Dymple, a Novel Dynamin-like High Molecular Weight GTPase
Lacking a Proline-rich Carboxyl-terminal Domain in Mammalian
Cells*
Takahiro
Kamimoto,
Yasuo
Nagai,
Hiroshi
Onogi,
Yoshinao
Muro
,
Takashi
Wakabayashi, and
Masatoshi
Hagiwara§¶
From the Departments of Anatomy and
Dermatology,
Nagoya University School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya
466 and § Department of Endocrinology, Medical Research
Institute, Tokyo Medical and Dental University, Tokyo 113, Japan
 |
ABSTRACT |
We have cloned human dymple, a novel
dynamin family member. The full-length cDNA sequence encodes a
protein composed of 736 amino acids with a molecular mass of 80 kDa.
This amino acid sequence most resembles yeast DNM1P and VPS1P. Dymple
lacks a proline-rich carboxyl-terminal domain through which dynamin
binds to SH3 domains to be activated. Northern blot analysis revealed
two transcript sizes of 2.5 and 4.2 kilobases with alternative
polyadenylation at the highest levels in brain, skeletal muscle, and
testis. It was further established that there are three patterns of
alternative splicing producing in-frame deletions in the coding
sequence of dymple in a tissue-specific manner. When overexpressed,
wild-type dymple exhibited a punctate perinuclear cytoplasmic pattern,
whereas an amino-terminal deletion mutant formed large aggregates
bounded by a trans-Golgi network marker. Since dynamin participates in clathrin-mediated endocytosis through a well-characterized mechanism, the existence of a dynamin-like molecule in each specific vesicle transport pathway has been predicted. Our findings suggest that dymple
may be the first example of such a subfamily in mammalian cells other
than dynamin itself, although its precise role and membrane
localization remain to be resolved.
 |
INTRODUCTION |
Dynamin was originally isolated from cow brain because of its
ability to cross-link and bundle microtubules in vitro in a nucleotide-dependent manner (1). Its extensive sequence
similarity with the interferon-inducible Mx proteins (2) and the yeast VPS1 (3) gene product revealed the existence of a
superfamily of dynamin-related high molecular weight GTP-binding
proteins (4). A variety of recently found proteins has been classified into this family (5).
Different forms of Mx, first identified as a murine gene that confers
selective resistance to influenza viruses related to interferon
(IFN-
/
), have been cloned in vertebrates, including a nuclear
protein murine Mx1 (6, 7) and cytoplasmic examples human MxA and MxB
(8). The yeast VPS1, one of more than 50 genes that are
required for soluble vacuolar protein sorting (9), encodes an Mx-like
protein (3) shown to play a direct role in the trafficking of vacuolar
and Golgi membrane proteins from the late Golgi apparatus to the
prevacuolar compartment (10). VPS1 was also found to be the
same gene as SPO15 in mutated yeasts not able to sporulate
because of a defect in meiotic spindle pole separation (11). Two
dynamin family genes have been identified in yeast: MGM1
required for mitochondrial genome maintenance (12) and DNM1,
involved in endosomal trafficking (13). Both the primary sequence and
function of Mgm1p are least similar to those of other dynamin family
members. Although DNM1P shares high sequence homology with VPS1P, its
function is distinct, since it participates in endocytosis at a novel
step after internalization and before delivery to the vacuole (13). In
plant cells, the dynamin-like protein phragmoplastin is associated with
the formation of cell plates, disc-like membrane-bound structures, by
fusion of Golgi-derived vesicles (14).
Dynamin has been isolated from Caenorhabditis elegans,
Drosophila, and a number of mammals with almost 68%
identity across species and appears to function in vesicular transport
during endocytosis elucidated from analysis of the shibire phenotype of
Drosophila in which a temperature-sensitive mutation renders the flies paralyzed because of an inability to recycle synaptic vesicles at nerve terminals (15, 16). In mammals, dynamin seems to
exist as at least two distinctive isoforms, brain-specific dynamin I
(17-19) and ubiquitous dynamin II (20, 21). The most pronounced
diversity between the sequences of dynamin isoforms and other
dynamin-related proteins resides in the carboxyl-terminal span, where a
basic proline-rich domain exists. Through this region, dynamin GTPase
activity is stimulated by various factors in vitro such as
the binding of microtubules and SH3 domains (22), acidic phospholipids
(23), or phosphorylation by protein kinase C (19, 24).
Recent findings have shown that interaction of the dynamin proline-rich
carboxyl-terminal domain with SH3 domains is responsible for in
vivo function in clathrin-mediated endocytosis (25-27). Electron
microscopic studies revealed that the necks of invaginated clathrin-coated pits are collared by dynamin oligomers and that pinching-off of vesicles requires dynamin GTPase activity (28, 29).
Although clathrin-coated vesicles originate from both the plasma
membrane and TGN1 membrane,
respectively, specified by their adaptor complex AP2 and AP1, dynamin
has been shown to be specifically associated with endocytic
clathrin-coated vesicles at the plasma membrane (30, 31). Within the
emerging superfamily of high molecular weight GTPases, VPS1P and DNM1P
in yeast cells resemble dynamin in their involvement with protein
sorting. Thus dynamin/VPS1P-like molecules also might be expected to
exist in vertebrates.
Human autoantibodies have proven to be useful reagents in elucidating
the structure and function of eukaryotic cellular components. Several
nuclear and cytosolic proteins have been identified as autoimmune
antigens (32), and recently we isolated a novel mitogen-activated protein kinase kinase, MAPKK6, using serum from a Behçet patient (33). In the present study, we aimed to identify the antigen targeted
by the serum of a scleroderma patient and screened by a HeLa cell
cDNA library. We obtained a human cDNA clone for dymple, a
dynamin family member lacking a proline-rich carboxyl-terminal domain
that exhibits overall homology to yeast VPS1P and DNM1P. Primary
characterization of this novel gene was also performed.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Autoimmune serum 114 was taken from a scleroderma
patient. Anti-
-adaptin mAb and brefeldin A were purchased from
Sigma, and anti-hemagglutinin (HA) mAb was from Boehringer Mannheim.
Alkaline phosphatase (AP)-conjugated goat anti-human IgG, fluorescein
isothiocyanate-conjugated goat anti-mouse IgG, and fluorescein
isothiocyanate-conjugated goat anti-human IgG were obtained from
Zymed Laboratories Inc. (San Francisco, CA). Texas
red-conjugated goat anti-mouse IgG and Texas red-conjugated goat
anti-human IgG were obtained from Southern Biotechnologies (Birmingham,
AL). The mouse Mx1 plasmid (pMxMN12) (34) was a kind gift from Dr. K. Nagata (Tokyo Institute of Technology).
Immunoscreening and cDNA Sequencing--
The UNI-ZAP HeLa
cDNA expression library was screened with serum 114 by standard
procedures as described previously (33) with more than 300,000 plaques
included in the first screening. The 20 positive clones obtained were
then subcloned into pBlueScriptII SK(
) with the in vivo
excision system (Stratagene, La Jolla, CA). Inserted cDNA sequences
were determined by the dye termination method with an ABI 373S DNA
sequencer (Perkin-Elmer).
5
-RACE--
Using oligo-DNA 5
-TATCATCCACGGGTTCACCG-3
(1096-1077 bp) and 5
-GCCTCTGATGTTGCCATATC-3
(698-679 bp),
respectively, as gene-specific primers 1 and 2, 5
-RACE was carried out
following manufacturer protocol (Life Technologies, Inc.). A 700-bp
major band was recovered from 1% SeaKem agarose gels and subcloned
into a pCRII TA cloning vector (CLONTECH, Palo
Alto, CA). The insert cDNA sequence, determined as above, showed
overlap with the original clone, being 300-bp longer at the 5
end.
RACE and original clones were ligated using the internal
HindIII (408 bp) site to give a full-length clone.
Preparation of Vectors, Recombinant Proteins, and Polyclonal
Antibody--
The dymple and murine Mx1 full-length inserts were
amplified as SalI-NotI fragments having a
SalI site before the initial codon and a NotI
site after the stop codon and subcloned into the mammalian transfection
vector pME-HA (33) or the bacterial expression vector pGEX 5X-3
(Pharmacia Biotech Inc.). Induction and batch purification of
recombinant dymple and murine Mx1 as GST fusion proteins were performed
as detailed in manufacturer protocols. The amino-terminal deletion
mutant N246 was created by the deletion of the HincII
fragment (1-245 amino acids) of the dymple insert in
pBlueScriptIISK(
) and subcloned as a
XhoI-NotI fragment into the
SalI-NotI site of the pME-HA vector. A
site-directed point mutation was introduced into dymple cDNA at
serine 39 to give isoleucine, and the resultant clone S39I was
subcloned into the pME-HA vector. For polyclonal antibody preparation,
the coding sequence of 570 amino acids to the carboxyl terminus (736 amino acids) of dymple were amplified as a
BamHI-PstI fragment and subcloned into the pQE31
vector (QIAGEN, Chatsworth, CA). His-tagged protein was purified in the
native condition following manufacturer protocols and used to immunize
a Japanese White rabbit.
GTPase Assay--
The assays of GTPase activity were performed
at 37 °C for 1 h in 25 µl of reaction mixture containing 50 mM Tris-HCl (pH 8.0), 5 mM MgCl2,
0.1 mM dithiothreitol, 130 µM cold GTP, 13 nM [
-32P]GTP (1 µCi, 3,000 Ci/mmol)
(Amersham, Buckinghamshire, UK), and 0.1 µg of purified GST fusion
proteins. Aliquots (1 µl each) of the reaction products were resolved
by chromatography on polyethyleneimine cellulose plates in 1.6 M LiCl (34).
Cell Culture and Transfection--
COS7 cell lines were cultured
at 37 °C in Dulbecco's modified Eagle's medium (Nissui, Tokyo,
Japan) supplemented with 10% fetal calf serum (Life Technologies,
Inc.) and transfected using the DEAE-dextran method as described
previously (33). Plasmid DNA for transfection was prepared by the
cesium chloride ultracentrifugation method. Cells were grown to
40-60% confluence, washed once with PBS, and incubated in the
transfection medium (80 µl of 20 mg/ml DEAE-dextran + 20 µg of
plasmid DNA/4 ml of serum-free medium) for 4 h. Cells were then
incubated in 10% Me2SO/PBS for 1 min, washed with PBS, and
re-fed in complete medium until the assay.
Western Blotting--
Approximately 48 h after
transfection, COS7 cells were washed three times with ice-cold PBS,
collected, and lysed in radioimmune precipitation buffer (10 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Nonidet
P-40, 0.1% sodium deoxycolate, 1 µM EDTA, 0.1 µM dithiothreitol, 5 µg/ml trypsin inhibitor, 5 µg/ml
leupeptin) at 4 °C. 10 µl aliquots of 1-ml-lysed samples were
resolved on SDS-polyacrylamide (10%) gels followed by transfer to
nitrocellulose membranes. For visualization, blots were blocked at room
temperature for 1 h with 5% skim milk, PBS, briefly rinsed with
Tween 20/TBS, and then incubated at 4 °C overnight with anti-HA mAb
at a dilution of 1/1,000 or with human serum 114 at a dilution of 1/200
in 1% bovine serum albumin, PBS and then washed four times each for 15 min with Tween 20/TBS. Bound antibodies were detected using
AP-conjugated goat anti-mouse IgG or anti-human IgG.
Immunolocalization--
For immunofluorescence, anti-dymple pAb
and anti-
-adaptin mAb were diluted with 1% bovine serum albumin,
PBS to 1/2,000 and 1/100, respectively. Fluorescent second antibodies
were used at 1/100 dilution. COS7 cells were plated out on coverslips,
transfected, and cultured for 36 h. They were then washed twice
with PBS, fixed with ice-cold methanol for 4 min at 4 °C, and washed
twice with PBS. The coverslips were then incubated at room temperature
for 1 h with primary antibody, washed twice with PBS for 5 min,
incubated at room temperature for 30 min with fluorescent (Texas red or fluorescein isothiocyanate)-conjugated second antibody, and washed twice again with PBS for 5 min. The coverslips were then dried, mounted
onto glass slides, and viewed by MRC-1024 confocal microscopy (Bio-RAD).
Northern Blot Analysis--
A 607-bp fragment (1564-2171 bp) of
dymple was amplified by PCR and labeled with
[
-32P]dCTP using a random priming kit (Boehringer
Mannheim). Labeled probe was hybridized to multiple tissue Northern
blot in ExpressHyb hybridization solution
(CLONTECH). As a control, the same multiple tissue
Northern blot was rehybridized with the 2.0-kb
-actin cDNA
control probe supplied with the kit. The blot was exposed to an imaging
plate overnight and analyzed with a BAS-2000 Image Analyzer (Fujix,
Tokyo, Japan).
Reverse-transcriptase (RT)-PCR--
Total RNAs were isolated
from ICR mouse brain, skeletal muscle, and testis using Isogen (Nippon
Gene, Tokyo, Japan). RT reactions were performed on 1 µg of total RNA
in 12 µl of 1 × PCR buffer (Life Technologies, Inc.) containing
3 mM MgCl2, 10 µM dithiothreitol, 0.4 mM dNTPs, 250 ng of oligo(dT) primer (Promega), 0.5 µl of RNase inhibitor (Toyobo, Tokyo, Japan), and Superscript reverse transcriptase (Life Technologies, Inc.). RT reactions were conducted at
70 °C for 5 min, 42 °C for 40 min, and 70 °C for 15 min. To terminate the reaction, 0.5 µl of RNase H (Toyobo) was added to the
reaction mixture, followed by incubation for 15 min at 55 °C. The
primers used were 5
-TTAGTGGCAATTGAACTGGC-3
and
5
-GGAGGCCAATTAGCTTGAAG-3
. A 1-µl aliquot of RT reaction solution
was used for the amplification of dymple cDNA by PCR cycling in a
40-µl reaction solution containing 1 × EX-Taq buffer
(Takara; Tokyo, Japan), 10 pmol of each primer, 0.2 mM
dNTPs, and 0.3 µl of EX-Taq polymerase (Takara). The PCR involved denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, and extension at 72 °C for 2 min for a total of 38 cycles. Amplified products were analyzed by agarose gel electrophoresis and
ethidium bromide staining. The cDNA fragments were cloned into the
pGEM-T TA cloning vector (Promega, Madison, WI).
In Situ Hybridization--
A 607-bp fragment was amplified by
RT-PCR from mouse brain as described above and subcloned into the
pGEM-T TA cloning vector (Promega). A digitonin-labeled antisense
mRNA probe was produced by in vitro transcription with
SP6 RNA polymerase using the NcoI-linearized plasmid as the
template, and a sense mRNA probe was made with SpeI
digest and T7 RNA polymerase using the same plasmid. The preparation of
samples and hybridization were performed as described previously
(35).
 |
RESULTS |
Isolation and Characterization of a Novel Dynamin-like Gene,
Dymple--
More than 300,000 plaques of UNI-ZAP HeLa cDNA
expression library was screened with serum 114 taken from a scleroderma
patient to obtain 20 positive clones. Partial sequence analysis showed seven of these to be overlapping (03, 04, 14, 31, 33, 51, and 61 in
Fig. 1A). These clones had
significant homology with dynamin family GTPases in the 5
sequence. To
obtain an extended 5
sequence, 5
RACE was carried out. The obtained
699-bp-length fragment (D1) had a 308-bp overlap with clone 04 and
extended 391 bp beyond its 5
end. D1 and 04 were ligated using an
internal HindIII site, resulting in a full-length clone
(D104, 2524 bp) with an open reading frame of 2208 bp (736 amino
acids). The initiation codon was found 84 bp downstream of the in frame
stop codon. A putative polyadenylation signal (AATAAA) was found at bp
2502 of this clone.

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Fig. 1.
Primary structures of dymple. A,
diagrammatic representation of the structures of the dymple mRNA
(top) and the immunoscreened cDNA clones
(below). The open box indicates the coding
sequence, and the stippled boxes show the in-frame deletions
found in clones 14 and 31. The consensus GTP binding motifs (I, II, III) adottre enclosed by
vertical lines. The corresponding region used as the probe
for the Northern blot analysis and the in situ hybridization experiment are indicated below the mRNA (A)n represents a
putative polyadenylation signal. B, comparison of the amino acid sequences of human dymple, dynamins I and II, and yeast DNM1P and
VPS1P. Dashes represent gaps inserted to facilitate
alignment. Identical residues to those in dymple are indicated by
reverse-contrasted letters. The locations of consensus GTP
binding motifs (I, II, III) are indicated by double
underlining. Alternative splicing sites in dymple between the (>)
and (<) and in dynamin I underlined with (^) are also
indicated. The PH domain of dynamin I is designated as (:) for
consensus and (.) for spacer regions. The membrane targeting motif in
dynamin I (25) is underlined with (#), and the binding site for SH3
domain (PXXPPXXPX) is indicated.
C, multiple alignment of the amino acid sequences between
the first and second consensus of the GTP binding domain. Region 2 of
the self-assembly motif found in the Mx family of proteins (7)
corresponds to the variable region in the dynamin superfamily, whereas
region 1 of the same motif is highly conserved. Note that each
subfamily has a typical sequence pattern and that dymple and the
C. elegans clone T12E12.4 have identical spacing sizes. The
indicated sequences are L07807 (human (h) dynamin I), L36983
(human dynamin II), L40588 (yeast DNM1), M33315 (yeast VPS1), M12279
(mouse (m3) Mx1), M30817 (human MxA), M30818 (human MxB),
U61944 (C. elegans cosmid T12E12).
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A homology search in the GenbankTM data base revealed that
the deduced amino acid sequence of the clone shares high homology with
dynamin family GTPases, especially yeast DNM1P and VPS1P (Fig.
1B). The sequence similarity was pronounced in the
amino-terminal half around the three conserved GTP binding domains
(indicated as I, II, and III in Fig. 1B) but less so in the
carboxyl-terminal remainder. The most remarkable difference from known
dynamin isoforms was found to be the lack of proline-rich
carboxyl-terminal domain, so we named this clone dymple for dynamin
family member proline-rich carboxyl-terminal domain less. This
carboxyl-terminal region is known to regulate dynamin GTPase activity
(22, 25, 36) and is also absent in all other dynamin-related
proteins.
Other marked discrepancies in the sequences between dymple and known
dynamin-related proteins were found in two regions, downstream of the
first consensus GTP binding domain and in the region corresponding to
the PH domain (37). The former involves an insert of unique sequences
of about 44 amino acids and 16 amino acids in the yeast DNM1P/VPS1P and
dymple, respectively (Fig. 1C). This corresponds to region 2 of the self-assembly motif found in Mx family proteins (Fig. 7 in Ref.
7), and region 1 of the same motif is perfectly identical among
mammalian Mx proteins and is also highly conserved among the dynamin
superfamily members. The divergence in the PH domain was even greater,
dymple most resembling yeast DNM1P, but actually no sequence similarity
was evident with the primary homology searches.
To examine tissue-specific expression of dymple mRNA, human
multiple tissue Northern blots were hybridized with the carboxyl terminus of clone 04 (Fig. 1A) as a specific probe. As shown
in Fig. 2 (upper panel),
dymple demonstrated two major transcript sizes of 2.5 and 4.2 kb.
Ubiquitous expression of both of them at low level was found with high
level, the smaller one in brain, skeletal muscle, and testis. The
larger form also proved to be abundant in the testis. Because the
middle size of the 3
sequence in multiple overlapping cDNA clones
(clone 14 and 51 in Fig. 1A) appeared to arise from
mis-annealing of the oligo-(dT) primer during the library synthesis,
the two transcript sizes correspond well to the alternative
polyadenylation found in the cDNA clones described above. The
111-bp in-frame deletion in the coding sequence (described below) could
not be analyzed in this experiment.

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Fig. 2.
Expression patterns of the dymple mRNA in
human tissues. Two major transcripts sizes of 2.5 and 4.2 kb were
apparent in all tissues analyzed. Abundant expression was observed for the smaller transcript in brain, skeletal muscle, and testis and for
the larger one in the testis (upper box). Blots were
stripped and reprobed with a -actin control probe (lower
box). Size markers are indicated on the left in
kb.
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HA-tagged dymple and mouse Mx1 were transiently expressed in COS7
cells, and the expressed proteins were detected with anti-HA mAb and
the human serum 114 on nitrocellulose membranes. Both gave bands at 80 kDa, corresponding to their deduced amino acid sequences (Fig.
3). Only dymple was visualized by the
human serum 114, suggesting that this recognizes a nonconserved region
of dynamin-related proteins.

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Fig. 3.
Immunoblotting analysis of dymple protein
overexpressed in COS7 cells. Total cell lysates from COS7 cells,
the control vector, mock-transfected (left), transfected
with dymple (middle), and murine Mx1 (right)
expression vectors were resolved on a 10% polyacrylamide gel. Blots
were incubated with anti-HA (left panel) or autoimmune serum
114 (right panel) and detected with alkaline phosphatase-conjugated secondary antibody.
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Because high molecular weight GTPases are known to have relatively high
basal GTP hydrolysis activity (5, 34, 38), bacterially expressed GST
fusion dymple was purified, and its GTPase activity was examined. Like
the murine Mx1 protein, dymple could hydrolyze GTP without additive
modifications or coactivators (Fig.
4).

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Fig. 4.
GTPase activity of recombinant dymple.
Bacterially expressed and purified GST (lane 2), GST-dymple
(lane 3), and GST-Mx1 (lane 4) proteins (0.1 µg) were added to 25 µl of assay mixture and incubated at 37 °C
for 1 h. The sample in lane 1 does not contain any
protein. Aliquots (1 µl) of the reaction products were resolved on
polyethyleneimine cellulose plates in 1.6 M LiCl for 2 h at room temperature.
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Intracellular Localization of Dymple--
The anti-dymple pAb
raised against a carboxyl-terminal 166-amino acid peptide of human
dymple allowed investigation of the distribution of wild-type and
mutant forms by confocal beam scanning laser fluorescence microscopy
(Fig. 5). The wild type, a form with a
point mutation in the first consensus GTP binding domain and an
amino-terminal deletion mutant lacking the entire GTP binding domain
were transiently expressed in COS7 cells and detected with the
anti-dymple pAb. To address the question of whether the TGN counterpart
of dynamin is dymple, cells were double-labeled with
-adaptin, a
component of the AP1 complex (39).

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Fig. 5.
Immunofluorescence localization of wild-type
and mutant forms of dymple. COS7 cells were transfected with wild
type (A, D, and G), a Ser39 to Ile
point mutant (B, E, and H), and an amino-terminal
deletion mutant (N246, C, F, and I) of dymple and
cultured for 36 h. Cells were labeled with anti-Dymple pAb
followed by Texas red-conjugated secondary antibody (red in
panels A, B, and C) and with anti -adaptin mAb
followed by fluorescein isothiocyanate-conjugated secondary antibody
(green in panels D, E, and F).
Overlapping images are shown in panels G, H and
I. In the wild -type cells, cytoplasmic and perinuclear
staining was observed similar to that for dynamin. In S39I cells,
the perinuclear signals were reduced. Three different expression levels
in N246 cells are shown. Note the large aggregates of dymple with
quite little -adaptin overlapping.
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Overexpressed wild-type dymple localized to the perinuclear zone with a
punctate cytoplasmic pattern, but neither interference nor obvious
overlapping were observed with
-adaptin, as seen in a similar
experiment with dynamin (30). A serine 39 to isoleucine mutation in the
first consensus GTP binding domain (S39I) resulted in decreased signal
in the perinuclear region with no alteration in the localization of
-adaptin. The amino-terminal deletion mutant (N246) lacking the
entire GTP binding domain, however, made large aggregates surrounded by
-adaptin-labeling with quite little overlapping, although weak
uniform cytoplasmic staining also remained. An analogous dynamin mutant
(N272 construct) also forms aggregates but throughout the cytoplasm
(30). Brefeldin A treatment altered the localization of
-adaptin as
shown previously (39), but no changes in the staining pattern for the
overexpressed wild-type and dymple mutants were detected when used in
the present study (data not shown). Endogenous dymple protein in COS7
cells can be recognized with this pAb in Western blots (data not shown) but was not visualized in immunofluorescence at the applied
dilution.
Tissue-specific Splicing and Distribution of Dymple--
We found
two types of "in-frame deletions" within multiple overlapping
cDNA clones. As indicated in Fig. 1B, clones 14 and 31 had 87- and 111-bp deletions, resulting in 29- and 37-amino acid
deletions, respectively. This was also proved by RT-PCR analysis of the
carboxyl terminus of dymple (1564-2171 bp) using mRNA prepared from murine brain, testis, and skeletal muscle (Fig.
6A). Sequence analysis of the
cDNAs recovered from the major product bands (a 607-bp band for
brain, a 529-bp band for testis, and a 496-bp band for skeletal muscle)
revealed their deduced amino acid sequences to be almost identical to
the corresponding parts of clones 04, 14, and 31, respectively, with
exactly the same splicing patterns (summarized in Fig. 6B
and C). These results indicate that these variants are
caused by natural 3
alternative splicing and are not a cDNA
synthesis artifact, strongly suggesting the existence of
tissue-specific alternative splicing. Dynamin isoforms also have
alternative exon use of the middle region that is identical in both
dynamin I and II, whereas these two have alternative termination and an
in-frame deletion of 4 amino acids, respectively (21, 40), although the
sites and patterns of alternative splicing are different between dymple
and dynamin isoforms.

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Fig. 6.
Alternative splicing of dymple and its tissue
specificity. A, RT-PCR analysis of mRNA from murine
brain, skeletal muscle, and testis. Lane M is the 1-kb DNA
ladder size marker. Lanes 1-3 are PCR products amplified
from brain, testis, and skeletal muscle, respectively. B,
schematic summary of the alternative splicing of dymple mRNA. The
positions of the PCR primers for RT-PCR are indicated by
half-arrows. Compared with the brain-form of dymple mRNA
(or human cDNA clone 04), testis (clone 14) and skeletal muscle
(clone 31) mRNAs lack 1705-1815 bp (532-568 amino acids) and
1705-1782 bp (532-557 amino acids) sequences, respectively. C, the deduced amino acid sequences of human (h)
and mouse (m) dymple. Nonidentical residues are marked with
asterisks. The alternatively spliced regions are indicated
as in Fig. 1B. Conceivable proline-rich sequence motifs are
found both within the alternatively skipped region and in its
downstream.
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Cell- and site-specific expression of dymple was examined using murine
brain sections with the murine brain-form fragment of dymple as the
probe. Abundant expression of dymple mRNA was noted in the
cerebellum and in the several regions of the cerebrum and diencephalon
(Fig. 7A). Prominent signals
were observed in the cerebellar Purkinje cells (Fig. 7B) and
in the pontile giant neurons (Fig. 7C).

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Fig. 7.
In situ hybridization analysis of
dymple in the mouse brain. A, sagittal section of a brain
hybridized with the antisense probe, showing signals in several areas.
B and C, cerebellum and pons sections at higher
magnification, respectively. The color development after nitro blue
tetrazolium/5-bromo-4-chloro-3-indolyl phosphate incubation is shown as
a negative image for better contrast. The brain-specific form of murine
dymple shown in Fig. 6B was used as the template for the
riboprobe. The same section of the brain hybridized with the sense
probe showed no signals (data not shown). Pkj, Purkinje
cells; IGR, inner granule cell layer. Scale bars:
A, 2 mm; B and C, 10 mm.
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DISCUSSION |
In the present study, we isolated a novel GTPase clone, dymple,
from a HeLa cell cDNA library by immunoscreening with serum from an
autoimmune patient. The epitope recognized by the antiserum was
concluded to be located in the carboxyl terminus (460-736 amino
acids), since all immunopositive clones overlapped in this region (Fig.
1A). Its low level of sequence conservation as compared with
dynamin superfamily members (5) presumably contributed to the observed
specificity of the serum.
The dynamin superfamily is subdivided into groups, each named for a
prototypic member: dynamin, VPS1P, Mx, and Mgm1p (13). The primary
sequence structure of dymple indicated that it should be classified
into the DNM1P/VPS1P subgroup because of the much closer similarity
than with the Mx family proteins or the yeast Mgm1p. Furthermore, a
BLAST search of the full-length amino acid sequence of dymple scored
slightly higher against the yeast DNM1P and VPS1P than human dynamin I. Both dymple and DNM1P/VPS1P lack the proline-rich carboxyl-terminal
domain, which is the most obvious difference between dynamin isoforms
and other related proteins found so far.
Comparison of the sequence revealed two regions with least homology and
thus, most likely to be responsible for specific function. The
diversity of carboxyl-terminal halves especially around the region
corresponding to the PH domain of dynamin is very pronounced, as noted
elsewhere (5). This also applies to dymple, since in this respect it
resembles neither known members of the dynamin superfamily nor other
sequences in the GenbankTM data base. In the sequence after
the first consensus of the GTP binding domain (Fig. 1C),
which corresponds to domain 2 of the self-assembling motif in mouse Mx1
(Fig. 7 in Ref. 7), the yeast DNM1P/VPS1P possess larger spacer regions
than dynamin or Mx family proteins (13). Dymple also has a unique
sequence here, although its length differs from those of DNM1P/VPS1P or
dynamin. The functional significance of these differences still remains to be elucidated, but the identical spacing size among dymple and the
C. elegans clone T12E12.4 (Fig. 1C) suggests that
these variable lengths may characterize each of the dynamin superfamily subgroups.
Recent studies have revealed multiple regions that target dynamin to
both clathrin-coated and non-clathrin-coated plasma membrane elements
(25). The sequence 786PAVPPARPG794 in rat
dynamin is one expected binding site for SH3 domains necessary for
colocalization with clathrin on coated pits, and the sequence 733ALKEALSIIGDIN746 is involved in membrane
binding. The latter sequence motif corresponds to the extreme carboxyl
terminus of the dymple and DNM1P/VPS1P sequences. Up to 100 amino acids
upstream of this motif, but not the additional upstream sequence where
dynamin PH domain resides, is sufficient for membrane binding in the
case of dynamin and appears to be highly conserved among dynamin family
members including dymple (Fig. 1B), implying that dymple
might target membrane elements other than clathrin-coated plasma
membranes.
An immunofluorescence study revealed that dymple and dynamin might have
similar characteristics for cycling between the soluble and
membrane-bound states and common functional property among amino-terminal GTP binding and carboxyl-terminal putative domains for
protein interaction. Dynamin has been found to be abundant in the
cytosolic pool, with binding sites at the plasma membrane being
saturable; thus, mutant forms exert dominant interference (30). In our
experiment, wild-type dymple was also found to be distributed like
dynamin, conceivably for analogous reasons, but amino-terminal deletion
resulted in aggregation at different places for the two proteins:
inside areas of TGN with dymple and throughout the cytoplasm in the
dynamin case. The different accumulation patterns may reflect variation
in the membrane localization targeted by specific binding factors,
although there remains a possibility that the large aggregates were
merely caused by inclusion into some other structure. Both in
vitro and in vivo interactions between dynamin and AP2
complex were confirmed, and expression of mutant dynamin was shown to
alter the distribution of
-adaptin (30). In the case of dymple,
neither interference nor obvious overlapping with
-adaptin were
observed, so clathrin-coated TGN membranes can be dismissed as possible
targets.
The expression pattern of dymple is reminiscent of the tissue
specificity of dynamin isoforms in rat (40). Dynamin I is exclusively
expressed in neuronal tissues (17, 18), whereas dynamin II is reported
to be ubiquitously expressed (20, 21), and dynamin III is
testis-specific (41), each having different alternative splicing
patterns. This suggests that the similarities in tissue distribution
patterns may reflect a functional relationship between dymple and
dynamin isoforms. Although it is possible that we found a ubiquitous
type and that another tissue-specific isoform exists, the same dymple
gene is strongly expressed in both neurons and testis. On the other
hand, we demonstrated tissue-specific alternative splicing patterns
within the coding sequence of dymple, resulting in an in-frame deletion
(Fig. 6B). The physiological significance of this phenomenon
is unknown, but an attractive speculation is that such variation causes
differential specificity of interaction. Interestingly, there are
conceivable proline-rich sequence motifs around the deletion (Fig.
6C).
Since tubulation and vesiculation seem to be innate properties of the
intracellular membranes responsible for protein transport (42), it is
tempting to speculate that each of the transport pathways utilize
dynamin-related molecules. Previously, the potential association of a
dynamin-like protein with the Golgi apparatus in mammalian cells was
discussed (43). Immunofluorescence, subcellular fractionation, and
electron microscopy have demonstrated that pAbs raised against three
different peptides from the amino-terminal-conserved domain of dynamin
labeled the Golgi complex. These peptide sequences are generally
conserved in dymple, with LTLVDLPGMTKV sequences in the second GTP
binding motif being identical. The corresponding pAb MC12 was found to
recognize an additional faint 80-90-kDa protein in rat brain and
cultured human fibroblasts other than the 100-kDa major band of dynamin
(Fig. 2 in Ref. 43), which seems to be consistent with our findings. It
is likely that different anti-dynamin antibodies give apparent various
staining patterns in immunofluorescence studies, so our result with
overexpressed dymple (Fig. 5) would not be inconsistent with previous
data whether the Golgi-associated dynamin-like protein includes dymple
or not. Further electron microscopic analysis is needed to determine
the precise localization, especially whether the Golgi is involved.
Dynamin participates in the budding of plasma membrane-derived
clathrin-coated vesicles with the AP2 complex but not TGN-derived ones
associated with AP1 (31). Thus the most likely position where
dynamin-like molecules might exist is the clathrin-coated pit at the
TGN membrane. Dymple, however, cannot be a simple TGN counterpart of
dynamin for the reasons outlined above. This would likely require a
dynamin isoform with a proline-rich carboxyl-terminal domain, since the
latter appears necessary for the mechanism of AP2 complex recruitment
of dynamin to clathrin-coated plasma membranes (26). Furthermore, the
sequences are similar between
-adaptin and
-adaptin (39),
subunits of AP2 and AP1 complexes that seem to serve for the
specificity of adaptor complexes for the plasma membrane and TGN,
respectively. Dymple does not complement VPS1 mutation in yeast
cells,2 so it can also not be
a cognate homologue of yeast VPS1P. The available data thus
indicate that there are several as yet unidentified members
of the dynamin/VPS1P subfamily of high molecular weight GTPases that participate in each specific vesicle transport
pathway. To test if dymple is really involved in some pathway of
protein transport in a manner similar to dynamin, functional studies
are required. We found the C. elegans clone T12E12.4 from
the cosmid data base to be most similar to dymple. Mutational analysis
should allow insight into the functional properties of this
protein.
 |
ACKNOWLEDGEMENTS |
We thank Dr. K. Nagata (Tokyo Institute of
Technology) for the murine Mx1 plasmid. We are also grateful to Dr. K. Nakayama (Tsukuba University) for sharing information before
publication. We acknowledge E. Conibear, T. H. Stevens (Oregon
University), and M. S. Robinson (Cambridge University) for helpful
discussion and critical reading of the manuscript.
 |
FOOTNOTES |
*
This work was supported by grants from the Ministry of
Education, Science, and Culture and the Ministry of Health and Welfare of Japan.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: Endocrinology
Dept., Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113, Japan. Tel.: +81-3-5803-5836; Fax.: +81-3-5803-0248; E-mail: m.hagiwara.end{at}mri.tmd.ac.jp.
1
The abbreviations used are: TGN, trans-Golgi
network; mAb and pAb, monoclonal and polyclonal antibodies,
respectively; AP, alkaline phosphatase; GST, glutathione
S-transferase; HA, hemagglutinin; RACE, rapid amplification
of cDNA ends; PBS, phosphate-buffered saline; RT, reverse
transcription; PCR, polymerase chain reaction; bp, base pair(s); kb,
kilobase pairs; PK domain, pleckstrin homology.
2
E. Conibear and T. H. Stevens, personal
communication.
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