From the Department of Cell Biology, University of
Massachusetts Medical School, Worcester, Massachusetts 01605 and
the § Department of Biochemistry and the Medical Research
Council Group in Protein Structure and Function, University of Alberta,
Edmonton, Alberta T6G 2H7, Canada
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ABSTRACT |
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Dynamin, a 100-kDa GTPase, has been implicated to
be involved in synaptic vesicle recycling, receptor-mediated
endocytosis, and other membrane sorting processes. Dynamin
self-assembles into helical collars around the necks of coated pits and
other membrane invaginations and mediates membrane scission. In
vitro, dynamin has been reported to exist as dimers,
tetramers, ring-shaped oligomers, and helical polymers. In this
study we sought to define self-assembly regions in dynamin. Deletion of
two closely spaced sequences near the dynamin-1 C terminus abolished
self-association as assayed by co-immunoprecipitation and the yeast
interaction trap, and reduced the sedimentation coefficient from 7.5 to
4.5 S. Circular dichroism spectroscopy and equilibrium
ultracentrifugation of synthetic peptides revealed coiled-coil
formation within the C-terminal assembly domain and at a third,
centrally located site. Two of the peptides formed tetramers,
supporting a role for each in the monomer-tetramer transition and
providing novel insight into the organization of the tetramer. Partial
deletions of the C-terminal assembly domain reversed the dominant
inhibition of endocytosis by dynamin-1 GTPase mutants. Self-association
was also observed between different dynamin isoforms. Taken altogether,
our results reveal two distinct coiled-coil-containing assembly domains
that can recognize other dynamin isoforms and mediate endocytic
inhibition. In addition, our data strongly suggests a parallel model
for dynamin subunit self-association.
Dynamin is a high molecular mass GTPase (1, 2) that has been
implicated in various aspects of endocytosis, including synaptic
vesicle recycling, the endocytosis of assorted receptors, internalization of caveolae, and more recently budding from the trans-Golgi network (reviewed in Refs. 3 and 4). Dynamin belongs to a
growing family of functionally diverse, large GTPases (4). In mammals,
multiple isoforms of dynamin itself have been identified (4, 5), and
functional dynamin homologs have been cloned from the lower eucaryotes
Drosophila melanogaster (shibire; Refs. 6 and 7)
and Caenorhabditis elegans (dyn-1; 8).
Dynamin self-assembly is critical to its function. It has been shown to
form helical collars around the necks of coated pits (8) and is
postulated to be involved in the subsequent budding of coated vesicles
and other early endocytic intermediates. Structures corresponding to
the helical collars can be formed in vitro from purified
protein (9) as well as in combination with acidic substrata including
microtubules (10), phospholipid tubules (11), and native synaptosomal
membranes (12). Polymerization is concentration-dependent
(13, 14) and inhibited in high ionic strength buffers (9).
Self-assembly does not require guanine nucleotides (15, 16), although
it is either stimulated or inhibited by them depending upon whether the
protein is free or membrane-bound (15, 16). Once polymerized, dynamin
can undergo a nucleotide-dependent conformational change
that results in membrane fission in vitro (11). In the
depolymerized state, dynamin has been reported to exist as either a
dimer (16) or a tetramer (17) based on cross-linking studies.
Analytical ultracentrifugation has favored tetramer as the predominant
species (18).
The regions of dynamin that are important in self-association and in
the interaction with lipids and other proteins have been a topic of
considerable interest. Dynamin contains a highly conserved, N-terminal
GTPase domain of ~300 aa1
and a centrally located pleckstrin homology (PH) domain that has been
reported to bind to phosphatidyinositol 4,5-bisphosphate (19, 20) and
G-protein Dynamin self-association stimulates its GTPase activity. Ligands that
bind to the proline-rich domain stimulate both assembly and GTP
hydrolysis, and removal of the domain inhibits both activities (13, 22,
25). More recently, proteolytic dynamin fragments missing the
proline-rich domain were shown by electron microscopy to be capable of
self-assembly when in the presence of GDP and metallofluorides at
physiological salt conditions (27) or on lipid bilayer tubes (11).
The role of the putative coiled-coil sequences in dynamin
self-association is uncertain. Coiled-coil probability is only ~60% (see "Results"), and whether this region actually forms coiled-coil structure, amphipathic A potential role for the putative coiled-coil region of dynamin emerged
from our previous analysis of the inhibitory endocytic phenotype in
mammalian cells transiently transfected with a dominant negative,
C-terminal deletion mutant (30, 31). Inhibition was unaffected by
removal of the proline-rich domain but was reversed by further deletion
into the putative coiled-coil region, suggesting that this part of the
molecule might mediate the interaction of mutant dynamin with the
endogenous wild-type protein (31). To gain further insight into the
mechanism underlying the inhibitory phenotype and to understand the
mechanism of dynamin self-association, we have used a series of
deletion mutants to define sites of dynamin self-association and
identify two distinct regions. Analysis of peptides reveals that at
least three sequences in dynamin-1 are capable of self-association.
These results suggest a critical role for the coiled-coil region in
dynamin self-assembly.
cDNA Constructs--
The dynamin-1ab isoform (1) was used
for most experiments. C-terminal and internal deletion mutants were
made via polymerase chain reaction and subcloned into the mammalian
expression vector, pSVL (Amersham Pharmacia Biotech). Polymerase chain
reaction was also used to fuse the HA (32) or Myc epitope (33) to the N terminus, and all constructs were sequenced to ascertain that the
mutations and epitope tags were correct. The dynamin-2bb isoform (31)
was also Myc-tagged at the N-terminal end using polymerase chain reaction.
Co-Immunoprecipitation Assay for Dynamin
Self-association--
For the co-immunoprecipitation assays, COS-7
cells were transiently transfected with both HA- and Myc-tagged
dynamin-1 constructs using the LipofectAMINE reagent (Life
Technologies, Inc.) as directed by the manufacturer. Cytosolic extracts
of the co-expressed proteins were prepared by lysing the transfected
cells in RIPA buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, 1 mM
phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1 µg/ml
leupeptin) and clearing the lysates at 30,000 × g and
4 °C for 20 min to remove any cellular debris. After preclearing the
supernatants with protein A beads (Amersham Pharmacia Biotech) for
several hours, fresh protein A beads were added along with either
anti-HA monoclonal antibody (Babco, Berkeley, CA) or anti-Myc
polyclonal antibody (a gift of Dr. Melissa Gee, University of Mass.
Medical School, Worcester, MA). Co-immunoprecipitation assays were
carried out overnight at 4 °C with end-over-end rotation, after
which the beads were washed several times in NET gel buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 0.25% gelatin, 0.1% Nonidet P-40) and then resuspended in an
equal volume of SDS sample buffer for SDS-polyacrylamide gel
electrophoresis. Western analysis was done using either the HA
monoclonal antibody (Babco) or the Myc monoclonal antibody (9E10,
ATCC), and chemiluminescence was used to detect any protein interactions (Kirkgaard and Perry Laboratories).
Yeast Interaction Trap--
The LexA-based interaction trap (34)
was kindly provided by Dr. Erica Golemis (Fox Chase Cancer Center,
Philadephia, PA) and used to assay for dynamin-dynamin interactions in
yeast. Wild-type dynamin-1 was subcloned into the JG202 bait expression
vector, which contains a nuclear localization signal. Wild-type and
deletion mutants of dynamin were constructed in the JG4-5 prey vector. We tested the suitability of the wild-type dynamin-1 in JG202 as bait
and found that it did not activate transcription alone. The lithium
acetate method was used to make yeast competent cells and for
transformations (35). Dynamin-dynamin interactions were scored positive
if transcription of the lacZ and LEU2 reporter genes were activated.
Sucrose Density Gradient Centrifugation--
Sucrose density
gradients (5-20%) were prepared in 20 mM Tris, pH 7.4, 50 mM KCl buffer. Cytosolic extracts of COS cells transfected with Myc-tagged dynamin constructs were prepared as described above.
For each gradient, 500 µg of the extract and 10 µg of each molecular mass marker (Sigma) were loaded. Centrifugation was at 26,500 rpm and 4 °C for 17 h. Fractions were collected at 20-s intervals and analyzed by SDS-polyacrylamide gel electrophoresis. Detection of dynamin was by chemiluminescence using a Myc monoclonal antibody. The positions of the molecular mass markers in the gradients were detected by Coomassie stain. Densitometric measurements were made
on a Bio-Rad model GS-700 Imaging Densitometer scanner interfaced to
the Bio-Rad Multi-analyst computer program (version 1.1). Protein concentrations were determined by the Bradford assay (Bio-Rad), and the
S values for the markers are as follows: bovine serum albumin, 4.5 S;
alcohol dehydrogenase, 7.5 S; Endocytosis Assays--
Transferrin uptake was measured in COS
cells as described previously (30) with the following modifications.
After transient transfection of the cells with Myc-tagged dynamin
constructs, the cells were grown in serum-free medium for 24 h.
Transferrin endocytosis was then stimulated by adding 20 µg/ml of
fluorescein isothiocyanate-labeled transferrin (Molecular Probes) for
10 min at 37 °C, after which uptake was stopped by placing the cells on ice and washing several times with ice-cold phosphate-buffered saline. Cells were then fixed with 4% paraformaldehyde for 15 min at
room temperature and subsequently extracted with either ice-cold
methanol for 5 min or 0.5% Triton X-100 for 2 min. Cell staining was
done as described previously (30), and immunofluorescent imaging was
with a Zeiss Axiophot microscope.
Peptide Synthesis and Purification--
Synthetic peptides were
prepared by solid phase synthesis methodology using a
4-benzylhydrylamine hydrochloride resin with conventional
N-t-butyloxycarbonyl chemistry on an Applied
Biosystems model 430A peptide synthesizer as described by Sereda
et al. (36). Peptides were cleaved from the resin by
reaction with hydrogen fluoride (20 ml/g resin) containing 10% anisole
and 2% 1,2-ethanedithiol for 1 h at Circular Dichroism Spectroscopy--
CD spectra were recorded on
a Jasco J-720 spectropolarimeter (Jasco, Inc.) interfaced to an Epson
Equity 386/25 computer running the Jasco DP-500/PS2 system software
(version 1.33a). The temperature-controlled cuvette holder was
maintained at 20 °C with a Lauda model RMS circulating water bath.
The instrument was calibrated with an aqueous solution of
recrystallized d-10-(+)-camphorsulfonic acid at 290.5 nm.
Results are expressed as the mean residue molar ellipticity [ Sedimentation Equilibrium Analytical
Ultracentrifugation--
Sedimentation equilibrium ultracentrifugation
was performed on a Beckman model XLI Analytical Ultracentrifuge and
interference optics as described in the instrument manual (Beckman
Instruments). Samples were dialyzed against and run in a 50 mM potassium phosphate, 100 mM KCl, 2 mM dithiothreitol, pH 7 buffer. Six-sector charcoal-filled epon sample cells were used, allowing three concentrations of sample to
be run simultaneously. Runs were performed at two or three speeds
between 20,000 and 32,000 rpm and were continued until there was no
significant difference in scans taken 2 h apart. The data were
evaluated using a nonlinear least squares curve-fitting algorithm (37)
contained in the NonLin analysis program. The program Sednterp
(Sedimentation Interpretation Program, version 1.01) was employed to
calculate the partial specific volume of the protein from the amino
acid composition as well as the solvent density and viscosity using
known values from physical tables.
Assays for Dynamin Self-association--
As a first step in
defining a self-association domain in dynamin-1, we tested for
interactions between epitope-tagged dynamin-1 constructs co-expressed
in COS cells. Full-length Myc- and HA-tagged wild-type dynamin-1 (C851;
Fig. 1, A and B;
see also Fig. 7) were observed to co-immunoprecipitate using antibody
recognizing either epitope. Co-immunoprecipitation was still observed
with C-terminal deletions that were lacking all or part of the
proline-rich region (C750; Fig. 1, A and B; see
also Fig. 7) as well as with mutants missing either the GTPase (N272;
see Fig. 7) or the PH domain (
As an alternative approach to defining interaction sites, we also used
a yeast two-hybrid system. Interactions were assayed between a
wild-type dynamin-1 bait and a panel of dynamin-1 deletion mutant prey
constructs. In agreement with the co-immunoprecipitation results,
dynamin-1 preys that were missing part of the proline-rich region still
interacted with the wild-type dynamin-1 bait (C761; Fig.
2; see also Fig. 7), although deletion of
the entire proline-rich region resulted in little, if any, interaction
(C750; Fig. 2; see also Fig. 7). We also detected a reduction in
interaction intensity with a C-terminal deletion mutant (C733; Fig. 2;
see also Fig. 7) that had previously been shown to reverse the dominant negative endocytic effect of K44E (30). As had been observed in the
co-immunoprecipitation experiments, interactions persisted in prey
devoid of either the GTP-binding domain (N272; Fig. 2; see also Fig. 7)
or the PH domain (
These data were consistent with our hypothesis that dominant inhibitory
dynamin polypeptides act through interaction with endogenous wild-type
dynamin protein (30). To test this possibility directly, we assayed for
transferrin uptake in cells overexpressing dominant negative dynamin-1
K44E mutants that also had deletions in the self-assembly region. Fig.
3 shows that removal of either of the two
self-association sequences identified in our co-immunoprecipitation and
yeast interaction trap assays resulted in reversal of the dominant
negative endocytic phenotype, i.e. the resumption of transferrin uptake. Because dynamin-1-K44E exhibits its inhibitory effects in cells containing primarily the dynamin-2 and dynamin-3 isoforms (30), we also tested for an interaction between isoforms. Interaction between dynamin-1 and dynamin-2 could be readily detected both by the yeast two-hybrid assay (Fig.
4A) and by
co-immunoprecipitation (Fig. 4B).
To learn more about the identity of the dynamin self-association
products, lysates of COS-7 cells expressing either epitope-tagged wild-type or mutant dynamin-1 were analyzed by sucrose density gradient
centrifugation. We established that the COS-expressed Myc-tagged
dynamin-1 isoform behaved similarly to rat brain dynamin-1 in the
sucrose density gradients (data not shown). Both ran as a 7.5 S peak,
indicating that they were not in a highly assembled state under these
conditions. The S value was unaffected by the removal of either the
proline-rich (Fig. 5A; see
also Fig. 7) or PH domains (Fig. 5B; see also Fig. 7).
However, deletions involving the self-assembly region identified by our
co-immunoprecipitation and yeast interaction trap experiments resulted
in a shift of the 7.5 S peak to 4.5 S (C695; Fig. 5A; see
also Fig. 7; and Peptide Analysis--
The domain implicated in dynamin-1
self-assembly contains two short, closely spaced sequences (aa 654-681
and aa 712-740) predicted by the Newcoils program (28) to have a
propensity for coiled-coil formation based on analysis of a data base
of known coiled-coil sequences (Fig.
8A). However, the probability is only ~60%, with no more than four heptad repeats predicted for
each region. Newcoils also predicts at even lower probability three
short sequences downstream of the GTPase domain between aa 295-470
(Fig. 8A). Paircoils, which is based on pairwise residue correlations (38) yielded a very different set of predictions, including a short sequence in near the center of the dynamin-1 polypeptide (Fig. 8B). Because of the disparity in the two
predictive approaches, we also examined dynamin-1 using a recently
developed nonstatistically based algorithm termed Stablecoils, which is based on the direct analysis of coiled-coil formation by synthetic peptides of systematically varied composition (39). The aa 651-675 region was judged to be the most hydrophobic with the highest propensity for amphipathic helix formation (Fig. 8C). A
lower, but significant, propensity for coiled-coil formation was
observed for aa 715-738, and additional sequences were identified in
the central portion of the polypeptide that differed from those
identified by Newcoils and Paircoils (Fig. 8).
To determine which of these short sequences in fact had properties
consistent with coiled-coil formation, we analyzed synthetic peptides
corresponding to five distinct regions by circular dichroism and
equilibrium ultracentrifugation (Table
I). The CD spectrum of the D1 peptide,
which was derived near the GTPase domain of the dynamin-1 polypeptide,
was predominantly random coil. We note, however, that in 50% TFE, a
helix-inducing hydrophobic solvent, it could be forced into an
Equilibrium ultracentrifugation of peptides D2, D3, and D4 revealed
that each is capable of oligomerization. The sedimentation data for D2
and D4 each fit to a homogenous species with mass average molecular
masses of 14,051 and 15,210 Da, respectively, in both cases consistent
with the formation of a tetramer (Fig. 11, A and B). D3
was observed to have a mass average molecular mass of 23,873 Da, which
approaches a hexameric species (Fig. 11C). This suggests
that D3 may not have a strictly defined oligomeric state but does have
the ability to form helical bundles.
Our results implicate two portions of dynamin in self-association,
a complex region located between the PH and proline-rich domains and a
region near the center of the polypeptide (Fig. 12A). We will refer to these
regions as the C-terminal and central self-assembly domain,
respectively. Our peptide analysis further identified coiled-coil
forming sequences within both regions (Fig. 12A) and
implicates coiled-coils as a primary mode of dynamin-dynamin interaction. Our results confirm our hypothesis that the C-terminal region mediates the interaction between dominant inhibitory dynamin mutants and endogenous wild-type protein. Furthermore, the
identification of two distinct coiled-coil domains has important
implications for dynamin structural organization, as discussed
below.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunits (21). Downstream of the PH domain are two
predicted regions of coiled-coil
-helix, and at the C terminus is a
100-aa basic, proline-rich region to which Src homology 3 domains
(22-24), acidic phospholipids (13), and microtubules (25) have been
shown to bind. This domain has been implicated in targeting dynamin to
coated pits through Src homology 3 domains (26).
-helix, or neither is uncertain. This region
is conserved among the dynamin isoforms and dynamin-related proteins
(4), and computer-based analysis (Newcoils; Ref. 28) predicts
comparable secondary structure in all of these proteins. The
dynamin-related Mx proteins exhibit clear heptad repeats within this
region consistent with a leucine zipper structure that, when fused to a
reporter gene, was capable of self-association (29).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-amylase, 9 S.
5 °C, washed with cold
ether several times, extracted from the resin with glacial acetic acid,
and then lyophilized. Purification of each peptide was performed by
reversed-phase chromatography on a Syn-Chropak semi-preparative C-8
column (inner diameter, 250 × 10 mm; 6.5-µm particle size;
300-Å pore size; SynChrom, Lafayette, IN) with a linear AB gradient
(ranging from 0.2 to 1.0% B/min) at a flow rate of 2 ml/min, where
solvent A is aqueous 0.05% trifluoroacetic acid and solvent B is
0.05% trifluoroacetic acid in acetonitrile. Homogeneity of the
purified peptides was verified by analytical reversed-phase
chromatography, amino acid analysis, and electrospray quadrapole mass spectrometry.
]
(degrees·cm2·dmol
1) calculated from the
following equation.
where
(Eq. 1)
obs is the observed ellipticity expressed
in millidegrees, M is the mean residue molecular mass
(molecular mass of the peptide divided by the number of amino acids),
l is the optical path length in centimeters, and
c is the final peptide concentration in mg/ml. The CD wave
scans were measured from 190 to 255 nm in benign buffer (0.1 M KCl, 0.05 M potassium phosphate, 2 mM dithiothreitol, pH 7) at two peptide concentrations, 1 and 0.1 mM, unless otherwise noted in the text. For samples
containing TFE, the above buffer was diluted 1:1 (v/v) with TFE.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
PH; Fig. 1C; see also Fig.
7). In contrast, co-immunoprecipitation was abolished by a C-terminal
deletion which removed the second of the two potential coiled-coil
forming segments (C695; Fig. 1, A and B; see also
Fig. 7). Co-immunoprecipitation was also eliminated by internal
deletions that removed the first of the two regions or both together
(
CC1 or
CC12; Fig. 1C; see also Fig. 7). These data
suggested that the sequence between aa 654 and aa 741 containing the
two regions may represent an important self-assembly domain.
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Fig. 1.
Co-immunoprecipitation of HA- and Myc-tagged
dynamin-1 constructs. Co-immunoprecipitations (i.p.) of
HA- and Myc-tagged dynamin-1 constructs were performed using a Myc
polyclonal antibody. Interactions were detected with either the Myc or
HA monoclonal antibodies. The blots in panel A
are probed with the HA mAb (lot L), those in panel B are
probed with the Myc mAb, and those in panel C are probed
with the HA mAb (lot N). The cross-reacting band observed in
panel C was lot-specific. In panels A and B,
co-immunoprecipitation was assayed between HA- and Myc-tagged
C-terminal deletion mutants of similar lengths, whereas in panel
C, Myc-tagged dynamin-1 internal deletion mutants were
co-immunoprecipitated with full-length HA-tagged dynamin-1. The
boundaries for the internal deletion mutants shown in panel
C are as follows: PH, aa 514-629;
CC1, aa 654-681;
CC12, aa 654-741.
C851, full-length dynamin-1; B, beads alone,
control.
PH; Fig. 2; see also Fig. 7). C-terminal deletions
missing some or all of the potential coiled- coil forming sequences
resulted in no interaction with the full-length bait (C695, C648, and
C514; Fig. 2; see also Fig. 7) as did partial or complete removal of
the coiled-coil region between aa 654-741 (
CC1,
CC2, and
CC12; Fig. 2; see also Fig. 7), consistent with our
co-immunoprecipitation data.
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Fig. 2.
Assay for dynamin-dynamin interaction using
the yeast interaction trap. Dynamin-dynamin interactions were
assayed in the yeast strain EGY48 using a full-length dynamin-1 bait
and dynamin-1 deletion mutant preys. Interactions were assayed on
galactose plates lacking uracil, histidine, and tryptophan in the
presence of 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside. Blue colonies are
positive; non-blue colonies are negative. The nomenclature
of the various mutants are the same as in Fig. 1. JG4-5
refers to the prey vector alone.
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Fig. 3.
Reversal of endocytic inhibition
by deletion of assembly sequences. Fluorescein
isothiocyanate-transferrin internalization was measured in
COS-7 cells transiently transfected with wild-type dynamin-1
(WT), a dominant inhibitory mutant form of dynamin-1
(K44E), or internal deletions of either of the latter
construct (K44E/ CC1 or K44E/
CC2). All
constructs were Myc-tagged, and their expression was detected by the
Myc monoclonal antibody 9E10. Transferrin internalization is inhibited
in the cell overexpressing the K44E mutant construct but is
restored in cells expressing the doubly mutant constructs.
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Fig. 4.
Heterotypic interaction between the dynamin-1
and dynamin-2 isoforms. A, dynamin-2 (Dyn-2)
bait was tested for an interaction with the dynamin-1
(Dyn-1) prey or with the JG4-5 vector as a control.
Selection was on galactose lacking uracil, histidine, and tryptophan in
the presence of 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside medium. B,
co-immunoprecipitation of dynamin isoforms. Myc-dynamin-2 and
HA-dynamin-1 were co-expressed in COS-7 cells and immunoprecipitated
using the Myc polyclonal antibody. C, beads alone, control.
D, anti-Myc immunoprecipitants blotted with Myc
(left) polyclonal or HA (right) monoclonal
antibody. The arrow points to the dynamin isoform in each
blot. IgG, cross-reacting antibody heavy chain.
CC12; Fig. 5B; see also Fig. 7), consistent with
dissociation to a smaller species. No such shift was produced by
deletion to aa 733 (C733; Figs. 6 and
7), which reverses the dominant
inhibitory endocytic effect (31), despite removal of several amino
acids from the C terminus of the self-assembly domain. However, partial
dissociation was observed in a different gradient buffer (20 mM Hepes, pH 7, 50 mM KCl; Figs. 6 and 7).
These results indicated a decrease in the affinity between
polypeptides, although the ability to self-associate was not abolished
completely.
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Fig. 5.
Sucrose density gradient centrifugation of
various dynamin-1 C-terminal deletion mutants. COS-7-expressed
Myc tagged dynamin-1 constructs were analyzed in 5-20% sucrose
density gradients and immunoblotted using anti-Myc antibody. The blots
were quantified by densitometry. The densitometric scans were
normalized for expressed protein concentration for each construct. S
values for markers used are shown at the top. A,
data for full-length dynamin-1 (C851) and C-terminal
deletions C750 and C695. B, data for
full-length dynamin-1 (C851) and the internal deletions
PH and
CC12.
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Fig. 6.
Buffer-dependent dissociation of
C733. The C-terminal dynamin-1 truncation construct C733 was
analyzed in 5-20% sucrose density gradients containing either the
standard gradient buffer (20 mM Tris, pH 7.4, 50 mM KCl) (A) or 20 mM Hepes, pH 7, 50 mM KCl (B). Other conditions are as described in
the legend to Fig. 5.
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Fig. 7.
Summary of the mapping results.
Schematic representation summarizing the cDNA constructs used to
map the C-terminal self-assembly region by co-immunoprecipitation
(Co-IP), yeast two-hybrid (2-Hybrid), and sucrose
density gradients (S-value). ++, very good interaction; +,
good interaction; +/ , detectable interaction;
, no interaction;
Pro-rich, proline-rich domain.
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Fig. 8.
Secondary structure analysis. Newcoils
(A), Paircoils (B), and Stablecoils
(C) plots of dynamin-1 are shown, along with sequences
selected for the design of synthetic peptides
(bottom).
-helical conformation, indicating that this region of dynamin has
the intrinsic ability to adopt a helical secondary structure (data not
shown). Similarly, the centrally derived D5 peptide exhibited a mixture
of random coil and
-sheet, but not
-helical, structure in benign
solution (Fig. 9A). In
contrast, peptides D2, D3, and D4 exhibited
-helical CD spectra,
with the characteristic minima at 208 and 222 nm (Fig. 9B).
An increase in
-helicity (10-20%) was observed for the D2 and D4
peptides at higher concentrations, whereas limited solubility of D3
precluded comparable analysis. CD spectra for a mixture of D2 and D4
were additive (data not shown). Curiously, D3/D4 or D2/D3 mixtures were
less than additive (data not shown). This may be due to nonspecific
interactions by D3, which also caused a similar, but smaller, decrease
in the mean residue molar ellipticity at 222 nm when mixed with a known
coiled-coil forming peptide from an unrelated protein, MMKif (Ref. 39
and data not shown). Peptides D2, D3, and D4 all exhibited low
stability and were completely unfolded at low concentrations of
guanidine HCl (0.75 M; Fig. 10). Table
II summarizes the CD data.
Amino acid sequences of the synthetic peptides used in this study
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Fig. 9.
CD spectra of peptides D1-D5. Dynamin-1
peptides D1 and D5 (100 µM), which correspond to aa
295-318 and aa 372-404, respectively (A), and peptides D2,
D3, and D4, which correspond to aa 323-352, aa 652-681, and aa
712-740, respectively (B), were analyzed at 20 °C in
benign buffer (0.1 M KCl, 0.05 M potassium
phosphate, 2 mM dithiothreitol, pH 7). Petides D2, D3, and
D4 showed characteristic -helical spectra.
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Fig. 10.
Denaturation of dynamin-1 peptides D2, D3,
and D4. CD spectra of the three helical dynamin peptides D2, D3,
and D4 in benign buffer at increasing molar concentrations of guanidine
HCl. Peptide concentrations were 0.32, 0.07, and 0.47 mg/ml for D2, D3,
and D4, respectively. The fraction folded of each peptide was
calculated as f = ([ ]obs
[
]u)/([
]f
[
]u) where f is the fraction folded,
[
]obs is molar ellipticity observed for a given
[guanidine HCl], [
]u is the molar ellipticity of the
unfolded state, and [
]f is the molar ellipticity of
the folded state.
Ellipticities and stabilities of the synthetic peptides
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Fig. 11.
Equilibrium ultracentrifugation of helical
peptides. A, peptide D2. B, peptide D4.
C, peptide D3. Peptide concentration gradients are shown and
fit to a tetramer (A and B), and hexamer for
C. Residuals (top) show deviation of measured
points from fit line. Insets, peptide concentration
gradients compared with theoretical lines for monomer (M),
dimer (D), tetramer (T), or hexamer
(H). The data for peptides D2 and D4 (A and
B) fit to a tetrameric species, whereas those for peptide D3
fit to a hexamer (C). Sedimentation runs were at 25,000, 22,000, and 25,000 rpm, for D2, D3, and D4, respectively.
r2/2, radial distance;
ln(y), natural log of fringe displacement
(concentration).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 12.
Models for the structural organization of
dynamin tetramers. A, summary of peptide analysis.
Dark bars, location of peptides that were -helical by CD
analysis; dotted bars, location of peptides that were random
coil or a mixture of random coil and
-sheet by CD;
Assembly, self-assembly regions identified in this paper;
R, shortest C-terminal deletion (aa 733) at which
receptor-mediated endocytosis is observed in a dominant negative
dynamin-1 mutant (see Ref. 31). B, dynamin-1 polypeptides
are shown either in a parallel (model I) or anti-parallel
(model II) orientation with coiled-coil sequences
corresponding to peptides D2, D3, and D4 linking the individual
polypeptides together.
Nature of Self-assembly Species-- A significant issue in this study is the stage in self-assembly in which these regions participate. Dynamin has been reported to exist in the unassembled state primarily as a tetramer (14, 16), although whether this species represents a stable, minimal assembly unit is unclear. Dynamin and the Mx proteins also assemble to partial rings, complete rings, and helical stacks of rings. Our analysis has dealt primarily, if not exclusively, with dynamin in the disassembled state. Our immunoprecipitation studies were performed at a sufficiently high ionic strength range to prevent assembly (14). The sucrose gradient analysis, although performed at lower ionic strength, revealed directly that the protein was in the disassembled state. The S value of 7.5 is consistent with a tetramer, albeit a highly assymetric structure. This species is clearly oligomeric in view of the sharp decrease in S value caused by small changes in C-terminal sequence. The state of self-association of dynamin-1 expressed in yeast is uncertain. The N-terminal fusion sequences could inhibit assembly, although N-terminal GFP-dynamin appears to function normally in cells (5).2 Because our mapping studies are so closely consistent with the results of our co-immunoprecipitation and centrifugation analysis, it is likely that the two-hybrid analysis primarily reflects monomer-dimer or monomer-tetramer formation, with one intriguing exception.
Previous studies of dynamin pointed to a role for the proline-rich domain in dynamin self-assembly (9, 14, 16). However, in these cases effects on polymer formation were assayed. Our data indicate that the proline-rich region is not required for self-association to the dimer or tetramer level; hence, it must be required for higher order assembly steps. We did observe diminution in the intensity of the yeast two-hybrid reaction using the C750 construct in the prey vector (Fig. 2), although the extent of co-immunoprecipitation was not affected by a comparable deletion, nor was the mutant protein observed to dissociate to the 4.5 S species by sucrose density gradient centrifugation. How the yeast result is to be interpreted is unclear. It may indicate greater sensitivity in detecting changes in affinity between dynamin subunits, or it may indicate that some extent of co-polymerization of bait and prey dynamin does actually occur in this system. We do note that although we tested for interactions between identical truncation mutant constructs in our other assays, full-length bait was used in the two-hybrid analysis. Thus, it is also possible that interaction between wild-type and the C750 mutant dynamin could be sterically affected by the proline-rich region.
The GTPase and PH domains were not required for dynamin self-association in our assays. The former result contrasts with those for the antiviral Mx proteins. Among the dynamin-related family members, the structure of these proteins has been the most extensively characterized. Three self-assembly regions have been mapped within the Mx1 protein. The monomer-to-polymer transition was assayed by gel filtration chromatography of full-length and truncation mutant Mx constructs. This analysis identified one self-assembly site within the GTPase domain (40) and a second near the C terminus (29). No role was detected for a putative leucine zipper domain further downstream at the extreme C terminus. In contrast, the latter region was implicated in dimerization in a separate study in which it was fused to a reporter enzyme. The basis for the difference in these results is unclear. The latter results, however, seem to be more nearly consistent with our findings for dynamin. Although we find no obvious sequence homology between the leucine zipper region of Mx and the C-terminal assembly domain of dynamin, their secondary structures seem to be related. Thus, it is likely that C-terminal coiled-coils may play a common role throughout the greater dynamin protein family, a conclusion supported by secondary structure predictions for other family members (41).
Role of Coiled-coils--
Secondary structure predictions for
dynamin-1 by the Newcoils, Stablecoils, or Paircoils algorithms
suggested several possible coiled-coil regions in dynamin-1, although
the predictions differed substantially. We tested the ability of five
of these regions to form interacting -helical structure using
synthetic peptides. Intriguingly, the predictive success of each
algorithm was variable. It is not yet clear whether this outcome
reflects the choice of peptide boundaries or features of the predictive
schemes. Three of the five peptides exhibited clear evidence for
-helix formation by CD spectroscopy: D3 and D4 within the C-terminal
self-assembly domain, identified in our analysis of recombinant dynamin
polypeptides, and D2, derived from the center of the polypeptide. D3
was predicted to have a 60% probability of coiled-coil formation by
Newcoils, only 11% probability by Paircoils, and the strongest
propensity for coiled-coil formation using Stablecoils. D4 also
exhibited 60% probability using Newcoils and approximately half the
propensity of D3 using Stablecoils. Results were most surprising for
the other peptides. D2 showed insignificant probability using Newcoils, only slightly positive probability using Stablecoils, and 11% using
Paircoils. We considered D1 worth examining because, despite moderate
predictive scores from Newcoils and Stablecoils, the homologous region
within dynamin-2 exhibited 95% probability using Newcoils.
Nonetheless, the D1 peptide was observed as a random coil by CD. It is
possible that the coiled-coil forming sequence within the complete
dynamin-1 polypeptide is more extensive, encompassing the D1 region and
other regions as well.
In the case of the C-terminal assembly domain our data support the
existence of two short stretches of -helix (D3 and D4) separated by
a 31-aa intervening sequence of undetermined structure (pI = ~5.3). Whether this region also participates in coil formation is
uncertain; however, the D3 coil cannot be much longer than predicted
because proline residues flank the sequence on either side. Although
the sequence of this C-terminal assembly domain is conserved among the
dynamin isoforms (79%), only the D3 region is consistently predicted
by the Newcoils program to form a potential coiled-coil, albeit with
varying probability (42-60% between rat dynamin isoforms). It is of
interest that the D4 region has very low coiled-coil probability in
dynamin-2. Inspection of the latter sequence suggests that a single
amino acid gap accounts for this difference. That dynamin-2
co-immunoprecipitated with dynamin-1 is significant; this result
implies that isoforms are interchangeable in the cell. It helps to
explain the dominant inhibition of endocytosis by heterologous
isoforms. In addition, it raises questions about models that implicate
dynamin isoforms in distinct cellular functions (5). Conceivably,
targeting signals exist that override the tendency toward miscibility
and segregate isoforms to different membranous compartments. However,
dynamins appear to be predominantly cytoplasmic (26), where our results
suggest they would be free to intermix. Finally, deletion of either of
these regions in the dynamin-1-K44E mutant reversed the dominant
negative endocytic effect and resulted in transferrin uptake,
indicating that these regions are indeed functionally important.
CD spectra of mixtures revealed no obvious evidence for
heterodimerization. However, despite their relatively short lengths, peptides D2, D3, and D4 all clearly self-associated. D2 and D4 were
observed to be tetrameric, with no evidence for higher order aggregates. That self-association involved coiled- coil formation is
supported by the increase in -helical content with concentration for
at least two of the three peptides. D3 associated to a somewhat nonhomogenous mixture that was predominantly hexameric, with evidence of dissociation to smaller species in the lower concentration range.
Because this peptide exhibited other evidence of nonspecific binding,
caution must be exercised in interpreting its self-association behavior. It is possible that in the context of the entire protein, the
D3 region is constrained to a four-stranded coiled-coil like the
other regions. The self-association behaviors of D2 and D4 are
particularly interesting in light of evidence that dynamin may exist in
the depolymerized state as a tetramer. Our results not only add
strong support to the latter claim but also suggest that the
coiled-coil regions are sufficient for dynamin tetramerization.
Structural and Physiological Role of Assembly Domains--
How the
dynamin self-assembly regions are distributed within the folded
tetramer is unknown. Two general models may be envisioned depending on
whether the -helices are arrayed in parallel or anti-parallel. In
the first case, we imagine that each of the three coiled segments,
corresponding to D2, D3, and D4, participates in the formation of a
tetrameric backbone with uniformly oriented polypeptides. Such an
arrangement is clearly feasible (Fig. 12B, model
I) and has a number of interesting structural consequences. For
one, the tetramer would have structural polarity, a property required
for the formation of a polymer with distinct membranous and cytoplasmic
surfaces as may be expected for dynamin (10, 11). In addition, it may
be argued based on symmetry considerations that the coiled-coil regions
must be co-linear. Thus, we would predict that the dynamin tetramer is
organized about a central coiled-coil shaft. It is also possible that
the central and C-terminal self-assembly regions function at different
levels of dynamin self-association, although the fact that both D2 and
D4 form tetramers makes such models seem less likely. Our existing
evidence indicates that the C-terminal region is important in the
monomer-dimer or monomer-tetramer transition; conceivably, the central
assembly domain might be involved in polymer formation. Although this
is an attractive possibility, it would lead to network formation among
dynamin polypeptides, inconsistent with the known features of the
dynamin self-assembly pathway. Similar problems arise for models in
which the peptides are proposed to interact in anti-parallel (Fig. 12B, model II). In particular, it is
difficult to see how interaction of both assembly domains could
occur between polypeptides in an antiparallel manner. Thus, we strongly
favor the parallel model.
It is of interest that the C-terminal self-assembly domain appears to
lie within the boundaries of a previously identified 13-kDa proteolytic
fragment of dynamin-1 implicated in GTPase regulation. This fragment,
termed the GED (GTPase effector
domain) was found to bind to the GTPase domain and
stimulate its activity (18). Whether the coiled-coil sequences we have
identified within this fragment or additional sequences within this
region are involved in GTPase effector activity is uncertain, and how
self-association influences this interaction remain to be seen.
However, it now seems clear that the coiled-coil segments of dynamin
represent a new, functionally important element. Further analysis of
these regions should be of considerable value in understanding how
assembly of this remarkable protein is controlled.
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ACKNOWLEDGEMENTS |
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We thank Dr. Chantal Gamby for the schematic diagram of dynamin organization and for helpful comments and discussion on the manuscript. We also thank other members of the Vallee lab, Dr. Wayne Zhou, and Dr. Steve Lambert for valuable comments and discussion.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM26701.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.
¶ To whom correspondence should be addressed. Tel.: 508-856-8988; Fax: 508-856-8987; E-mail: Richard.vallee{at}ummed.edu.
2 R. J. Vasquez and R. B. Vallee, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: aa, amino acid(s); TFE, trifluoroethanol; PH, pleckstrin homology; HA, hemagglutinin.
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