxyphtalic
acid. No additional products are formed when an unrelated protein
(carbonic anhydrase) is added to the reaction. The experiments showed
that the assembly domain spontaneously dimerizes in solution, thereby
supporting the yeast 2-hybrid results.
In Vitro Binding between Separated Protein Fragments--
To
further investigate the interactions between the dynamin GTPase domain
and the assembly domain, we performed in vitro binding experiments with dynamin protein fragments. Binding was achieved by
incubating radiolabeled dynamin fragments together with a GST fusion
protein and glutathione-Sepharose beads. Binding was detected by
autoradiography of the glutathione eluates after they were fractionated
by SDS-PAGE.
The GTPase domain fused to GST pulls down a small fraction of
radiolabeled assembly domain (Fig. 6),
which indicates that in vitro binding is weak. However, the
amount of assembly domain that was pulled down by GST-GTPase was
increased by EDTA, which inhibits GTP binding by chelating the excess
Mg2+ in the in vitro transcription/translation
mixture. The strength of the binding interactions was determined by
competition with unlabeled assembly domain. The amount of radiolabeled
assembly domain, pulled down in binding reactions with EDTA, was
reduced by half when approximately 1 µM unlabeled
assembly domain was added to these reactions (data not shown).
Introducing the K44A mutation into the GST-GTPase construct caused
increased binding between the assembly domain and the GTPase domain
(Fig. 6), similar to results obtained with the 2-hybrid system (Fig.
2). We conclude that binding interactions between the GTPase domain and
the assembly domain are regulated by the nucleotide-bound state of the
GTPase domain. In vitro binding between the middle domain
and the assembly domain was tested in pull-down experiments. The middle
domain was pulled down with GST-assembly domain but not with
GST-clathrin light chain, which was used as a negative control (Fig.
7). We conclude that the three protein
domains that are present in all dynamin family members are capable of
binding to each other both in vitro and in the 2-hybrid
system.
A wealth of literature describes the interactions of the dynamin
PH domain and PRD with other molecules. The PH domain and PRD may
cooperate to localize dynamin to clathrin-coated pits (22, 23).
However, the ability of dynamin to self-assemble in vitro
without the addition of other proteins indicates that all the binding
interactions necessary for spiral formation are contained within
dynamin. Here, we describe the discovery of three binding interactions
between different domains of dynamin, which may account for the
assembly process.
Two of the binding interactions were anticipated. Binding of the
assembly domain to itself is consistent with the weak coiled coil
predicted by the Coils program (21), and the homologous domain in Mx
proteins contains leucine zippers (24). Our yeast 2-hybrid experiments
and cross-linking demonstrate that the dynamin assembly domain
dimerizes. The second binding interaction, between the GTPase domain
and the assembly domain, is consistent with previous results obtained
with proteolytic fragments of dynamin (14) and C-terminal deletions of
Mx proteins (25). Our results add to those previous reports by
delineating the binding sequences and investigating the nucleotide requirements.
The C-terminal 20 amino acids of the GTPase domain might form a coiled
coil (21), but this cannot be the sole determinant for binding between
the GTPase domain and the assembly domain, which is regulated by
nucleotides. GTP weakens binding of the GTPase domain to the assembly
domain, whereas the GDP-bound and nucleotide-free states strengthen
binding between the GTPase domain and the assembly domain. We could not
determine whether the assembly domain induces nucleotide release or,
alternatively, promotes GTP hydrolysis, because hydrolysis and release
occur at very high rates even without assembly domain (data not shown).
However, binding between the GTPase and the assembly domain might
resemble binding between other GTP-binding proteins and guanine
nucleotide release proteins, which is also stronger in the
nucleotide-free state or with mutations like K44A and S45N (26, 27).
Tight binding between the K44A mutant GTPase domain and the assembly
domain might lock up the dynamin spiral. This is reflected by the
potent dominant negative properties of dynamin K44A mutants in
transfection experiments (3). Intact dynamin with the K44A mutation can
assemble into a multimeric spiral in vitro. It can also
retain co-assembled wild type dynamin, which would otherwise disassemble after GTP hydrolysis (17).
Binding between the middle domain and the assembly domain was
unexpected. Little or nothing is known about the function of the middle
domain. In fact, we were unable to determine the exact boundaries
between the GTPase domain and the middle domain by sequence
comparisons, because the GTPases of dynamin-related proteins are quite
different from other GTP-binding proteins. We therefore determined the
boundary between the GTPase domain and the middle domain with
deletions, which retained the binding activities of the GTPase domain
or the middle domain. Our experiments clearly indicate that both
domains have independent binding activities.
The discovery of independent binding activities provides an explanation
for the complex complementation patterns observed with lethal mutations
in the Drosophila shibire gene (the dynamin homologue). One
complementation group has mutations in the GTPase domain, whereas a
second group has mutations in the middle domain (28). Animals that are
heteroallelic for these two complementation groups are viable,
suggesting that the GTPase domain and the middle domain have
independent functions. Those independent functions are most likely the
different binding activities that we discovered.
Binding of the assembly domain to itself suggests that the basic unit
within a dynamin complex is a dimer. Other domains might also
contribute to dimerization. For example, the C-terminal segment of the
GTPase domain might form a short coiled coil (21), and the PH domain
might dimerize, too (29). Linking dimeric dynamin into a chain requires
a different binding interaction. We found two possibilities. The
assembly domains could bind either to the GTPase domains or to the
middle domains of an adjacent dimer. It seems likely that dynamin
dimers are linked in a head to tail configuration, similar to the
filaments formed by septins, which are GTP-binding proteins with a
C-terminal coiled coil analogous to dynamin (30). Head to tail
interactions might also contribute to the formation of tetramers, which
is the predominant state of cytosolic dynamin (14).
Two different binding interactions are needed to account for
dimerization and head to tail linkage. A third binding interaction might provide structural support, for example by joining the rungs of
the dynamin spiral (Fig. 8). It was
previously proposed that dynamin spirals are constricted by concerted
conformational changes in which the subunits are shortened when they
bind or hydrolyze GTP (Fig. 8B) (8). Our results suggest an
alternative model in which dynamin spirals are constricted by
ratcheting one rung of the spiral along the next rung of the spiral
(Fig. 8C). The lengths of the individual subunits need not
change if the contacts between the rungs are moved up one subunit at a
time. Movement might occur when the GTPase domains of one rung bind and
subsequently release the assembly domains of the next rung. Such
stepwise movement would be similar to the movement of ATP-driven motor
molecules such as kinesins or myosins. Indeed, it had been previously
noted that there is significant structural similarity between
GTP-binding proteins and the ATP binding domains of kinesin and myosin
(31). The lengths of the steps along the dynamin spiral are similar to
the steps taken by kinesin (32). In this ratchet hypothesis, dynamin
combines the enzymatic properties of GTP-binding proteins with the
motor properties of kinesin and myosin.
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S1 (a
nonhydrolyzable GTP analog) (4). Purified dynamin also assembles into
spirals or rings in the absence of membrane (5). These structures are
strikingly similar to electron-dense collars that were observed many
years earlier in neurons of Drosophila shibire mutants (6).
Recent experiments demonstrated the ability of brain cytosol and
purified dynamin to form lipid tubules (7, 8) Purified dynamin can
sever the lipid tubules by GTP-dependent constriction (8).
It is therefore likely that the dynamin spirals at the necks of budding
vesicles catalyze the pinching-off of clathrin-coated vesicles.
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-galactosidase activity
(CLONTECH Laboratories).
80 °C.
GST fused to the yeast clathrin light chain protein was a gift from
Diane Chu.
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-galactosidase), suggesting that this
colony contained a target protein that binds to the dynamin GTPase
domain. The target plasmid isolated from this colony was sequenced and,
much to our surprise, found to encode the C-terminal half of dynamin.
The cDNA starts at amino acid position 545, which is in the dynamin
PH domain, and then extends into the 3'-untranslated region (Fig.
1). This was a first indication that the
dynamin GTPase domain binds to C-terminal sequences.
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Fig. 1.
Positions of dynamin fragments that were
tested with the yeast 2-hybrid system. The dynamin protein domains
are the GTPase domain, the middle domain (M), the PH domain,
the assembly domain (Asm), and the PRD. The boundaries of
the fragments are indicated by the positions in the dynamin amino acid
sequence. The + and symbols indicate whether these fragments bind to
the assembly domain in yeast 2-hybrid experiments.
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Fig. 2.
Binding between the dynamin GTPase domain and
the assembly domain detected with the yeast 2-hybrid system. The
streaks show yeast transformed with 2-hybrid plasmids and stained with
5-bromo-4-chloro-3-indolyl b-D-galactopyranoside. The
streaks on the left have target plasmid containing the dynamin assembly
domain (Asm) and on the right have vector alone
(pAct). From top to bottom, the streaks have bait vector
alone (pAS) or they have bait vector with the wild type
GTPase domain (GTPase), with the K44A GTPase mutant
(K44A), with the S45N GTPase mutant (S45N), or
with the assembly domain (Asm). The staining patterns
indicate that the assembly domain binds weakly to the wild type GTPase
domain, strongly to K44A and S45N mutant GTPase domains, and strongly
to itself.
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Fig. 3.
Binding between the dynamin middle domain and
the assembly domain detected with the yeast 2-hybrid system. The
grid shows streaks of yeast that were transformed with bait
plasmids (pAS), shown along the y axis, and
target plasmids (pAct), shown along the x axis.
Dynamin fragments and homologous fragments from rat Mx1 were paired in
different combinations to test the specificity of the binding
interactions. The homologous combinations are boxed in
black. The heterologous combinations are boxed in
gray. Binding was detected between the dynamin middle
(D-Mid) and assembly domains (Asm), between the
Mx1 middle (M-Mid) and assembly domains (Asm),
and between the dynamin assembly domain and itself but not between the
Mx1 assembly domain and itself nor with any of the heterologous
combinations.
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Fig. 4.
Delineation of the boundary between the
GTPase domain and the middle domain using the yeast 2-hybrid
system. The top panel shows GTPase fragments of
increasing size in the bait vector tested with the assembly domain
(Asm) or with the target vector without insert
(pAct). The GTPase fragment used here contained the S45N
mutation, which was shown to increase the strength of binding (Fig. 2).
The middle panel shows a similar series with middle domain
fragments of decreasing size. The lower panel shows controls
for the staining reaction. On the left is a streak of yeast transformed
with the assembly domain cloned into both bait and target vectors, and
on the right is a streak of yeast transformed with vectors alone. The
numberings correspond to the fragments depicted in Fig.
1.
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Fig. 5.
In vitro cross-linking of the
dynamin assembly domain. Dimerization of the assembly domain was
tested with the cross-linking reagent magnesium monoperoxyphtalic acid
(MMPP), which activates the nickel complexed with the
C-terminal His tag of the assembly domain fragments. The reaction
products were size-fractionated by SDS-PAGE and stained with Coomassie
Brilliant Blue. The lanes show assembly domain without
cross-linker (1st lane), assembly domain with cross-linker
(2nd lane), assembly domain with cross-linker and chicken
albumin (Alb.), which was used as a nonspecific competitor
(3rd lane), chicken albumin with cross-linker (4th
lane), and chicken albumin without cross-linker (5th
lane). The arrows indicate the sizes of monomeric
and dimeric assembly domain fragments.
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Fig. 6.
In vitro binding between the
dynamin GTPase domain and the assembly domain detected with GST-pull
down. The assembly domain (Asm) was pulled down by the
wild type (WT) GTPase or the K44A mutant fused to GST. The
interaction with the wild type GTPase domain was enhanced by EDTA,
which inhibits GTP binding. The interactions with the K44A mutant
GTPase domain were not influenced by EDTA, because the K44A mutant
already has a GTP binding defect. Radiolabeled protein fragments were
made by in vitro transcription/translation with
[35S]methionine. To obtain larger quantities of the GST
fusion proteins, unlabeled methionine was also added to the
transcription translation reaction (see "Experimental Procedures").
The reaction products were then incubated with glutathione-Sepharose,
pelleted, washed, and eluted with glutathione. The glutathione
eluates were separated by SDS-PAGE and detected by
autoradiography.
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Fig. 7.
In vitro binding between the
dynamin middle domain and the assembly domain detected with GST-pull
down. The middle domain was pulled down by assembly domain fused
to GST (GST-Asm). A fusion protein containing GST and
clathrin light chain (GST-CLC) was used to test whether the
binding reaction was specific. This fusion protein did not pull down
the radiolabeled middle domain. Binding, pull-down, and detection
conditions were the same as in Fig. 6. The middle domain was
synthesized by in vitro transcription/translation with
[35S]methionine. The GST fusion proteins were
unlabeled.
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Fig. 8.
A, summary of binding interactions
between different dynamin domains. The following binding interactions
were described here: the assembly domain (Asm, A) binds to
itself (1), the assembly domain binds to the middle domain
(M, 2), and the assembly domain also binds to the
GTPase domain (3). The PH domain binds to
phosphatidylinositol 4,5-bisphosphate (PIP2), and
the PRD binds to proteins containing SH3 domains. B,
conventional view of dynamin spiral undergoing constriction by a
conformational change. The dynamin spiral is presumably formed by
dynamin dimers linked into a chain. This chain might be in a head to
tail configuration in which the extra binding interaction could be used
to stabilize the spiral by forming connections between the rungs. As
previously suggested, the dynamin spiral might constrict by concerted
conformational changes in each of the subunits (5). C,
ratcheting as an alternative mechanism for constriction. We propose
that the dynamin dimers are linked into a chain in which the middle
domains of one dimer bind to the assembly domains of the next dimer.
The GTPase domains of one rung could then bind to and release assembly
domains of an adjacent rung, thereby ratcheting the rungs of the
spirals along each other. The GTPase cycle would thereby provide the
force needed for constriction using a mechanism similar to the movement
of myosin along actin filaments. For simplicity, the PH domains and the
PRDs were omitted from the drawings.
The ratchet hypothesis is consistent with previous electron microsopy data. Some of the spirals made with purified dynamin appear to be partly constricted, as if caught in the act of sliding shut (5). The earliest report of dynamin isolation also describes the ability of dynamin to decorate microtubules, cause microtubule bundling, and then cause the microtubules to slide along each other (33). Microtubule bundling might occur when the dynamin GTPases on one microtubule binds to the dynamin assembly domains on an adjacent microtubule. Runaway GTP hydrolysis by the dynamin molecules may then cause the decorated microtubules to slide along each other. The ratchet hypothesis departs from the classic perception of GTP-binding proteins as regulatory molecules. If this hypothesis is born out, then ATP-driven motor molecules, such as myosin and kinesin, might provide more meaningful paradigms (31). However, the debate on whether dynamin truly is a severing enzyme or, alternatively, acts as a regulatory GTPase needs to be settled first (34). Future experiments will decide between the different hypotheses that describe dynamin function.
The three domains that bind to each other are conserved throughout the
dynamin family and are therefore likely to reflect common structural
features (9). While this work was in progress, it was shown that the
middle and assembly domains of Mx proteins bind to each other, similar
to the interactions described in this paper for dynamin (35).
Furthermore, purified Mx protein can form spirals as shown by electron
microscopy (36). Sequence similarity, the similar binding interactions,
and the ability to form spirals raise the possibility that dynamin and
Mx proteins perform similar functions but with different targets. Other
dynamin family members were not yet tested in vitro nor have
their cellular functions been characterized in detail. Nevertheless,
the high degree of sequence similarity suggests that those other
proteins might also form spirals in vitro. It is therefore
possible that all these proteins use assembly and constriction
mechanisms similar to dynamin.
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ACKNOWLEDGEMENTS |
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We thank G. Payne, A. Labrousse, D. Rube, and I. Davydov for their many helpful suggestions.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM51866, American Heart Association Grant 965084, and Muscular Dystrophy Association Grant WB931203 (to A. M. v. d. B).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by the Myasthenia Gravis Foundation and the American Heart Association, Western states affiliate.
¶ Supported by the Jonsson Comprehensive Cancer Center, UCLA. Present address: Dept. of MCD Biology, University of California, Santa Barbara, CA 93106.
To whom correspondence should be addressed: Dept. of Biological
Chemistry, UCLA School of Medicine, P. O. Box 951737, Los Angeles, CA
90095-1737. Tel.: 310-825-9779; Fax: 310-206-5272; E-mail:
avan{at}mednet.ucla.edu.
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ABBREVIATIONS |
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The abbreviations used are:
GTPS, guanosine
5'-3-O-(thio)triphosphate;
PRD, proline-rich domain;
PH
domain, pleckstrin homology domain;
GST, glutathione
S-transferase;
PAGE, polyacrylamide gel
electrophoresis.
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REFERENCES |
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