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J. Biol. Chem., Vol. 277, Issue 14, 12432-12436, April 5, 2002
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§,, and¶
From the Laboratory for Developmental Neurobiology,
Brain Science Institute, RIKEN (The Institute of Physical and Chemical
Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan and the
¶ Division of Molecular Neurobiology, Department of Basic Medical
Science, The Institute of Medical Science, The University of Tokyo,
4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received for publication, January 3, 2002, and in revised form, February 5, 2002
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
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Myosin Va is a member of the
unconventional class V myosin family, and a mutation in the
myosin Va gene causes pigment granule transport defects in
human Griscelli syndrome and dilute mice. How myosin Va
recognizes its cargo (i.e. melanosomes), however, has
re- mained undetermined over the past decade. In this
study, we discovered Slac2-a/melanophilin to be the "missing
link" between myosin Va and GTP-Rab27A present in the melanosome.
Deletion analysis and site-directed mutagenesis showed that the
N-terminal Slp (synaptotagmin-like protein) homology domain
of Slac2-a specifically binds Rab27A/B isoforms and that the
C-terminal half directly binds the globular tail of myosin Va. The
tripartite protein complex (Rab27A· Slac2-a·myosin Va) in
melanoma cells was further confirmed by immunoprecipitation. The
discovery that myosin Va indirectly recognizes its cargo through Slac2-a, a novel Rab27A/B effector, should shed light on molecular recognition of its specific cargo by class V myosin.
The synaptotagmin-like protein
(Slp)1 family is classified
as a subfamily of C-terminal-type tandem C2 proteins (1-7) and was originally defined as an N-terminal Slp homology domain (SHD) (5)
and C-terminal tandem C2 domains, putative Ca2+ binding
motifs (known as the C2A domain and C2B domain) (8). To date, four
members of the Slp family (Slp1/Jfc1, Slp2-a, Slp3-a, and
Slp4/granuphilin-a) have been described in the mouse and human (4, 5,
9, 10), and several alternatively splicing isoforms have been
identified in Slp2, Slp3, and Slp4 (5, 10). An additional
slp gene (slp5) has been found on chromosome X
(Xp21.1) in the human genome (5).
The Slp family contains two conserved domains at the N terminus,
referred to as SHD1 and SHD2 (5). The SHD1 and SHD2 of Slp3-a and Slp4
are separated by a sequence containing two zinc-finger motifs, whereas
Slp1 and Slp2-a lack such zinc-finger motifs, and their SHD1 and SHD2
are linked together (5). The SHD has also been found in other proteins,
including Slac2-a (Slp homologue lacking
C2 domains-a) and Slac2-b/KIAA0624 (11),
suggesting a general role of the SHD in cellular signaling.
Two very recent important discoveries have been made concerning the
functional relationship between the SHD and Rab27A, one of the small
GTP-binding proteins believed to be essential for membrane trafficking
in eukaryotic cells (12). The first was our discovery that the SHD of
the Slp family and Slac2-a directly interact with the GTP-bound form of
Rab27A both in vitro and in intact cells (13). Since
mutations in the rab27A gene cause hemophagocytic syndrome
(Griscelli syndrome), an uncontrolled T lymphocyte and macrophage
activation syndrome in humans (14, 15), and defects in granule
exocytosis in cytotoxic T lymphocytes and melanosome transport in
ashen mice (16-19), we hypothesized that the Slp family and
Slac2 are involved in such membrane trafficking (13). The second very
recent important discovery is the identification of Slac2-a as
melanophilin and that a mutation in the mlph gene causes
defects in melanosome transport in leaden mice (20). Interestingly ashen mice, which carry a mutation in the
rab27A gene, and dilute mice, which carry a
mutation in the myosin Va gene, which encodes one of the
actin-based motor proteins (21, 22), have shown similar defects in
pigment granule transport (i.e. clumping of melanosomes in
the perinuclear region), and as a result ashen,
leaden, and dilute mice all exhibit a similarly lighter coat color (14, 16, 19, 23-25). In addition, genetic analysis
has shown that these three proteins function in the same or overlapping
transport pathways (20), but the functional relationships between these
three molecules, Rab27A, Slac2-a/melanophilin, and myosin Va, in
melanosome transport remained to be clarified (25).
In this study, we report on two domain structures of Slac2-a, the
N-terminal SHD, which specifically interacts with the Rab27A and Rab27B
isoforms, and the C-terminal domain, which directly interacts with the
globular tail of myosin Va, and we discuss the role of the tripartite
protein complex in melanosome transport based on our findings.
Molecular Cloning of Mouse Rab27B, Rab34, and Myosin Va
cDNAs--
cDNA encoding a full open reading frame of mouse
Rab27B, Rab34, and myosin Va was amplified from Marathon-Ready adult
brain cDNA (CLONTECH) by reverse transcriptase
PCR as described previously (26). The purified PCR products were
directly inserted into the pGEM-T Easy vector (Promega, Madison, WI).
Both strands were completely sequenced using a Hitachi SQ-5500 DNA
sequencer. We identified one deletion as a result of alternative
splicing (deletion of amino acids 1387-1411) and several amino acid
differences (R695A, D904E, and V905L) compared with the reported myosin
Va sequences (24), and they were unlikely to be PCR-induced errors
because we found the same differences in at least two independent
clones. Addition of the FLAG tag (or T7 tag) to the N terminus of Rab34 (pEF-FLAG-Rab34) or to the N terminus of myosin Va (pEF-T7- or -FLAG-myosin Va) and construction of the expression vector were performed as described previously (26-29). pEF-T7-Slac2-a,
-FLAG-Slac2-a, and other -FLAG-Rabs were prepared as described
previously (13).
Construction of Deletion Mutants of Slac2-a and Myosin Va
and Site-directed Mutagenesis--
Deletion mutants of Slac2-a
(pEF-T7-Slac2-a-SHD and -
pEF-T7-Slac2-a-(A4) carrying a SLEWY-to-ALEAA substitution (Fig.
1A) was constructed by two-step PCR techniques as described previously using the following mutagenic oligonucleotides (31): 5'-GGCGGCCGCCAGAGCACCGATCTTCAC-3' (A4 primer 1; antisense)
and 5'-GCGGCCGCCTACCAGCACGTGAGGGCT-3' (A4 primer 2; sense).
Direct Interaction between Slac2-a and Myosin
Va--
pEF-T7-GST-Slac2-a-
The purified FLAG-myosin Va-GT protein and Rab27A were incubated with
glutathione-Sepharose beads (wet volume, 20 µl) either coupled with
T7-GST-Slac2-a or T7-GST alone in 50 mM HEPES-KOH, pH 7.2, 100 mM NaCl, 1 mM MgCl2, and 0.2%
Triton X-100 for 1 h at 4 °C. After washing three times with 1 ml of the binding buffer without recombinant proteins, proteins trapped
with the beads were analyzed by 10% SDS-PAGE followed by
immunoblotting with horseradish (HRP)-conjugated anti-FLAG tag antibody
(Sigma) and anti-Rab27 mouse monoclonal antibody (Transduction
Laboratories, Lexington, KY) as described previously (26, 30).
Antibody Production and Immunoprecipitation--
cDNA
encoding the C terminus of Slac2-a (amino acids 401-590) was amplified
by conventional PCR and subcloned into the pGEX-4T-3 vector (named
pGEX-4T-3-Slac2-a
Melanoma cell line B16-F1 derived from the wild-type mouse was
cultured as described previously (13). Cells (10-cm dish) were
homogenized in a buffer containing 1 ml of 50 mM HEPES-KOH, pH 7.2, 150 mM NaCl, and protease inhibitors, and proteins
were solubilized with 1% Triton X-100 at 4 °C for 1 h. After
removal of insoluble material by centrifugation, the supernatant was
incubated with either anti-Slac2-a IgG (10 µg/ml) or normal rabbit
IgG for 1 h at 4 °C followed by incubation with protein
A-Sepharose beads (Amersham Biosciences) for 1 h at 4 °C. After
washing the beads five times with 50 mM HEPES-KOH, pH 7.2, 150 mM NaCl, 0.2% Triton X-100, and protease inhibitors,
the immunoprecipitates were subjected to 10 or 7.5% SDS-PAGE followed
by immunoblotting with anti-Slac2-a (1:250 dilution), anti-myosin Va
(1:100 dilution), or anti-Rab27 monoclonal antibody (1:250 dilution) as
described previously (26, 33).
Miscellaneous Procedures--
Cotransfection of pEF-T7-Slac2-a,
pEF-FLAG-Rab27A/B, and/or pEF-T7-myosin Va into COS-7 cells (5 × 105 cells (the day before transfection)/10-cm dish) was
performed as described previously (30). Proteins were solubilized with a buffer containing 1% Triton X-100, 250 mM NaCl, 1 mM MgCl2, 50 mM HEPES-KOH, pH 7.2, and protease inhibitors at 4 °C for 1 h. T7-Slac2-a and
FLAG-Rab27A/B were immunoprecipitated with anti-T7 tag
antibody-conjugated agarose (Novagen, Madison, WI) and anti-FLAG M2
antibody-conjugated agarose (Sigma), respectively, as described previously (26, 29, 35). SDS-PAGE and immunoblotting analyses with
HRP-conjugated anti-FLAG and anti-T7 tag antibodies (Novagen) were also
performed as described previously (26, 30). The blots and gels shown in
this paper are representative of at least two or three independent experiments.
The SHD of Slac2-a Specifically Interacts with Rab27A/B
Isoforms--
We recently discovered that the SHD of Slac2-a/b
directly binds the GTP-bound form of Rab27A in vitro and in
intact cells (13). However, it remained undetermined whether the
full-length Slac2-a protein specifically recognizes the Rab27A molecule
and whether Rab27A interacts with the SHD alone or also with the large Slac2-a C-terminal domain with unknown function. To address these issues, we first investigated the specific interaction of full-length Slac2-a with various Rab proteins (Rab1, Rab2, Rab3A, Rab4A, Rab5A, Rab6A, Rab7, Rab8, Rab9, Rab10, Rab11A, Rab17, Rab18, Rab20, Rab22, Rab23, Rab25, Rab27A, Rab27B, Rab28, Rab34, or Rab37) in intact cells
by co-expression assay (30, 35). In brief, T7-tagged Slac2-a and each
of the FLAG-tagged Rabs were co-expressed in COS-7 cells, and
T7-Slac2-a protein was immunoprecipitated with the anti-T7
antibody-conjugated agarose (26, 30). As expected, T7-Slac2-a protein
specifically co-immunoprecipitated with the FLAG-Rab 27A and -Rab27B
isoforms but not with other Rabs (Fig. 1,
B, lane 18, and
D, lane 1). The faint signal observed in Fig. 1B, lane 20, was probably attributable to
nonspecific interaction of Rab34 with the agarose beads.
Next we investigated the possible involvement of the C terminus of
Slac2-a in Rab27A/B binding in the same co-transfection assay. The SHD
domain alone efficiently co-immunoprecipitated with both the Rab27A and
Rab27B isoforms (Fig. 1, C and D, lane 2), but the C-terminal domain lacking the SHD ( Two Domain Structures of Slac2-a: the N-terminal SHD Responsible
for Rab27A/B Binding and the C-terminal Domain Responsible for Myosin
Va Binding--
The results of a genetic analysis comparing
dilute, ashen, and leaden mice have
indicated that myosin Va, Rab27A, and Slac2-a function in the same or
overlapping transport pathways in melanosome transport (20). Consistent
with this, myosin Va in extracts from melanocytes has been shown to
co-immunoprecipitate with Rab27A (19). However, when FLAG-Rab27A and
T7-myosin Va were co-expressed in COS-7 cells, no Rab27A-myosin Va
complex was detected in the cell lysates (Fig.
2A, lane 8),
indicating that an additional protein must link Rab27A and myosin Va.
Since the SHD of Slac2-a specifically binds Rab27A (Fig. 1), we
hypothesized that Slac2-a is the missing link between Rab27A and myosin
Va in melanosome transport. To test this hypothesis, three proteins
(FLAG-Rab27A, T7-Slac2-a, and T7-myosin Va) were co-expressed in COS-7
cells, and their associations were analyzed by immunoprecipitation as described above (26, 35). As expected, in the presence of full-length
T7-Slac2-a, T7-myosin Va co-immunoprecipitated with FLAG-Rab27A
(Fig. 2A, lane 5), whereas in the absence of
T7-Slac2-a, T7-myosin Va was undetectable in the anti-FLAG antibody
immunoprecipitants (Fig. 2A, lane 8).
Interestingly the SHD alone (Slac2-a-SHD) or the C-terminal half alone
(Slac2-a-
We next sought to identify the myosin Va-binding site in Slac2-a by
dual tag (T7 and FLAG) co-expression assay. When T7-Slac2-a deletion
mutants and FLAG-myosin Va were co-expressed in COS-7 cells,
T7-Slac2-a- Direct Interaction between the Globular Tail of Myosin Va and the
C-terminal Domain of Slac2-a--
Since the globular tail of myosin Va
was thought to be essential for cargo recognition (21) (Fig.
3A), we investigated the interaction between Slac2-a and the myosin Va globular tail by using
the same dual tag (T7 and FLAG) co-expression assay, and as shown in
Fig. 3B, Slac2-a- The Tripartite Protein Complex (Rab27A·Slac2-a·Myosin Va) in
Melanoma Cells--
Lastly, immunoprecipitation analysis was performed
to investigate whether the tripartite protein complex
(Rab27A·Slac2-a·myosin Va) is formed at physiological conditions.
As shown in Fig. 3F, both myosin Va and Rab27A were
co-immunoprecipitated with anti-Slac2-a-specific antibody (Fig.
3E), but not control IgG, from melanoma cell lysates. Thus,
the tripartite protein complex (Rab27A·Slac2-a·myosin Va) demonstrated by in vitro binding experiments should be
physiologically relevant.
Conclusions--
The results of a recent biochemical
analysis have suggested that the tail domain of myosin Va (or Vb)
recognizes its cargo by directly binding to the proteins present in the
cargo (e.g. Rab11, Rab25, and synaptobrevin·synaptophysin
complex) (37-39). However, since myosin Va did not directly recognize
Rab27A in the melanosomes, an additional linker protein was proposed to assist melanosome recognition in melanosome transport (25). In the
present study, we discovered that Slac2-a is a missing link between
Rab27A and myosin Va in melanosome transport and demonstrated how
myosin Va recognizes its specific cargo (i.e. melanosomes)
by its globular tail domain. The possible role of the tripartite
protein complex (Slac2-a, Rab27A, and myosin Va) in melanosome capture
in actin-rich cell periphery is summarized in Fig.
4. The SHD of Slac2-a specifically binds
the GTP-Rab27A in the melanosomes, and the C terminus of Slac2-a binds
the globular tail of myosin Va, which binds actin filament via the head
domain. Since Slac2-a is expressed in various tissues, including the
brain (20), the Slac2-a·Rab27·myosin Va complex may be involved in other types of membrane trafficking. For instance,
Slac2-a·Rab27B·myosin Va may be involved in endoplasmic reticulum
transport to dendrites in neurons because the inositol
1,4,5-trisphosphate receptor on the endoplasmic reticulum does not
migrate to the postsynaptic spines in dilute mice (40, 41).
Further work is necessary to define the universality and/or
specialty of the tripartite protein complex, Slac2-a, Rab27A/B,
and myosin Va, in membrane trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
146) and of myosin Va (pEF-FLAG-myosin
Va-GT (globular tail)) were essentially constructed by conventional PCR
as described previously (28, 30) using the following oligonucleotides
with restriction enzyme sites (underlined) or stop codons (in bold):
5'-CGGATCCGGTGGAGGTGGATCTGAGCC-3' (Slac2-a-146 primer;
sense),
5'-GCGAATTCATGGCTCAGATCCACCTCCAC-3' (Slac2-a-SHD 3'-primer), and
5'-CGGATCCGAAAAGCAGGATAAAACTGT-3' (myosin Va-1417
primer; sense).
146, -T7-GST-Slac2-a, -T7-Rab27A,
-T7-GST-FLAG-myosin Va-GT, and pEF-T7-GST vectors were constructed by PCR essentially as described
elsewhere.2 COS-7 cells
(5 × 105 cells (the day before transfection)/10-cm
dish) were transfected with 4 µg of plasmids as described previously
(30). Three days after transfection, cells were harvested and
homogenized in 1 ml of a buffer containing 50 mM HEPES-KOH,
pH 7.2, 250 mM NaCl, 0.1 mM
phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A in a glass-Teflon Potter homogenizer with
10 strokes at 900-1000 rpm. After solubilization with 1% Triton
X-100, insoluble material was removed by centrifugation at 15,000 rpm
for 10 min. Expressed GST fusion proteins were affinity-purified on
glutathione-Sepharose beads (wet volume, 20 µl; Amersham Biosciences)
according to the manufacturer's recommendations. After extensively
washing the beads with 10 mM HEPES-KOH, pH 7.2, 100 mM NaCl, and 0.2% Triton X-100, thrombin (1 unit, Sigma)
digestion was performed on the same column at 25 °C for 1 h.
The eluate containing FLAG-myosin Va-GT protein (or Rab27A) was then
incubated with benzamidine-Sepharose 6B (wet volume, 20 µl; Amersham
Biosciences) to remove the thrombin. Protein concentrations were
estimated by 10% SDS-PAGE or determined with a Bio-Rad protein assay
kit using bovine serum albumin as a reference.
400) (Amersham Biosciences) as described previously
(28). GST fusion proteins were expressed and purified on
glutathione-Sepharose beads by the standard method (32). New Zealand
White rabbits were immunized with purified GST-Slac2-a400 (or
FLAG-myosin Va-GT), and anti-Slac2-a antibody was affinity-purified by
exposure to antigen-bound Affi-Gel 10 beads (Bio-Rad) as described
previously (33). Specificity of the antibody was checked by
immunoblotting using recombinant T7-tagged Slac2-a expressed in COS-7
cells (34).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
View larger version (35K):
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Fig. 1.
Mapping of the domain responsible for Rab27A
and Rab27B binding in Slac2-a. A, schematic
representation of deletion mutants of Slac2-a. The N-terminal SHD
(SHD1 and SHD2, black boxes) are
separated by two Zn2+-finger motifs (5). The Rab27A/B
binding activity of each mutant is indicated after its name (+, ±, or
). T7-Slac2-a-(A4) contained an Ala-based substitution in the
putative Rab binding domain (arrowhead) (36). B,
specific interaction of Slac2-a with Rab27A. pEF-T7-Slac2-a and
pEF-FLAG-Rab were cotransfected into COS-7 cells. The proteins
expressed were immunoprecipitated by anti-T7 tag antibody-conjugated
agarose. Co-immunoprecipitated (IP) FLAG-Rabs were first
detected with HRP-conjugated anti-FLAG tag antibody (1:10,000 dilution)
(middle panel, Blot: anti-FLAG,
IP: anti-T7). The same blots were then stripped
and reprobed with HRP-conjugated anti-T7 tag antibody (1:10,000
dilution) to ensure that the same amounts of T7-Slac2-a proteins had
been loaded (bottom panel, Blot:
anti-T7, IP: anti-T7). The upper
panel indicates the total expressed FLAG-Rabs (1/80 volume
of the reaction mixture) used for immunoprecipitation. The positions of
the molecular weight markers (× 10
3) are shown on the
left. C and D, mapping of the domains
of Slac2-a responsible for Rab27A and Rab27B binding, respectively.
pEF-T7-Slac2-a deletion mutants and pEF-FLAG-Rab27A/B were
cotransfected into COS-7 cells. Co-immunoprecipitated (IP)
FLAG-Rabs and immunoprecipitated T7-Slac2-a are shown in the
upper panels (Blot: anti-FLAG,
IP: anti-T7) and lower panels
(Blot: anti-T7, IP:
anti-T7), respectively. The far right lanes in
the upper panels in C and D indicate
the total expressed FLAG-Rab27A/B (1/80 volume of the reaction mixture)
used for immunoprecipitation. The positions of the molecular weight
markers (× 103) are shown on the right.
146) did not
(Fig. 1, C and D, lane 3). In
addition, the Slac2-a mutant carrying the SLEWY-to-ALEAA substitutions
in SHD2 (referred to as Slac2-a(A4), see Fig. 1A)
dramatically reduced the Rab27A/B binding activity (Fig. 1,
C and D, lane 4). Consistent with
this, crystallographic analysis has shown the corresponding sequence
(SGAWFF) in rabphilin-3 directly interacts with Rab3A (36). These
findings indicated that the SHD of Slac2-a specifically binds Ra27A/B
isoforms but that the large C-terminal domain is not involved in the
recognition of Rab27A/B molecules and might have different functions.
146) failed to mediate co-immunoprecipitation of T7-myosin
Va with FLAG-Rab27A (Fig. 2A, lanes 6 and
7), suggesting that different domains of Slac2-a may be
involved in Rab27A binding and myosin Va binding. Similar results were
obtained when FLAG-Rab27B was used instead of FLAG-Rab27A (data not
shown).
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Fig. 2.
The tripartite protein complex formed by
Slac2-a, Rab27A, and myosin Va. A, Slac2-a bridges the
gap between Rab27A and myosin Va. pEF-FLAG-Rab27A, pEF-T7-Slac2-a
mutants, and/or pEF-T7-myosin Va were cotransfected into COS-7 cells.
The proteins expressed were immunoprecipitated by anti-FLAG tag
antibody-conjugated agarose. Co-immunoprecipitated (IP)
T7-myosin Va and -Slac2-a were first detected by HRP-conjugated anti-T7
antibody (top and middle panels). The same blots
were then stripped and reprobed with HRP-conjugated anti-FLAG tag
antibody (bottom panel). Lanes 1-4 show the
total expressed T7-myosin Va and -Slac2-a (1/80 volume of the
reaction mixture) used for immunoprecipitation. Note that only the
full-length Slac2-a (closed arrowhead) forms the link
between Rab27A and myosin Va (lane 5), although
T7-Slac2-a-SHD (arrow), but not T7-Slac2-a- 146
(open arrowhead), efficiently immunoprecipitated with
FLAG-Rab27A. B, two domain structures of Slac2-a.
FLAG-Slac2-a deletion mutants and T7-myosin Va were co-expressed in
COS-7 cells, and their associations were analyzed by
immunoprecipitation as described above. Note that the C-terminal half
of Slac2-a, but not the SHD, is essential for myosin Va binding. The
positions of the molecular weight markers (× 103) are
shown on the left.
146, but not T7-Slac2-a-SHD, co-immunoprecipitated with
FLAG-myosin Va (Fig. 2B), indicating the two domain
structures of Slac2-a: the N-terminal SHD involved in the GTP-bound
form of Rab27A binding and the C-terminal domain involved in myosin Va binding.
146 interacted with the globular tail of
myosin Va in intact cells. We used purified recombinant proteins
(GST-Slac2-a (or -146), Rab27A, and FLAG-myosin Va-GT) for an
in vitro binding assay to further confirm the direct
interaction between the C terminus of Slac2-a and the globular tail of
myosin Va as well as the in vitro formation of a tripartite
protein complex from purified components. As expected, FLAG-myosin
Va-GT bound GST-Slac2-a-146 but not GST alone (Fig. 3C,
lane 2, arrow), and full-length Slac2-a bound
both FLAG-myosin Va-GT and Rab27A (Fig. 3D,
arrows).
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Fig. 3.
Mapping of the domain responsible for Slac2-a
binding in myosin Va. A, schematic representation of
mouse myosin Va. Myosin Va consists of an N-terminal head (motor)
domain (hatched box), a neck domain (open box)
including the IQ motif, and a proximal tail, medial tail (shaded
boxes), and globular tail (black box) (see also Fig. 4
and Ref. 21). The position of the PEST sequence is indicated by
PEST. B, the globular tail of myosin Va interacts with
Slac2-a- 146. pEF-T7-Slac2-a and pEF-FLAG-myosin Va-GT were
cotransfected into COS-7 cells, and their associations were analyzed by
immunoprecipitation (26, 30). Co-immunoprecipitated (IP)
FLAG-myosin Va-GT and immunoprecipitated T7-Slac2-a-146 are shown in
the middle panel (Blot: anti-FLAG,
IP: anti-T7) and bottom panel
(Blot: anti-T7, IP:
anti-T7), respectively. The top panel shows the
total expressed FLAG-myosin Va-GT (1/80 volume of the reaction
mixture) used for immunoprecipitation. C, direct interaction
between the GST-Slac2-a C terminus and FLAG-myosin Va-GT visualized by
anti-FLAG antibody (upper panel) and Amido Black staining
(lower panel). D, in vitro formation
of the tripartite protein complex from purified components
(GST-Slac2-a, FLAG-myosin Va-GT, and Rab27A) visualized by anti-FLAG
antibody (top panel), anti-Rab27 antibody (middle
panel), and Amido Black staining (bottom panel).
GST-Slac2-a (open arrowhead) directly interacted with both
FLAG-myosin Va-GT (closed arrow) and Rab27A (open
arrow). E, expression of Slac2-a in melanoma (B16-F1)
cells. Total homogenate of melanoma cells were subjected to 7.5%
SDS-PAGE followed by immunoblotting with the anti-Slac2-a-specific
antibody. F, formation of the tripartite protein complex
(Rab27A·Slac2-a·myosin Va) in melanoma cells. Note that both myosin
Va (upper panel) and Rab27A (bottom panel)
co-immunoprecipitated with Slac2-a from melanoma cell lysates. The
positions of the molecular weight markers (× 103) are
shown on the left.
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Fig. 4.
Schematic representation of the tripartite
protein complex responsible for melanosome transport. Rab27A is
associated with the melanosome (15, 19, 42). Slac2-a binds the
GTP-bound form of Rab27A through its SHD (Fig. 1) (13) and also
directly binds the globular tail of myosin Va through the C-terminal
domain (Fig. 3). The head (motor) domain of myosin Va interacts with
actin filaments. Thus, the tripartite protein complex of Rab27A,
Slac2-a/melanophilin, and myosin Va is essential to the capture and
local movement of melanosomes in the actin-rich cell periphery
(25).
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ACKNOWLEDGEMENTS |
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We thank Eiko Kanno and Yukie Ogata for expert technical assistance.
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FOOTNOTES |
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* This work was supported in part by grants from the Science and Technology Agency to Japan (to K. M.) and Grant 13780624 from the Ministry of Education, Science, and Culture of Japan (to M. F.).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.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail: mnfukuda@brain.riken.go.jp.
Published, JBC Papers in Press, February 20, 2002, DOI 10.1074/jbc.C200005200
2 M. Fukuda, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are: Slp, synaptotagmin-like protein; GST, glutathione S-transferase; GT, globular tail; HRP, horseradish peroxidase; SHD, Slp homology domain; Slac2, Slp homologue lacking C2 domains.
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TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
|
|---|
| 1. |
Marquèze, B.,
Berton, F.,
and Seagar, M.
(2000)
Biochimie (Paris)
82,
409-420[Medline]
[Order article via Infotrieve] |
| 2. |
Duncan, R. R.,
Shipston, M. J.,
and Chow, R. H.
(2000)
Biochimie (Paris)
82,
421-426[Medline]
[Order article via Infotrieve] |
| 3. |
Fukuda, M.,
Saegusa, C.,
Kanno, E.,
and Mikoshiba, K.
(2001)
J. Biol. Chem.
276,
24441-24444 |
| 4. |
Fukuda, M.,
and Mikoshiba, K.
(2001)
Biochem. Biophys. Res. Commun.
281,
1226-1233[CrossRef][Medline]
[Order article via Infotrieve] |
| 5. |
Fukuda, M.,
Saegusa, C.,
and Mikoshiba, K.
(2001)
Biochem. Biophys. Res. Commun.
283,
513-519[CrossRef][Medline]
[Order article via Infotrieve] |
| 6. |
Fukuda, M.,
and Mikoshiba, K.
(2001)
FEBS Lett.
503,
217-218[CrossRef][Medline]
[Order article via Infotrieve] |
| 7. |
Fukuda, M.,
and Mikoshiba, K.
(2001)
Biochem. J.
360,
441-448[CrossRef][Medline]
[Order article via Infotrieve] |
| 8. |
Nalefski, E. A.,
and Falke, J. J.
(1996)
Protein Sci.
5,
2375-2390[Abstract] |
| 9. |
McAdara Berkowitz, J. K.,
Catz, S. D.,
Johnson, J. L.,
Ruedi, J. M.,
Thon, V.,
and Babior, B. M.
(2001)
J. Biol. Chem.
276,
18855-18862 |
| 10. |
Wang, J.,
Takeuchi, T.,
Yokota, H.,
and Izumi, T.
(1999)
J. Biol. Chem.
274,
28542-28548 |
| 11. |
Ishikawa, K.,
Nagase, T.,
Suyama, M.,
Miyajima, N.,
Tanaka, A.,
Kotani, H.,
Nomura, N.,
and Ohara, O.
(1998)
DNA Res.
5,
169-176[Abstract] |
| 12. |
Zerial, M.,
and McBride, H.
(2001)
Nat. Rev. Mol. Cell. Biol.
2,
107-117[CrossRef][Medline]
[Order article via Infotrieve] |
| 13. |
Kuroda, T. S.,
Fukuda, M.,
Ariga, H.,
and Mikoshiba, K.
(2002)
J. Biol. Chem.
277,
9212-9218 |
| 14. |
Menasche, G.,
Pastural, E.,
Feldmann, J.,
Certain, S.,
Ersoy, F.,
Dupuis, S.,
Wulffraat, N.,
Bianchi, D.,
Fischer, A., Le,
Deist, F.,
and de Saint Basile, G.
(2000)
Nat. Genet.
25,
173-176[CrossRef][Medline]
[Order article via Infotrieve] |
| 15. |
Bahadoran, P.,
Aberdam, E.,
Mantoux, F.,
Busca, R.,
Bille, K.,
Yalman, N.,
de Saint-Basile, G.,
Casaroli-Marano, R.,
Ortonne, J. P.,
and Ballotti, R.
(2001)
J. Cell Biol.
152,
843-850 |
| 16. |
Wilson, S. M.,
Yip, R.,
Swing, D. A.,
O'Sullivan, T. N.,
Zhang, Y.,
Novak, E. K.,
Swank, R. T.,
Russell, L. B.,
Copeland, N. G.,
and Jenkins, N. A.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
7933-7938 |
| 17. |
Stinchcombe, J. C.,
Barral, D. C.,
Mules, E. H.,
Booth, S.,
Hume, A. N.,
Machesky, L. M.,
Seabra, M. C.,
and Griffiths, G. M.
(2001)
J. Cell Biol.
152,
825-834 |
| 18. |
Haddad, E. K., Wu, X.,
Hammer, J. A., III,
and Henkart, P. A.
(2001)
J. Cell Biol.
152,
835-842 |
| 19. |
Hume, A. N.,
Collinson, L. M.,
Rapak, A.,
Gomes, A. Q.,
Hopkins, C. R.,
and Seabra, M. C.
(2001)
J. Cell Biol.
152,
795-808 |
| 20. |
Matesic, L. E.,
Yip, R.,
Reuss, A. E.,
Swing, D. A.,
O'Sullivan, T. N.,
Fletcher, C. F.,
Copeland, N. G.,
and Jenkins, N. A.
(2001)
Proc. Natl. Acad. Sci. U. S. A.
98,
10238-10243 |
| 21. |
Reck-Peterson, S. L.,
Provance, D. W., Jr.,
Mooseker, M. S.,
and Mercer, J. A.
(2000)
Biochim. Biophys. Acta
1496,
36-51[Medline]
[Order article via Infotrieve] |
| 22. |
Sellers, J. R.
(2000)
Biochim. Biophys. Acta
1496,
3-25[Medline]
[Order article via Infotrieve] |
| 23. |
Moore, K. J.,
Seperack, P. K.,
Strobel, M. C.,
Swing, D. A.,
Copeland, N. G.,
and Jenkins, N. A.
(1988)
Proc. Natl. Acad. Sci. U. S. A.
85,
8131-8135 |
| 24. |
Mercer, J. A.,
Seperack, P. K.,
Strobel, M. C.,
Copeland, N. G.,
and Jenkins, N. A.
(1991)
Nature
349,
709-713[CrossRef][Medline]
[Order article via Infotrieve] |
| 25. |
Marks, M. S.,
and Seabra, M. C.
(2001)
Nat. Rev. Mol. Cell. Biol.
2,
738-748[CrossRef][Medline]
[Order article via Infotrieve] |
| 26. |
Fukuda, M.,
Kanno, E.,
and Mikoshiba, K.
(1999)
J. Biol. Chem.
274,
31421-31427 |
| 27. |
Mizushima, S.,
and Nagata, S.
(1990)
Nucleic Acids Res.
18,
5332 |
| 28. |
Fukuda, M.,
Aruga, J.,
Niinobe, M.,
Aimoto, S.,
and Mikoshiba, K.
(1994)
J. Biol. Chem.
269,
29206-29211 |
| 29. |
Fukuda, M.,
and Mikoshiba, K.
(2000)
J. Biol. Chem.
275,
28180-28185 |
| 30. |
Fukuda, M.,
Kanno, E.,
Ogata, Y.,
and Mikoshiba, K.
(2001)
J. Biol. Chem.
276,
40319-40325 |
| 31. |
Fukuda, M.,
Kojima, T.,
Aruga, J.,
Niinobe, M.,
and Mikoshiba, K.
(1995)
J. Biol. Chem.
270,
26523-26527 |
| 32. |
Smith, D. B.,
and Johnson, K. S.
(1988)
Gene (Amst.)
67,
31-40[CrossRef][Medline]
[Order article via Infotrieve] |
| 33. |
Fukuda, M.,
and Mikoshiba, K.
(1999)
J. Biol. Chem.
274,
31428-31434 |
| 34. |
Fukuda, M.,
Martin, T. F. J.,
Kowalchyk, J. A.,
Zhang, X.,
and Mikoshiba, K.
(2002)
J. Biol. Chem.
277,
4601-4604 |
| 35. |
Fukuda, M.,
and Mikoshiba, K.
(2001)
J. Biol. Chem.
276,
27670-27676 |
| 36. |
Ostermeier, C.,
and Brunger, A. T.
(1999)
Cell
96,
363-374[CrossRef][Medline]
[Order article via Infotrieve] |
| 37. |
Prekeris, R.,
and Terrian, D. M.
(1997)
J. Cell Biol.
137,
1589-1601 |
| 38. |
Cheney, R. E.,
and Rodriguez, O. C.
(2001)
Science
293,
1263-1264 |
| 39. |
Lapierre, L. A.,
Kumar, R.,
Hales, C. M.,
Navarre, J.,
Bhartur, S. G.,
Burnette, J. O.,
Provance, D. W., Jr.,
Mercer, J. A.,
Bahler, M.,
and Goldenring, J. R.
(2001)
Mol. Biol. Cell
12,
1843-1857 |
| 40. |
Takagishi, Y.,
Oda, S.,
Hayasaka, S.,
Dekker-Ohno, K.,
Shikata, T.,
Inouye, M.,
and Yamamura, H.
(1996)
Neurosci. Lett.
215,
169-172[Medline]
[Order article via Infotrieve] |
| 41. |
Miyata, M.,
Finch, E. A.,
Khiroug, L.,
Hashimoto, K.,
Hayasaka, S.,
Oda, S. I.,
Inouye, M.,
Takagishi, Y.,
Augustine, G. J.,
and Kano, M.
(2000)
Neuron
28,
233-244[CrossRef][Medline]
[Order article via Infotrieve] |
| 42. |
Wu, X.,
Rao, K.,
Bowers, M. B,
Copeland, N. G.,
Jenkins, N. A.,
and Hammer, J. A., III
(2001)
J. Cell Sci.
114,
1091-1100[Abstract] |
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