From the Institute for Molecular Pathology, Dr.
Bohrgasse 7, A-1030 Vienna, Austria and the § Basel
Institute for Immunology, Grenzacherstraße 487, Ch-4005 Basel, Switzerland
Received for publication, February 16, 2001
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ABSTRACT |
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Delivery of transport vesicles to their receptor
compartment involves tethering, priming, and fusion. Soluble NSF
attachment protein- Vesicles mediate the transport between membrane-bound compartments
of eukaryotic cells (1). To maintain the functional and morphological
integrity of the cell, protein and lipid transport has to follow a
selective and precise mechanism. After entry of cargo into the budding
vesicle, it pinches off and is destined for the target membrane. Short
range movement of vesicles is mediated by diffusion, whereas motor
proteins move vesicles over a long distance on cytoskeletal tracks.
Finally, vesicles dock to and fuse with the target membrane, resulting
in the delivery of the cargo. Vesicular transport is a cyclic process
accounting for the vectorial flux of cargo while maintaining the
structural and functional integrity of the individual compartments (for
reviews see Refs. 2-5).
Docking is the morphologically defined event of the association of a
vesicle with a target membrane. Tethering is the biochemical equivalent
and involves the formation of a stable interaction of a vesicle with a
membrane. Priming refers to the activation of the membrane surfaces to
obtain fusion competence (6). The SNARE1 hypothesis (7)
provides an explanation for specificity
in vesicular transport; during targeting, the membrane-bound
v-SNARE protein of a vesicle interacts with the corresponding
t-SNARE of the target membrane forming a SNARE complex. SNAREs were
initially identified in the neuronal system (7), and subsequently
homologous proteins involved in other transport steps in the mammalian
cell and in yeast were identified. SNAREs of the synapse and of the yeast vacuole are involved in docking and fusion of proteoliposomes and
vacuolar membranes, respectively (8-10). Several soluble proteins regulate the v-t-SNARE interaction. Members of the SNAP family of
proteins, consisting of the ubiquitously expressed Several proteins involved in transport through the Golgi apparatus have
been identified. The v-SNARE GOS28 was the first SNARE implicated in
intra-Golgi protein transport (21, 22). Subsequent studies identified
GS27/membrin (23, 24), rbet1 and rsec22b (24, 25), GS32 (26), syntaxin
5 (27), syntaxin 6 (28), syntaxin 10 (29), and syntaxin 16 (30, 31) as
additional Golgi SNAREs. Rab family proteins such as Rab6 (32), Rab10
(33), and Rab12 (34) have been localized to the Golgi complex. Rab6 has
been shown to be involved in intra-Golgi protein transport (35, 36) and
retrograde transport to the endoplasmic reticulum (37). Cytosol
fractionation studies revealed p115(TAP) as a protein involved in
intra-Golgi transport (38). It forms a homodimer and is composed of a
globular head domain and a rodlike tail domain (39). p115 binds to the
Golgi-associated proteins GM130 (40) and giantin (41). GM130 is
mitotically phosphorylated, and this dissociates the p115-GM130
interaction (40). This is concomitant with the vesiculation of the
Golgi complex during mitosis.
Although many proteins involved in the targeting of vesicles have been
identified, no consensus exists about the order of involvement of these
proteins (42, 43). This study focused on the interaction of
Golgi-derived transport vesicles with their target membrane in
intra-Golgi transport. GOS28 was chosen as a representative of the
SNARE family, and Rab6 was chosen as a representative of the Rab
family. Both proteins and p115 are examined for their contribution in
intra-Golgi transport relative to Recombinant Material--
Bovine His6- Antibodies--
The following antibodies were used:
anti-syntaxin antibody HPC-1 (47), anti-NSF antibody 6E6 (48),
anti- Subcellular Fractions and Intra-Golgi Transport
Assay--
Extract from bovine brain membranes was prepared as
described in Ref. 7, and GDI was purified as described in Ref. 51. 1 M KCl-treated (K-Golgi) membranes were prepared
according to Ref. 52. Intra-Golgi protein transport assays were carried
out at 37 °C for 60 min as described (53, 54). Reactions containing K-Golgi membranes were carried out in a volume of 100 µl. For the
two-stage assay, transport reactions were incubated with 500 ng of 2F10
on ice for 20 min and transferred to 37 °C for 20 min. The reactions
were placed on ice, and the cytoplasmic domains of GOS28, GDI, and
anti-Rab6 were added, respectively. After the addition of 500 ng of
His6- Domain Analysis of
The deletion mutants of
The anti- Development of a Two-stage Intra-Golgi Transport Assay--
An
in vitro transport assay that reconstituted vesicular
intra-Golgi transport was developed by Rothman and co-workers (54, 58).
Subsequent characterization of the transport reaction resulted in a
consensus that this assay allows measurement of the fusion of
Golgi-derived transport vesicles with early Golgi elements (59, 60),
establishing this assay as a tool to identify and characterize
components of the transport machinery (61). The antibody 2F10 inhibits
the intra-Golgi transport reaction.2 This inhibition is
caused by a depletion of The Contribution of p115, GOS28, and Rab Proteins to Intra-Golgi
Protein Transport--
The proteins p115, GOS28, and Rab6 have been
implicated in intra-Golgi protein transport (for references see the
Introduction), and we studied their role in the one- and two-stage
transport assays. A polyclonal antiserum against the globular head
domain of p115 was generated. Affinity-purified antibodies recognized a
single band of 115 kDa of Chinese hamster ovary whole cell lysate in
Western analysis (data not shown). We added increasing amounts of these
antibodies to the transport reactions between 1 M
KCl-treated Golgi membranes. As shown in Fig.
4 (top panel), only the
one-stage transport reaction but not the two-stage reaction was
inhibited by the anti-p115 antibody. The inhibition reached a
saturation at 400 ng of antibody/100-µl reaction that could be
reversed by the addition of antigen (data not shown). Initially, the
antibodies were tested in the standard intra-Golgi transport assay and
did not inhibit protein transport (data not shown). Subsequently, K-Golgi membranes were used in the assay. Salt treatment removes p115
quantitatively from Golgi membranes, and cytosol becomes the only
source of p115 (38). 100 ng of NSF had to be added to the transport
reaction in these experiments because the 1 M KCl wash
removes NSF likewise. The degree of GTP
We then examined the inhibition pattern of the cytoplasmic domain of
GOS28 in the one- and two-stage transport assays. The cytoplasmic
domain of GOS28 containing a hexahistidine motif at the N terminus was
expressed in E. coli. The protein was purified from
inclusion bodies after solubilization in urea. The protein was bound to
a nickel-nitrilotriacetic acid matrix, renatured by applying a gradient
of a decreasing concentration of urea, and eluted by a gradient of
increasing imidazole concentration. In SDS-PAGE a single band of 26 kDa
was observed (data not shown). The renatured cytoplasmic domain of
GOS28 was added to the one- and two-stage transport assay using
standard membranes. In both cases, a similar degree of 70% inhibition
was observed. In the two-stage reaction, a 2.5-fold smaller amount of
GOS28 was sufficient to achieve inhibition (Fig.
5). Two control reactions for the specificity of GOS28 were carried out. 1) The effect of GOS28 can be
neutralized by incubation with antibodies directed against GOS28 (data
not shown); 2) the cytoplasmic domain of the plasma membrane SNARE
synaptobrevin/VAMP was applied. Soluble recombinant synaptobrevin/VAMP
was confirmed to be active by virtue of the fact that it forms a
complex with the cytoplasmic domain of syntaxin 1a and SNAP25 that can
be disassembled by
The contribution of Rab proteins to the intra-Golgi transport was
determined by adding GDI or antibodies against Rab6 (3A6) to the one-
and two-stage transport assays. GDI depletes the transport reaction of
all Rab proteins. Both GDI and anti-Rab6 inhibit the one- and two-stage
reactions in a saturable manner by 50% (Fig. 6A and data not shown).
Because GDI inactivates Rab proteins, the specific effect of both
reagents was determined by simultaneous addition. The combination of
GDI and anti-Rab6 exhibits a comparable inhibition that does not differ
in a statistically significant manner when applied separately (Fig.
6B), suggesting that both proteins act specifically.
Our results define the biochemical sequence of proteins involved in the
targeting of vesicles during transport through the Golgi apparatus. The
addition of recombinant The SNARE hypothesis predicted that SNAP and NSF bind to SNAREs
paired in trans as the result of vesicle docking (7). This prediction placed SNAP and NSF at a defined step of the targeting mechanism. Meanwhile, it was shown for the fusion of yeast vacuoles that only t-SNAREs require priming (66) and that priming can take place
without tethering (10). In this study, we examined protein transport
through the Golgi apparatus, the central organelle of constitutive
secretion. In interpreting our data and the data obtained in the yeast
system, we propose that the function of A promiscuous interaction of bacterially expressed SNAREs involved in
unrelated transport steps has been observed (72, 73). We compared the
effect of the cytoplasmic domain of GOS28 and the neuronal SNARE
synaptobrevin/VAMP in the standard intra-Golgi transport reaction. The
neuronal SNAREs synaptobrevin/VAMP, syntaxin 1a, and SNAP25 are, to our
knowledge, the only SNAREs that, after expression in E. coli, have been shown to form a complex that can be disassembled
with p115 is involved in transcytosis in hepatocytes and is found on
vesicles (45). Independently, it was isolated from cytosol in an assay
measuring the transport of proteins between high salt-treated Golgi
membranes (38, 39). High salt treatment of membranes removes peripheral
membrane proteins, including coatomer, resulting in an increased amount
of uncoupled homotypic fusion (64). The antibody against p115
inhibited intra-Golgi transport only after removal of p115 by high salt
treatment of the membranes. An inhibition was observed in the one-stage
assay but not in the two-stage assay. This result suggests that p115 is
recruited to the membrane in an GDI depletes membranes of Rab proteins resulting in a partial
inhibition of the one- and two-stage transport assays. An involvement of Rab6 in intra-Golgi transport has been demonstrated (35, 36), and
the anti-Rab6 antibody showed an inhibition pattern comparable with
GDI. This result suggests that Rab proteins, in particular Rab6, act
after or parallel to (
SNAP) mediates the disruption of
SNAREs by N-ethylmaleimide sensitive factor (NSF)
and was employed to determine the hierarchy of proteins responsible for
intra-Golgi protein transport. The N-terminal 23 amino acids of
SNAP
are necessary for SNARE binding. The antibody 2F10 recognizes this
SNARE interaction domain of
SNAP and inhibits intra-Golgi protein
transport reversibly. This antibody was applied to modify the transport
assay to determine the protein requirements relative to the action of
SNAP and NSF. We found that 1) p115 acts independently of
SNAP
and NSF, 2) SNAREs are required after tethering and interact
selectively after activation by
SNAP and NSF, and 3) Rab proteins
act after SNARE activation and before fusion.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- and
SNAP and
the neuronal
SNAP, bind to SNAREs (11, 12). Subsequently, the ATPase
NSF binds to SNAREs via the SNAPs (13), and ATP hydrolysis results in
the disruption of SNARE complexes (7). Another family of proteins
involved in vesicular transport is the Rab family of GTP-binding
proteins (14). They cycle between a GTP- and a GDP-bound form, and both
forms can be membrane-associated (15). The GDP dissociation inhibitor
(GDI) forms a complex with Rabs in their GDP-bound form and removes
them from the membrane (16-18). GDP is exchanged for GTP by a guanine
nucleotide exchange factor (19, 20).
SNAP and NSF.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SNAP was
purified from Escherichia coli lysate according to Ref. 12,
and GST-VAMP was purified as described in Ref. 44. The truncated
forms of GST
SNAP designated GST
SNAP-(1-156), GST
SNAP-(24-295), and GST
SNAP-(35-295) were obtained by
amplification of the corresponding DNA fragment of
SNAP with
oligonucleotides introducing a BamHI site at the 5' end and
an EcoRI site at the 3' end followed by ligation into the
E. coli expression plasmid pGEX4T-1 (Amersham Pharmacia
Biotech). GST
SNAP and its deletion mutants were purified as
described in Ref. 21, and His6-NSFmyc was purified
according to Ref. 7. To bacterially express the head domain of p115
from rat (45), a DNA fragment coding for amino acids 1-651 was
amplified by polymerase chain reaction introducing a
BamHI site at the 5' end and an EcoRI site at the
3' end. The EcoRI site in the open reading frame was removed
without affecting the translated amino acids. This fragment was ligated
into pGEX4T-1. The p115 head domain was expressed as a fusion protein
with GST and was purified by thrombin cleavage according to the
manufacturer's instruction. The cytoplasmic domain of
His6-GOS28 was expressed in E. coli (21).
Insoluble protein (inclusion bodies) was dissolved in buffer A (8 M urea, 100 mM sodium phosphate, pH 8.0) and
cleared by centrifugation. Soluble protein was bound to a
nickel-nitrilotriacetic acid column (Qiagen) equilibrated with buffer
A. After washing with 50 column volumes of buffer A, a linear gradient
against buffer B (100 mM KCl, 10% (w/v) glycerol, 1 mM 2-mercaptoethanol, 20 mM imidazole,
50 mM Tris-HCl, pH 8.0) was applied. Refolded protein was
eluted with a linear gradient of buffer B containing 500 mM
imidazole. Protein concentration was determined by the method of
Bradford (46).
SNAP antibody 2F10,2
anti-Rab6 antibody 3A6 (49), and anti-SNAP25 antibody (50). Polyclonal
GST antibody was purchased from Santa Cruz Biotechnology, and
anti-synaptobrevin/VAMP serum was from Wako. For the preparation of an
antibody directed against p115, a rabbit was immunized with the head
domain of p115. Antibody was affinity-purified from antigen coupled to
CNBr-activated Sepharose 4B (Amersham Pharmacia Biotech). Bound
antibody was eluted with 0.1 M glycine (pH 2.8),
neutralized, concentrated to 1.4 µg/µl in a dialysis tubing placed
on dry polyethylene glycol 35000, and dialyzed against 50 mM KCl, 25 mM HEPES (pH 7.4), 5% glycerol, 0.5 mM
-mercaptoethanol.
SNAP, the samples were incubated at 37 °C for
1 h. The two-stage assays containing K-Golgi membranes were
inhibited with 500 ng of 2F10 as described above, and anti-p115 antibody and 400 ng of His6
SNAP were added. Error
bars indicate standard deviations of experiments carried out as
triplicates (see Fig. 4) or as duplicates (see Figs. 5 and 6).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SNAP--
SNAP is required for the
disassembly of SNARE complexes by NSF. This dissociation is a key event
in vesicular protein transport; therefore, we chose
SNAP as a target
to examine its role and, in conclusion, that of NSF and SNAREs in
protein targeting. To determine the domain structure of
SNAP we
carried out limited proteolysis with subtilisin, a protease of low
sequence specificity. Protein domains of compact and rigid conformation
are more resistant to proteolytic degradation by subtilisin, whereas
flexible parts of a protein become preferentially hydrolyzed (55). To
identify core domains of
SNAP we incubated His6-
SNAP
with increasing amounts of subtilisin. The proteolytic fragments were
separated by SDS-PAGE. Fig. 1A
shows a typical result of limited proteolysis of
SNAP. After
transfer to a polyvinylidene fluoride membrane, partial N-terminal
amino acid sequencing was performed on the four proteolytic
intermediates indicated in Fig. 1A. The largest of the four
intermediates starts at position 32 with the smaller fragments
beginning at positions 93, 140, and 157 of
SNAP, respectively (Fig.
1B). A comparison of the apparent molecular weight of the proteolytic fragments with the calculated molecular weight suggests that the C terminus was not subject to proteolytic degradation (data
not shown). The hydrophilic amino acid serine found in positions 24 and
35 flanks the site of the first proteolytic cut at position 32. Truncated GST
SNAP fusion proteins were expressed in E. coli that lacked either the first 23 or 31 amino acids of
SNAP.
In addition, amino acids 1-156 of
SNAP were fused to GST and
expressed in E. coli.
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Fig. 1.
Limited proteolysis of
SNAP. A, subtilisin digest of
SNAP. In each reaction, 60 µg of
SNAP were incubated with 0, 0.025, 0.075, 0.25, 0.75, 2.5, 7.5, and 25 µg of subtilisin
(lanes 1-8) in 25 mM HEPES (pH 7.4), 50 mM KCl, 5 mM NaCl, 1 mM
dithiothreitol, 10% (v/v) glycerol in a volume of 25 µl at 4 °C
for 20 min. The reaction was stopped by the addition of 5 µl of 1 M phenylmethylsulfonyl fluoride, and an aliquot was
separated by SDS-PAGE on a 15% gel and stained with Coomassie
Brilliant Blue R. Lane 9 contains subtilisin only.
B, amino acid sequence of
SNAP. The arrowheads
indicate the sites of subtilisin hydrolysis. Forward arrows
indicate the beginning of N-terminally truncated mutants (24-295 and
35-295), and the reverse arrow indicates the end of the
C-terminally truncated form (1) of
SNAP expressed in E. coli as a GST fusion protein.
SNAP were examined for their ability to bind
to neuronal SNARE complexes. SNARE complexes were prepared from bovine
brain membranes by lysis with Triton X-100.
SNAP and its truncation
mutants were incubated with the membrane extract and purified with
glutathione-agarose beads. Neuronal SNAREs were detected by SDS-PAGE
and transfer to a nitrocellulose membrane followed by Western analysis.
Compared with full-length
SNAP, removal of 23 amino acids from the N
terminus results in a decreased affinity for SNAREs, and removal of 34 amino acids renders SNARE binding undetectable. However, amino acids
1-23 of
SNAP are not sufficient for SNARE binding because a mutant
SNAP consisting of amino acids 1-156 does not bind SNAREs (Fig.
2A).
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Fig. 2.
Functional characterization of
SNAP mutants. A, binding of
neuronal SNAREs to
SNAP. 2 µg of the indicated GST
SNAP was
incubated with 150 µg of bovine brain membrane proteins and 25 µl
of glutathione-agarose in 130 µl of 25 mM HEPES (pH 7.4),
50 mM KCl, 1 mM dithiothreitol, 10% (v/v)
glycerol for 2 h. The agarose beads were washed in the same buffer
containing 0.2% Triton X-100. An aliquot was applied to SDS-PAGE
followed by Western analysis with the antibodies listed. B,
epitope mapping of the monoclonal anti-
SNAP antibody 2F10. 200 ng of
full-length or truncated GST
SNAP were subjected to SDS-PAGE and
transfer to nitrocellulose. The membrane was developed with anti-GST
serum and the monoclonal antibody 2F10.
SNAP antibody 2F10 interferes with the binding of SNAREs to
SNAP.2 Considering the critical role of the N terminus
of
SNAP in SNARE binding, we applied the deletion mutants of
SNAP
in a Western analysis to determine the epitope of 2F10. As depicted in
Fig. 2B, the antibody 2F10 recognized full-length
SNAP
but none of the N-terminal truncation mutants. This coincidence
suggests that the N terminus of
SNAP is directly involved in binding
of neuronal SNAREs. The critical role of the N terminus of
SNAP in
binding SNAREs is also supported by studies of the interaction of
recombinant SNAREs with
SNAP (56) and electrophysiological
experiments. In this study, a peptide derived from the N terminus of
SNAP inhibited calcium-induced exocytosis after microinjection into the giant axon of squid (57).
SNAP from both cytosol and membranes. The
SNAP-2F10 complex is predicted to be soluble and to be minimally
invasive during the transport assay. In the first step we determined
the time span for which the transport assay is sensitive for 2F10.
Standard transport reactions were started, and 2F10 was added after
discrete time intervals. As shown in Fig.
3A, the transport signal
becomes independent of
SNAP within 20 min. The remaining incubation
time of 40 min at 37 °C is required for the glycosylation of the
vesicular stomatitis virus- encoded glycoprotein. In the second
step we tested whether the transport inhibition caused by 2F10 is
reversible. Transport was first inhibited by 2F10 for 20 min, and then
SNAP was added. As depicted in Fig. 3B, the inhibition by
2F10 is reversible. The reversibility of the inhibition by 2F10 made it
possible to divide the intra-Golgi transport reaction into two steps,
enabling us to resolve requirements before and after
SNAP. In the
first step, the reaction is inhibited by 2F10, allowing transport until the first
SNAP-dependent step is reached. Cells treated
with N-ethylmaleimide accumulate docked vesicles at the
Golgi apparatus (62).
SNAP and NSF were identified as protein
targets of N-ethylmaleimide (63), implying that the
transport reaction is inhibited after the accumulation of docked
transport intermediates. In the second step, this inhibition is
reversed by the addition of recombinant
SNAP. At this point,
substrates can be tested for their potential to inhibit the two-stage
transport reaction, and the quality and quantity of inhibition can be
compared with a standard one-stage transport reaction (Fig.
3C).
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Fig. 3.
SNAP requirement of the
intra-Golgi transport reaction. A, susceptibility of
the transport reaction for inhibition by the anti-
SNAP antibody
2F10. Intra-Golgi transport reactions consisting of 5 µl of donor and
5 µl of acceptor membranes, 12 µl of cytosol, 0.4 µM
[3H]UDP-GlcNAc, and an ATP-regenerating system in a total
volume of 50 µl (54) were incubated at 37 °C for 1 h. 500 ng
of 2F10 were added after the indicated time intervals. Then the
membranes were lysed by detergent, and the vesicular stomatitis
virus-encoded glycoprotein was immunoprecipitated. B,
reversibility of the inhibition by the antibody 2F10. An intra-Golgi
transport reaction was carried out for 1 h at 37 °C (lane
1). The same reaction was incubated with 500 ng of 2F10 on ice for
20 min and then for 1 h at 37 °C (lane 2). Another
transport reaction was incubated with 500 ng of 2F10 for 20 min on ice
and subsequently at 37 °C for 20 min. The reaction was transferred
back on ice, 500 ng of
SNAP were added, and the incubation at
37 °C was continued for 1 h (lane 3). C,
model for the two-stage intra-Golgi transport reaction. The antibody
2F10 depletes the transport assay of
SNAP, resulting in a block of
the assay at a step preceding the fusion of transport vesicles with the
acceptor membrane. The reaction proceeds after the addition of
SNAP,
and protein requirements relative to
SNAP and its interacting
proteins NSF and SNAREs can be distinguished.
S inhibition is an indicator
of to what extent vesicular transport as compared with uncoupled
fusion contributes to the transport reaction (64). In our assay design,
GTP
S inhibited the reaction by 60% (Fig. 4, bottom
panel), suggesting that most of the transport signal is due to
vesicular transport intermediates.
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Fig. 4.
Involvement of p115 in intra-Golgi
transport. A, inhibition of intra-Golgi transport by
anti-p115 antibodies. A one-stage transport reaction containing 20 µg
of cytosol, 10 µl of K-Golgi donor, acceptor membranes, 250 ng of
NSF, and an ATP-regenerating system was incubated at 37 °C for
1 h (lane 1). Cytosol was incubated with 1.2 µg of
anti-p115 antibody on ice for 10 min, and the same reaction was
performed (lane 2). The two-stage reactions were incubated
with 500 ng of 2F10 on ice for 20 min and at 37 °C for 20 min. The
reactions were transferred on ice, and 400 ng of SNAP (lane
3) and 1.2 µg of anti-p115 (lane 4) were added, kept
on ice for 10 min, and incubated for 1 h at 37 °C.
B, incubation with a nonhydrolyzable analogue of GTP.
One-stage transport reactions were carried out in the absence
(lane 1) and the presence (lane 2) of 10 µM GTP
S.
SNAP and NSF as described in Ref. 65. No
inhibition by synaptobrevin/VAMP was observed in the transport reaction
(Fig. 5).
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Fig. 5.
Inhibition of intra-Golgi transport by
cytoplasmic domains of v-SNAREs. Standard transport reactions
(with one stage) as described in the legend for Fig. 3 were carried out
with the indicated amounts of the cytoplasmic domain of GOS28
(closed circles) and synaptobrevin/VAMP (open
squares). Two-stage transport reactions were incubated with 500 ng
of 2F10 for 20 min on ice and then for 20 min at 37 °C. The
reactions were transferred on ice to add 500 ng of SNAP and the
indicated amounts of the cytoplasmic domain of GOS28 (open
circles). The incubation was continued at 37 °C for 1 h.
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Fig. 6.
The involvement of Rab proteins in
intra-Golgi transport. A, inhibition of intra-Golgi
transport by GDI and anti-Rab6 antibodies. One-stage (lanes
1-3) and two-stage transport reactions (lanes 4-6)
were performed as outlined in the legend for Fig. 5. The one-stage
reactions were inhibited by 2.6 µg of GDI (lane 2) and 1.6 µg of anti-Rab6 antibodies (lane 3), and the two-stage
reactions were inhibited by the same amounts of GDI (lane 5)
and anti-Rab6 (lane 6). B, simultaneous
inhibition by GDI and anti-Rab6. A one-stage transport reaction
(lane 1) was incubated with 2.6 µg of GDI (lane
2), with 1.4 µg of anti-Rab6 antibodies (lane 3), or
with the same amounts of both reagents (lane 4).
SNAP to an intra-Golgi transport reaction
inhibited by the anti-
SNAP antibody 2F10 results in a complete
reversal of the inhibition. This observation enabled us to modify the
intra-Golgi transport assay to determine the contribution and temporal
action of p115, GOS28, and Rab6, three proteins known to be involved in
intra-Golgi transport, relative to
SNAP and its interaction partner
NSF. The data presented can be most easily explained by p115-mediated
binding of vesicles to the target membrane followed by the activation
of SNAREs on the vesicle and the target membrane, respectively. Rab
proteins, in particular Rab6, are required for a step between SNARE
activation and fusion.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
SNAP and NSF is to
continuously activate the SNAREs on all membranes involved in
constitutive transport. This activation would keep the machinery for
the recognition and fusion of membranes continuously active for the
consumption of newly delivered vesicles to ensure a high transport
rate. As a consequence, no sequential order of priming relative to
tethering or fusion can be assigned in constitutive secretion in
vivo. In regulated secretion, the cycle of generation and
consumption of vesicles becomes arrested and allows one to resolve
mechanistic intermediates. The model systems studied suggest a role for
SNAP and NSF before and after vesicle fusion; in neuroendocrine cells, a readily releasable pool of vesicles is docked to the plasma
membrane and fuses after the influx of calcium ions from the
extracellular medium (67, 68). SNAP and NSF are required for a priming
step that precedes the calcium-triggered fusion (69). This finding was
confirmed in the temperature-sensitive Drosophila NSF mutant
named comatose (6). Two lines of evidence suggest an additional role
for SNAP and NSF after fusion; the temperature-sensitive phenotype of
comatose is reversible, and the recovery kinetic corresponds to the
kinetic of the Drosophila dynamin mutant shibire known to be
required for the recycling of vesicles (70). In comatose mutants,
v-t-SNARE complexes accumulate on synaptic vesicles after a temperature
shift (71). This suggests that SNAP and NSF are required to separate v-
and t-SNAREs after the fusion to ensure that v-SNAREs enrich on
recycling vesicles and t-SNAREs remain at the target membrane.
SNAP and NSF (65). Therefore, the v-SNARE synaptobrevin/VAMP
was the protein of choice to examine the specificity of SNARE
interactions in the intra-Golgi transport assay. The cytoplasmic domain
of GOS28 inhibited the transport reaction in a
dose-dependent manner, confirming previous studies that
showed that an antibody against GOS28 inhibits intra-Golgi transport
(21, 22). No inhibition by the cytoplasmic domain of synaptobrevin/VAMP
was observed. Our data suggest that SNAREs can interact in a specific
manner, and we conclude that the structural organization of the cell is
sufficiently preserved in the cell-free intra-Golgi transport assay to
retain the key mechanisms in specific vesicular targeting. Compared
with the one-stage reaction, the same inhibition results from a lower
concentration of the cytoplasmic domain of GOS28 in the two-stage
reaction. A kinetic and a steric effect can account for the observed
difference.
SNAP binds to unpaired t-SNAREs (74), and a depletion by
the antibody 2F10 gives the cytoplasmic domain of GOS28 a better access
to syntaxin 5; alternatively, the simultaneous addition of
SNAP and
the cytoplasmic domain of GOS28 in the two-stage reaction results in a
higher association rate for the cytoplasmic domain of GOS28.
SNAP- and NSF-independent manner and
that the p115 N-terminal head domain does not become accessible to
antibody during the transport reaction. Ultrastructural analysis of
Golgi complexes reveals that vesicles are tethered to Golgi stacks by
fibrous structures, unable to diffuse freely (75). p115 mediates
binding of vesicles to Golgi membranes (41) and is a candidate for such a string protein. The partial inhibition of the intra-Golgi
transport reaction caused by the anti-p115 antibody is consistent with
the functional redundancy for interaction of vesicles and target
membrane observed in a yeast strain harboring mutated p115. This
conditional mutant yeast strain expresses a truncated form of Uso1p,
the yeast homologue of p115 (39, 45). The mutant Uso1p lacks the
vesicle-binding domain (40, 76), and the thermosensitive phenotype can
be suppressed by the overexpression of SNAREs (77), suggesting that
SNAREs can bypass the function of Uso1p and mediate the docking of vesicles.
SNAP and NSF. The GTPase Rab5 is involved in
the fusion of early endosomes (78), and several proteins interacting
with its GTP-bound form have been identified. One of them is the
protein EEA1 (79). Early endosome fusion can be inhibited by GDI and
can be stimulated by EEA1 (80). An increased concentration of EEA1 can
suppress the inhibition by GDI. The partial inhibition of the
intra-Golgi transport assay can be due to an EEA1-like activity that
bypasses the Rab6 requirement. A dual requirement for Rab proteins
has been resolved for the fusion of yeast vacuoles. Antibodies against
the
SNAP homologue Sec17p and the NSF homologue Sec18p have been
compared with GDI and an antibody against the Rab homologue Ypt7p in a
kinetic analysis. Under the assay conditions, GDI and Ypt7p act
downstream of Sec17p and Sec18p (81). However, when the concentration
of Sec18p present in the assay is increased 50-fold over the amount
necessary for saturation (82), the sensitivity of the fusion reaction
toward GDI and anti-Ypt7p changes, and a contribution of Ypt7p at a
step preceding Sec17p and Sec18p could be demonstrated (83, 84). Biochemical and genetic studies of the endoplasmic reticulum-Golgi transport in yeast corroborate these observations. A genetic analysis of proteins involved in endoplasmic reticulum-Golgi transport revealed
an interaction between the Rab homologue YPT1 and the p115 homolgue
USO1 (77). In addition, high amounts of GDI remove Ypt1p and Uso1p from
membranes (85). On the other hand, transport vesicles containing
mutated Ypt1p remain fusion-competent, whereas acceptor membranes
carrying the mutant of Ypt1p are fusion-incompetent (86). Two roles for
Rab proteins and their associated proteins emerge in the yeast system.
One precedes the requirement for Sec17p and Sec18p and catalyzes the
tethering of vesicles to membranes. Another not yet defined function of
Rab proteins is accomplished after the ATP hydrolysis by Sec18p. In the
case of the mammalian Golgi apparatus, vesicles are tethered to the
cisternae by vesicle-bound giantin linked via p115 to cisternae-bound
GM130. Giantin and GM130 are members of the golgin protein family.
Golgins share a Rab6-binding domain that may be required for targeting
to the cisternae (87).
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ACKNOWLEDGEMENTS |
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We thank N. Pavletich for advice concerning the proteolytic digestions, H. Erdjument-Bromage for amino acid sequencing, E. Sztul for the plasmid encoding p115, S. Pfeffer for GDI, and R. Scheller for the expression clone encoding GST-VAMP. The technical assistance of W. Eng is acknowledged. We are grateful to C. Hughes, T. Engel, T. Harder, and J. Pieters for critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported by a postdoctoral fellowship from the Max Kade Foundation (to M. J. S. G.) and a postdoctoral fellowship from the Deutsche Forschungsgemeinschaft (to C. W.). The Basel Institute for Immunology was founded and is supported by F. Hoffmann-La Roche Ltd., Basel, Switzerland.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.: 41616051351; Fax: 41616051364; E-mail: wimmer@bii.ch.
Published, JBC Papers in Press, March 12, 2001, DOI 10.1074/jbc.M101513200
2 C. Wimmer, M. Amherdt, L. Orci, T. H. Söllner, and J. E. Rothman, manuscript in preparation.
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ABBREVIATIONS |
---|
The abbreviations used are:
SNARE, soluble NSF
attachment protein receptor;
v-SNARE, vesicle localized SNARE;
t-SNARE, target membrane localized SNARE;
SNAP, soluble NSF attachment protein;
NSF, N-ethylmaleimide sensitive factor;
GDI, GDP dissociation
inhibitor;
VAMP, vesicle associated membrane protein;
GST, glutathione
S-transferase;
K-Golgi, 1 M KCl-treated Golgi;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
PAGE, polyacrylamide gel electrophoresis.
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