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INTRODUCTION |
Dynamin is a high molecular weight GTP-binding protein with an
intrinsic GTPase activity. Many proteins that belong to the dynamin
family have been identified in a variety of organisms ranging from
yeasts to mammals. These proteins have a highly conserved N-terminal
GTPase domain of ~300 amino acids (for reviews, see Refs. 1-4). This
family is divided into subfamilies on the basis of the structural
similarity (2). The dynamin subfamily consists of mammalian dynamins I,
II, and III, and the Drosophila shibire gene product. A
second subfamily consists of Saccharomyces cerevisiae Vps1p
and Dnm1p and a recently identified mammalian member designated DVLP1 (5), dymple (6), DLP1
(dynamin-like protein 1) (7), or DRP1 (dynamin-related protein 1) (8).
Typical structural features that discriminate between members of the
Vps1 and dynamin subfamilies are that the former lack the pleckstrin
homology and Pro-rich domains, which are involved in interactions with
phospholipids and proteins containing the SH3 domain, respectively. Two
plant homologs, phragmoplastin and aG68/ADL1, are grouped into another subfamily. Interferon-inducible Mx proteins and yeast Mgm1p constitute the most diverged subfamily.
Members of the dynamin and Vps1 subfamilies have been implicated in
intracellular vesicular transport. Analyses using cells transfected
with dynamin mutants and using Drosophila shibire mutants
demonstrated that dynamins are responsible for budding of endocytic
vesicles from the plasma membrane (9-12). It has also been reported
that dynamin II is involved in transport from the
trans-Golgi network (13). Morphological analysis and
in vitro experiments showed that dynamin self-assembles into
rings and/or spirals around the necks of invaginated clathrin-coated pits, thereby suggesting a model that GTP hydrolysis of dynamin pinches
off the budding vesicles (14, 15). On the other hand, Vps1p was
identified as one of the gene products required for vacuolar protein
sorting in yeast (16), and Dnm1p was then isolated as a homolog of
Vps1p and may participate in an endocytic process (17). Recently, the
mammalian protein most homologous to Vps1p and Dnm1p not only in the
primary structure but also in the domain organization has been
identified by us and others and designated DVLP (Dnm1p/Vps1p-like
protein) (5), dymple (6), DLP1 (7), or DRP1 (8). The most abundant form
of DVLP consists of 736 amino acids, but some splicing variants are
also present (6, 7). Indirect immunofluorescence microscopy revealed
that DVLP is localized to a punctate cytoplasmic structures around the
nucleus (5-8). Yoon et al. (7) showed by immunoelectron
microscopy that DLP1/DVLP-positive structures coalign with microtubules
and tubules of the endoplasmic reticulum and proposed that DLP1/DVLP may participate in the formation of nascent secretory vesicles from the
endoplasmic reticulum.
Velocity sedimentation and gel filtration analyses and cross-linking
experiments indicated that dynamin exists as homo-oligomers, mainly
tetramers, under physiological salt conditions (15, 18-21). Under low
salt conditions, however, it co-assembles into polymers to form rings
and/or spirals (15, 20, 21). During the course of our experiments, we
noticed a possibility that DVLP is also oligomeric. We therefore
analyzed this phenomenon in detail.
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EXPERIMENTAL PROCEDURES |
Plasmid Construction--
Construction of the expression vector
for human DVLP with an N-terminal hemagglutinin (HA) epitope sequence
(pcDNA3-HAN-DVLP) was described previously (5). An expression
vector for Myc-tagged DVLP (pcDNA3-MycN-DVLP) was constructed by
replacing the fragment encoding the HA epitope of pcDNA3-HAN-DVLP with
a double-stranded oligonucleotide for the Myc epitope. A vector for
Myc-tagged DVLP(278-736) was constructed by replacing the entire DVLP
portion of pcDNA3-MycN-DVLP with an MscI-NotI
fragment of the DVLP cDNA. For the use in the two-hybrid analysis,
bait and prey vectors for DVLP were constructed by ligation of a DNA
fragment for DVLP from pcDNA3-HAN-DVLP into pGBT9 and pGAD10
(CLONTECH), respectively. Bait and prey vectors for
deletion mutants (Fig. 1), except for the prey vector of DVLP(580-736) and DVLP(490-634), were constructed by utilizing the endogenous restriction sites of the DVLP cDNA; the DNA fragments of
DVLP(580-736) and DVLP(490-634) were prepared by a polymerase chain
reaction-based strategy.
Antibodies--
Sources of antibodies used were as follows:
monoclonal rat (3F10) and mouse (12CA5) anti-HA antibodies,
Boehringer Mannheim; monoclonal mouse anti-Myc antibody (9E10),
Berkeley Antibody Co.; peroxidase-conjugated anti-mouse IgG, Amersham
Pharmacia Biotech; other secondary antibodies, Jackson ImmunoResearch Laboratories.
Indirect Immunofluorescence Analysis--
A Clone 9 rat
hepatocyte cell line stably expressing HA-tagged DVLP (Clone 9/DVLP-HA)
was established by selection of clonal cell lines transfected with
pcDNA3-HAN-DVLP in the presence of 800 µg/ml Geneticin
(Wako Pure Chemicals). For immunofluorescence analyses, Clone 9 cells
grown in wells of eight-well Lab-Tek-II chamber slides (Nunc) were
transfected with pcDNA3-MycN-DVLP(278-736) alone or in combination
with pcDNA3-HAN-DVLP using a TransIT-LT1 transfection reagent
(PanVera Corp.), cultured for 20 h, and processed for indirect
immunofluorescence analysis as described previously (5, 22). Briefly,
cells fixed and permeabilized with methanol at
20 °C for 5 min or
those fixed with 4% paraformaldehyde and permeabilized with 50 µg/ml
digitonin for 5 min at 4 °C were incubated with anti-HA and/or
anti-Myc antibodies. The cells were then incubated with FITC-conjugated
and/or Cy3-conjugated secondary antibodies and observed with a
laser-scanning confocal microscope (TCS-NT, Leica Lasertechnik).
Preparation of Cytosol, Gel Filtration, and Western Blot
Analysis--
Clone 9/DVLP-HA cells were homogenized in two volumes of
HCB150 (20 mM HEPES-KOH, pH 7.2, 2 mM EGTA, 1 mM MgCl2, 1 mM dithiothreitol, 150 mM NaCl) by 5 sets of 20 strokes with a Dounce homogenizer. The homogenate was centrifuged at 200,000 × g for 60 min in a Beckman TLA 100.2 rotor, and the supernatant was used as a
cytosol fraction. The cytosol fraction containing ~500 µg of
protein was applied to a Superdex 200 column (1.0 × 30 cm;
Amersham Pharmacia Biotech) equilibrated with HCB150 and eluted at a
flow rate of 0.4 ml/min. Fractions of 0.2 ml that were collected were
precipitated with cold acetone, dissolved and boiled in
SDS-polyacrylamide gel electrophoresis sample buffer, electrophoresed
on a 7.5% SDS-polyacrylamide gel under reducing conditions, and
electroblotted onto an Immobilon-P membrane (Millipore). The blot was
incubated sequentially with anti-HA antibody (3F10) and
peroxidase-labeled anti-rat IgG, and detected using a Renaissance
Chemiluminescence reagent Plus (NEN Life Science Products)
according to the manufacturer's instructions.
Cross-linking Experiments--
Clone 9/DVLP-HA cells were washed
twice with phosphate-buffered saline and lysed in HCB150 containing 1%
Triton X-100 and 10% glycerol. The lysates were cleared by
centrifugation at 12,000 rpm for 10 min in a microcentrifuge and then
incubated at 4 °C for indicated time periods in the presence of 1 mM dithiobis(succinimidylpropionate) (DTSP) (Sigma). After
the incubation, residual DTSP was quenched by the addition of glycine
(final concentration, 100 mM). The mixture was then
electrophoresed on a 7% SDS-polyacrylamide gel under nonreducing
conditions and subjected to Western blot analysis with anti-HA antibody
as described above.
Sedimentation Assay--
The Clone 9/DVLP-HA cytosol in HCB150
was dialyzed overnight at 4 °C against HCB0 (no NaCl), HCB50 (50 mM NaCl), or HCB100 (100 mM NaCl). The samples
were then centrifuged at 200,000 × g for 15 min in the
TLA 100.2 rotor. The resultant pellet and supernatant were subjected to
SDS-polyacrylamide gel electrophoresis and Western blot analysis as
described above.
Yeast Two-hybrid Analysis--
Yeast transformation and
two-hybrid analysis were performed according to the instructions for
the MATCHMAKER two-hybrid system (CLONTECH).
Briefly, a yeast strain Y190 was co-transformed with a pGBT9-based bait
vector and a pGAD10-based prey vector and plated on a medium lacking
Trp, Leu, and His and containing 10-25 mM 3-aminotriazole
(Wako Pure Chemicals). After 5-7 days of incubation, colonies were
tested for
-galactosidase (
-Gal) activity by use of replica
filter assay.
-Gal activity was also measured by a liquid culture
assay using o-nitrophenylgalactoside as a substrate (23).
Co-immunoprecipitation Analysis--
Human embryonic kidney 293 cells transiently transfected with either pcDNA3-HAN-DVLP or
pcDNA3-MycN-DVLP, or both were washed twice with ice-cold
phosphate-buffered saline and lysed in ice-cold cell lysis buffer (50 mM Tris-HCl, pH 7.5, 1% Triton X-100, 2.5 mM
EDTA, 0.25 M NaCl containing a CompleteTM
protease inhibitor mixture (Boehringer Mannheim)). The lysates were
cleared by centrifugation at 12,000 rpm for 10 min in a
microcentrifuge. A half of the lysates containing ~20 µg of protein
were directly subjected to SDS-polyacrylamide gel electrophoresis and
Western blot analysis using anti-HA (3F10) or anti-Myc (9E10) antibody. The other half was immunoprecipitated with anti-HA antibody (12CA5) and
protein A-Sepharose and subjected to SDS-polyacrylamide gel electrophoresis and Western blot analysis using the monoclonal anti-Myc antibody.
In VitroTranscription/Translation and Immunoprecipitation
Analysis--
Either pcDNA3-HAN-DVLP(1-489) alone,
pcDNA3-MycN-DVLP(490-736) alone, or both was subjected to in
vitro transcription/translation in the presence of an EXPRESS
protein labeling mixture (NEN Life Science Products) using a TnT T7
transcription/translation system (Promega Corp.). The reaction product
was immunoprecipitated with anti-HA antibody (12CA5) and protein
A-Sepharose, electrophoresed on SDS-polyacrylamide gel, and analyzed
using a BAS2000 bioimaging analyzer (Fuji Film Co.).
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RESULTS |
Evidence for Intermolecular Interaction of DVLP in Cells--
By
indirect immunofluorescence analysis of Clone 9 cells transiently
transfected with HA-tagged DVLP, we found that it associated with
punctate cytoplasmic structures around the nucleus (6). There was a
possibility, however, that the staining pattern was caused by
overexpression. We therefore established cell lines stably expressing
HA-tagged DVLP at a moderate level. Fig. 2A shows a typical
staining pattern for DVLP in one (Clone 9/DVLP-HA) of the established
cell lines. As in transiently transfected cells, DVLP was localized to
punctate cytoplasmic structures in these stable transfectants. On the
other hand, Kamimoto et al. (6) have reported that a
dymple/DVLP mutant lacking the N-terminal GTPase domain forms large
cytoplasmic aggregates. In an attempt to reproduce their data, we made
a similar construct, DVLP(278-736) (Fig.
1), tagged with a Myc epitope and
transiently expressed in Clone 9 cells. As shown in Fig.
2B, we could reproduce the data of Kamimoto et al. (6): the N-terminal deletion mutant was localized to large cytoplasmic aggregates.

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Fig. 1.
Schematic representation of structures of
DVLP and its deletion constructs used in the present study.
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Fig. 2.
Indirect immunofluorescence analysis.
A, Clone 9/DVLP-HA cells were stained with anti-HA antibody.
B, Clone 9 cells transfected with the expression vector for
Myc-tagged DVLP(278-736) were stained with anti-Myc antibody. C,
C', D, and D', Clone 9 cells
co-transfected with the expression vectors for HA-tagged DVLP(FL) and
for Myc-tagged DVLP(278-736) were double-stained with anti-HA
(C and D) and anti-Myc (C' and
D') antibodies.
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During the course of this experiment, we met with unexpected findings.
In an attempt to discriminate unequivocally the localization of
DVLP(278-736) from that of full-length DVLP (DVLP(FL)), we transfected
the Myc-tagged DVLP(278-736) construct along with the HA-tagged
DVLP(FL) construct into Clone 9 cells and double-stained with anti-HA
and anti-Myc antibodies. We found that in some populations of the cells
with a relatively low expression level of DVLP(278-736), the staining
for the deletion mutant was superimposed on the punctate cytoplasmic
staining for DVLP(FL) (Fig. 2, C and C'). In
contrast, in another cell population expressing a relatively high level of DVLP(278-736) as evident from the intense staining for Myc, DVLP(FL) colocalized with DVLP(278-736) in large cytoplasmic
aggregates (Fig. 2, D and D'). These observations
suggest a possibility that DVLP may exist as oligomers/multimers intracellularly.
DVLP Is a Tetramer under Physiological Salt Conditions--
To
confirm the above speculation, a cytosolic extract was prepared from
Clone 9/DVLP-HA cells using buffer containing a physiological concentration of NaCl (HCB150) and subjected to gel filtration on a
Superdex-200 column, and the fractions were analyzed by Western blotting with anti-HA antibody. As shown in Fig.
3, DVLP-HA eluted from the gel with an
apparent molecular mass of ~320 kDa. Because the molecular mass of
DVLP-HA monomer is ~80 kDa (Fig. 3; also see Figs.
4 and 7), the species of ~320 kDa is
probably a tetramer.

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Fig. 3.
Gel filtration analysis of cytosol of Clone
9/DVLP-HA cells. Cytosol of Clone 9/DVLP-HA cells was fractionated
with a Superdex 200 column, and fractions were analyzed by Western
blotting with anti-HA antibody. Molecular mass markers used were bovine
thyroglobulin (669 kDa), sweet potato -amylase (200 kDa), and bovine
serum albumin (66 kDa).
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Fig. 4.
Cross-linking analysis. Clone 9/DVLP-HA
cell lysates were untreated or treated with DTSP for indicated time
periods and analyzed by Western blotting with anti-HA antibody.
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To corroborate the gel filtration data, we then performed cross-linking
studies. Clone 9/DVLP-HA cell lysates were treated with a cross-linker,
DTSP, in HCB150, and the extent of cross-linking was monitored on
Western blots. As shown in Fig. 4, a molecular species of ~300 kDa
appeared in a time-dependent manner, with disappearance of
the ~80-kDa band. It is also noteworthy that no dimer/trimer
intermediates of DVLP were detected during the incubation, in contrast
to cross-linking studies of dynamin (18, 19), in which not only a
tetramer but also dynamin species corresponding to a dimer and a trimer
were observed. Collectively, these data indicate that DVLP is probably
tetrameric in cells.
DVLP Aggregates under Low Salt Conditions--
Dynamin has been
reported to self-assemble into rings and/or spirals under low salt
conditions (15, 20, 21). The dynamin assembly can be quantified by a
sedimentation assay in which polymerized assembled dynamin appears in
the pellet after high speed centrifugation. To examine whether DVLP
also polymerizes under low salt conditions, the Clone 9/DVLP-HA cytosol
in HCB150 was dialyzed against lower salt buffers and subjected to
ultracentrifugation, and the resultant supernatants and pellets were
analyzed by Western blotting. As shown in Fig.
5, in higher salt buffers (HCB100 and
HCB150), the largest fraction of DVLP remained in the supernatant,
whereas in lower salt buffers (HCB0 and HCB50), a significant fraction sedimented. Densitometric analysis revealed that in HCB100 and HCB150
only ~10 and <5%, respectively, of DVLP sedimented, whereas in HCB0
and HCB50, ~80 and ~40%, respectively, of DVLP assembled into
sedimentable complexes.

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Fig. 5.
Sedimentation assay. Cytosol of Clone
9/DVLP-HA cells in HCB150 was dialyzed against HCB0, HCB50, or HCB100
and centrifuged at 200,000 × g for 15 min. The pellet
(P) and supernatant (S) fractions were analyzed
by Western blotting with anti-HA antibody.
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Intermolecular Interaction of DVLP Molecules--
The experiments
thus far suggest that, like dynamin, DVLP may exist as tetramers under
physiological salt conditions and assemble into sedimentable complexes
under low salt conditions. However, it was possible that such complexes
might be formed by the aid of an unknown adaptor protein(s), because we
used crude cytosolic extracts in the above experiments. To demonstrate
the direct intermolecular interaction, we then used the yeast
two-hybrid system. When bait and prey vectors fused to DVLP(FL) were
co-transformed into reporter yeast cells, the co-transformant grew well
on a plate lacking His (Fig.
6A) and showed a high level of
-Gal activity (Fig. 6B). Co-transformation of the
DVLP(FL) bait vector with either an empty prey vector (Fig. 6,
A and B) or a prey vector fused to an unrelated
protein (lamin; not shown) did not result in colony formation on the
His-free plate nor show a significant
-Gal activity. The data
therefore indicate a specific intermolecular interaction of DVLP.

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Fig. 6.
Two-hybrid analysis. Yeast cells were
co-transformed with a pGBT9-based bait vector and a pGAD10-based prey
vector fused to full-length DVLP or its deletion mutants as shown in
Fig. 1. A, the transformed cells were streaked on
His-containing (+His) or His-deficient ( His)
plates. The His plate contained 25 mM 3-aminotriazole.
B, the transformed cells were subjected to liquid -Gal
assay as described under "Experimental Procedures."
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The above data demonstrated the intermolecular interaction of DVLP
in vitro and in the heterologous yeast expression
system. To demonstrate such an interaction in cells, we
co-transfected cells with vectors for HA- and Myc-tagged
DVLP(FL) and examined whether anti-HA antibody was able to
co-immunoprecipitate Myc-tagged DVLP from lysate of the
co-transfectant. Fig. 7A shows
the expression of both HA- and Myc-tagged DVLP in the co-transfectant.
When the lysate of the co-transfectant was immunoprecipitated with
anti-HA antibody and then subjected to Western blotting with anti-Myc antibody, a specific band of Myc-DVLP was detected (Fig.
7B), demonstrating the intermolecular interaction of DVLP in
cells.

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Fig. 7.
Co-immunoprecipitation of HA- and Myc-tagged
DVLP expressed in cells. Cell lysates were prepared from 293 cells
transiently transfected with an expression vector for HA- or Myc-tagged
DVLP or both. A, the cell lysates were directly analyzed by
Western blotting (WB) with anti-HA or anti-Myc antibody.
B, the lysates were immunoprecipitated (IP) with
anti-HA antibody and analyzed by Western blotting with anti-Myc
antibody. A major band at ~49 kDa is derived from IgG heavy
chains.
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Interaction between Domains of DVLP--
To delineate domains of
DVLP responsible for the intermolecular interaction, various deletion
mutants were constructed and subjected to the two-hybrid analysis. We
anticipated that a C-terminal domain, named dynamin/Vps1p homology 2 (DVH2), could be responsible for such an interaction, because this
domain is significantly conserved in the dynamin and Vps1 subfamilies
and has the potential to form a coiled-coil structure (1, 5), which has
been shown to be involved in homo- and hetero-oligomeric
protein-protein interactions. Therefore, we first constructed bait and
prey vectors for DVLP(490-736), which includes the DVH2 domain (Fig.
1), and examined whether the homotypic interaction of the coiled-coil domain of DVLP occurred. However, we failed to detect such an interaction (Fig. 6). In contrast, when the prey vector for
DVLP(490-736) was co-transformed with a bait vector for the deleted
part, namely DVLP(1-489), a significant interaction was observed; the
co-transformant grew well on the His-free plate, and its
-Gal
activity approximated that of the DVLP(FL)-DVLP(FL) co-transformant
(Fig. 6). A similar result was obtained using a combination of a bait
vector for DVLP(490-736) and a prey vector for DVLP(1-489) (data not
shown). A homotypic interaction of DVLP(1-489) was not detected (Fig.
6). To corroborate the two-hybrid results, we examined whether in
vitro translated HA-tagged DVLP(1-489) and Myc-tagged
DVLP(490-736) can be co-immunoprecipitated. As shown in Fig.
8, the anti-HA antibody precipitated not
only HA-tagged DVLP(1-489) but also Myc-tagged DVLP(490-736).
Although the origin of a band between those of HA-DVLP(1-489) and
Myc-DVLP(490-736) is not clear, it may be a degradation product or a
not fully translated product of HA-DVLP(1-489), because the band was
also detected in the lane in which the HA-DVLP(1-489) construct alone
was subjected to in vitro translation and
immunoprecipitation with anti-HA antibody.

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Fig. 8.
Co-immunoprecipitation of in vitro
translated DVLP(1-489) and DVLP(490-736). A vector for
either HA-tagged DVLP(1-489) alone, Myc-tagged DVLP(490-736) alone,
or both were subjected to in vitro transcription/translation
in the presence of [35S]methionine. The products were
immunoprecipitated with anti-HA antibody, electrophoresed on a 10%
SDS-polyacrylamide gel, and analyzed with an imaging analyzer.
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DVLP(1-489) covers the N-terminal GTPase domain and another conserved
domain, named DVH1. In order to determine which region of DVLP(1-489)
is responsible for the interaction with DVLP(490-736), we successively
truncated the DVLP(1-489) construct from its C terminus (Fig. 1) and
examined whether the truncation mutant was able to interact with
DVLP(490-736) by the two-hybrid system. As shown in Fig.
6B, DVLP(1-424), which contains the largest part of DVH1
domain, was able to interact with DVLP(490-736). By contrast, DVLP(1-343), which lacks the DVH1 domain, was unable to interact with
DVLP(490-736). These data indicate that the DVH1 domain is required
for the interaction with the C-terminal DVH2-containing region.
However, in addition to the DVH1 domain, the GTPase domain appears to
be also required for the interaction, because DVLP(295-527), which
lacks the GTP-binding domain, failed to show a significant interaction
with DVLP(490-736).
We then examined which region of DVLP(490-736) is responsible for the
interaction with DVLP(1-489). DVLP(490-634) was unable to interact
with DVLP(1-489), indicating that the DVH2 domain is required for the
interaction with the N-terminal region. We then truncated successively
the DVLP(490-736) construct from its N terminus (Fig. 1). As shown in
Fig. 6B, although DVLP(542-736) was capable of interacting
with DVLP(1-489), DVLP(580-736) interacted no longer with the
N-terminal region. These data indicate that although it is not
conserved in the dynamin and Vps1 subfamilies, the region upstream of
the DVH2 domain is also required for the interaction with the
N-terminal region.
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DISCUSSION |
DVLP shares some primary structural features with dynamin. In this
study, we have shown that, as is the case for dynamin, DVLP may be
tetrameric under physiological salt conditions and aggregates into
sedimentable complexes under low salt conditions. The similarities not
only in the structure but also in the complex formation make it
tempting to speculate that DVLP plays a role in the formation of
carrier vesicles in a manner similar to that of dynamin, although it is
currently unclear which transport step(s) DVLP is involved in. Under
the appropriate conditions, DVLP tetramers in the cytosol may assemble
into rings and/or spirals at the necks of budding coated pits prior to
vesicle fission. In this context, a recent report of Carr and Hinshaw
(20) is noteworthy. They have shown that even under physiological salt
conditions, dynamin polymerizes into spirals in the presence of GDP and
-phosphate analogues (aluminum fluoride and beryllium fluoride) or
in the presence of GTP
S. We performed similar experiments for DVLP
but failed to detect its assembly into sedimentable complexes in the presence of the GTP analogues under physiological salt conditions (data
not shown). The reason for the difference between the behavior of
dynamin and the behavior of DVLP is unclear. One possibility is that
the difference may be caused by the difference in the used experimental
system; Carr and Hinshaw (20) used purified recombinant dynamin,
whereas we used crude cytosolic extract containing DVLP. Indeed, using
the cytosolic fraction of Clone 9/DVLP-HA cells, we could not detect
sedimentable complexes of dynamin II in the presence of the GTP
analogues under physiological salt conditions (data not shown). To
address this issue, experiments using purified DVLP will be required.
Attempts are under way to express and purify recombinant DVLP using a
baculovirus system.
Analyses using the two-hybrid system have shown the intermolecular
interaction of DVLP and delineated domains of DVLP responsible for the
interaction; the N-terminal region containing the GTPase and DVH1
domains can interact with the C-terminal DVH2-containing region.
Because these three domains are highly conserved in the dynamin and
Vps1 subfamilies, a common mechanism may govern the interdomain
interactions of dynamin and DVLP. Indeed, our data are in good
agreement with recent data on intermolecular and interdomain interactions of dynamin (19, 20, 24): (i) a dynamin mutant lacking the
C-terminal Pro-rich domain, which is not present in DVLP, can form
itself into tetramers (19) and assemble into spirals (20); (ii) an
N-terminal fragment of dynamin that is deduced to contain the GTPase
and DVH1 domains and a C-terminal fragment that is deduced to contain
the DVH2 domain, both of which are generated by limited
endoproteolysis, are co-purified by chromatography (19, 24); (iii) the
N-terminal and DVH2-containing proteolytic fragments can be
cross-linked with each other (19) and co-assemble into spirals (19,
20); and (iv) the pleckstrin homology domain, which is not present in
DVLP, is not required for the co-assembly nor cross-linked with the
N-terminal or DVH2-containing fragment (19). In this context, it is
also noteworthy that the DVH2-containing fragment appears to stimulate
the GTPase activity of the N-terminal fragment (19). The interaction
between the region containing the GTPase domain and the DVH2 domain may
regulate the assembly/disassembly of the dynamin family proteins.
On the basis of the present data, three models for the tetramer
formation of DVLP are possible (Fig. 9).
In the first model (model 1), an interaction between the N- and
C-terminal regions occurs in the same DVLP polypeptide, and the four
monomer units then assemble into a tetramer. In the second (model 2),
the interaction occur between two adjacent antiparallel polypeptides,
and the two dimer units then assemble into a tetramer. In the third
(model 3), the interactions sequentially occur between adjacent
parallel polypeptides, and a ring-like structure composed of four
polypeptides is thereby formed. We favor the last model, because we
could not detect any homotypic interactions between domains of DVLP by
the two-hybrid analysis or any dimeric intermediates by the gel
filtration and cross-linking analyses. In contrast, Muhlberg et
al. (19) favored model 2 for the tetramer formation of dynamin on
the basis of their analyses using its proteolytic fragments. A
difference between our data on DVLP using the two-hybrid system and the
data on dynamin using proteolytic fragments is that in the latter, homotypic interactions between the N-terminal fragments and between the
DVH2-containing fragments were also detected, although to a much lesser
extent. Despite the difference, the data of Muhlberg et al.
(19) appear to be also compatible with model 3, which they did not
propose. Otherwise, the mechanisms underlying the tetramer formation of
DVLP and dynamin may be slightly different from each other, because,
unlike dynamin (18, 19), no dimeric intermediates of DVLP were observed
in the cross-linking experiments (Fig. 4).