From the Department of Molecular, Cellular, and
Developmental Biology, University of Michigan, Ann Arbor, Michigan
48109 and the § Department of Biology, Haverford College,
Haverford, Pennsylvania 19041
Received for publication, January 21, 2003
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ABSTRACT |
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The endoplasmic reticulum/Golgi SNARE
rbet1 cycles between the endoplasmic reticulum and Golgi and is
essential for cargo transport in the secretory pathway. Although the
quaternary SNARE complex containing rbet1 is known to function in
membrane fusion, the structural role of rbet1 is unclear. Furthermore,
the structural determinants for rbet1 targeting and its cyclical
itinerary have not been investigated. We utilized protein interaction
assays to demonstrate that the rbet1 SNARE motif plays a structural
role similar to the carboxyl-terminal helix of SNAP-25 in the synaptic SNARE complex and demonstrated the importance to SNARE complex assembly
of a conserved salt bridge between rbet1 and sec22b. We also examined
the potential role of the rbet1 SNARE motif and SNARE interactions in
rbet1 localization and dynamics. We found that, in contrast to what has
been observed for syntaxin 5, the rbet1 SNARE motif was essential for
proper targeting. To test whether SNARE interactions were important for
the targeting function of the SNARE motif, we used charge repulsion
mutations at the conserved salt bridge position that rendered rbet1
defective for binary, ternary, and quaternary SNARE interactions. We
found that heteromeric SNARE interactions are not required at any step
in rbet1 targeting or dynamics. Furthermore, the heteromeric state of
the SNARE motif does not influence its interaction with the COPI
coat or efficient recruitment onto transport vesicles. We conclude that
protein targeting is a completely independent function of the rbet1
SNARE motif, which is capable of distinct classes of protein interactions.
Soluble N-ethylmaleimide-sensitive factor attachment
protein receptor (SNARE)1
complexes bridge opposing membrane bilayers and appear to mediate specific membrane fusion in the endomembrane system (1, 2). Although
SNARE complexes appear to represent a universal membrane fusion
machine, their role in determining the specificity of intracellular membrane fusion is still being established (3, 4). Each SNARE complex
characterized to date appears to consist of a thermostable parallel
helix bundle composed of four heptad repeat-containing SNARE motifs
(5-9). Because the parallel SNARE motifs are anchored via
transmembrane domains continuous with the carboxyl end of the SNARE
motifs, the amino-to-carboxyl-terminal "zippering up" of the
vesicle (v-) SNARE motif with the target membrane (t-) SNAREs draws the
two membranes into close apposition and apparently drives lipid mixing
and fusion between the opposing bilayers (3).
Although most of the residues that interact in the core of SNARE helix
bundles are hydrophobic, one well conserved interior layer of polar
residues lies at the center of each bundle. Interestingly, most SNAREs
structurally related to syntaxin 1A and SNAP-25 contain a glutamine at
this conserved "zero layer" position (called "Q-SNAREs"), whereas SNAREs related to VAMP 2 contain an arginine at this position ("R-SNAREs") (10). Based upon protein profiling analysis, Q-SNAREs can be further subdivided into QA-SNAREs related to
syntaxin and QB- and QC-SNAREs related to the
amino- and carboxyl-terminal SNARE motifs of SNAP-25, respectively
(11). Notably, all of the characterized SNARE complexes and fusogenic
subsets of SNAREs in liposome fusion to date contain one each of the
QA-, QB-, QC-, and R-SNARE motifs.
Several studies have investigated the function of this conserved
central layer; although aspects of its composition are essential for
proper SNARE function, its precise role(s) in complex formation,
membrane fusion and/or SNARE recycling still remains unclear (reviewed
in Ref. 2).
In several systems it appeared that a reproducible pattern emerged
concerning the subunit organization of SNARE four-helix bundles. The
exocytic SNARE complex is well known to be comprised of a set of three
interacting Q-SNARE helices anchored on one membrane that form a
binding site for an R-SNARE helix anchored on an opposing membrane. In
the endosomal complex, now known in atomic detail, the positions
occupied by the amino- and carboxyl-terminal SNAP-25 helices in the
exocytic complex are held by the QB-SNARE vti1b and the
QC-SNARE syntaxin 8, respectively, whereas the R-SNARE VAMP
8 superimposes upon the VAMP 2 position, and syntaxin 7 is spatially
equivalent to syntaxin 1A (6, 7). Thus, this crystal structure
demonstrated that SNARE motif positions are highly superimposable between complexes and that the structural roles of the QA-,
QB-, QC-, and R-SNARE motifs are likely to be
conserved, at least between the synapse and endosomes. A similar
assumption was made about the ER/Golgi quaternary complex because the
R-SNARE sec22b bound strongly only to the combination of the
QA-, QB-, and QC-SNAREs syntaxin 5, membrin, and rbet1. Thus, sec22b appeared to play an analogous role to
VAMP 2 and was modeled to oppose the Q-SNAREs syntaxin 5, membrin, and
rbet1 in ER/Golgi membrane fusion events (9, 12). However, results from
in vitro liposome fusion with purified recombinant yeast
ER/Golgi SNAREs suggested a different model of the putative first
fusion event in the secretory pathway, because fusion did not occur
when Sec22p opposed Sed5p, Bos1p, and Betlp on liposomes (13). Instead,
a membrane fusion signal was generated only when Bet1p opposed Sec22p,
Bos1p, and Sed5p. This result was interpreted to mean that in
vivo, Bet1p is the V-SNARE that opposes a t-SNARE complex composed
of Sec22p, Bos1p, and Sed5p. According to this interpretation, the
spatial organization and membrane topology of structurally related
SNAREs in membrane fusion complexes is not conserved and instead must
be determined on a case-by-case basis.
Because of the specific intracellular localizations displayed by SNAREs
and because their specific localizations and interactions appear to
encode at least one layer of specificity in vesicle transport reactions
(3), there has been considerable interest in understanding the
intracellular targeting determinants within the SNAREs themselves. The
results have been surprisingly variable. In the simplest cases, the
subcellular localization information is apparently contained entirely
within the transmembrane domain and may even be a simple function of
transmembrane domain length. This is the case for the plasma membrane
SNAREs syntaxin 3 and 4 as well as for the ER/Golgi syntaxin 5, whose
transmembrane domains are sufficient for proper localization (14-16).
Syntaxin 5 may also contain additional targeting information within its cytoplasmic domain, although results differ. On the other hand, syntaxin 6, which follows a complex itinerary that includes
steady-state localization to the trans-Golgi network
and endosomes (17) and undergoes constitutive cycling to the plasma
membrane, appears to be targeted by two distinct determinants within
its cytoplasmic domain (18), one within the SNARE motif and the other
within its amino-terminal domain, now known to represent a three-helix bundle (19). At least two other syntaxin family members also contain
essential localization determinants within their cytoplasmic domains
(16). Although it was speculated that the targeting via these
determinants was accomplished by protein interactions, the binding
partners required for localization were not identified. Similarly, the
SNARE motif was required for efficient VAMP 2 recruitment onto synaptic
vesicles (20). In this case, mutagenesis revealed that the capacity for
synaptic SNARE interactions did not correlate with targeting to
synaptic vesicles, implicating other potential SNARE motif binding
partners for targeting (20, 21). Very recently, the mammalian R-SNARE
ykt6 was demonstrated to be targeted to a very specific yet undescribed
vesicular localization by virtue of its profilin-like amino-terminal
domain, completely independently of its SNARE motif or hydrophobic
anchor in membranes (22). Thus, as of yet SNARE targeting does not
appear to follow a clear set of rules and may instead utilize several
distinct classes of protein and/or membrane interactions.
One question that has not been explored is whether SNAREs are
trafficked individually or as complexes. This would be particularly relevant to t-SNAREs that undergo constitutive cycling, for example within the Golgi, between the ER and Golgi or between the Golgi and
endosomes. Some cycling SNAREs may contain their own intrinsic targeting signals, whereas others could depend upon interactions with
these SNAREs for their targeting. If Q-SNARE complexes are indeed an
important intermediate in SNARE complex assembly, then one might expect
these complexes to be efficiently recruited onto newly forming
vesicles. On the other hand, one might expect used or aberrant SNARE
complexes to be excluded from newly forming vesicles.
Targeting determinants of the dynamically localized ER/Golgi
QC-SNARE Bet1p and its rat ortholog rbet1 have not been
defined. rbet1 is a small SNARE consisting of a carboxyl-terminal
transmembrane domain attached to a 95-amino acid cytoplasmic domain
with no predicted structure other than the SNARE motif of residues
27-90. In mammals, rbet1 displays a steady-state localization to
VTCs and early Golgi and undergoes rapid constitutive cycling
between the ER and Golgi (23). Direct interactions between the yeast Bet1p SNARE motif and the COPI and COPII coat machinery have been described (24, 25). Furthermore, interactions of Bos1p and Sec22p with
COPII components were sufficient to produce a 3-4-fold enrichment of
the SNAREs in COPII vesicles budded from chemically defined liposomes
(26). Thus, interactions between the rbet1 SNARE motif and COP
coats could be important for its steady-state localization and for its
constitutive ER/Golgi cycling. On the other hand, correct recruitment
to budding vesicles may be more complex in vivo than simple
interactions with coats; for example, it may also involve determinants
that localize the protein to vesicle budding sites or membrane domains.
This may be the case for the erv41p-erv46p complex, another rapidly
cycling vesicle membrane protein (27). Recent findings in yeast
indicated that ARF-GAP bound transiently to the Bet1p SNARE
motif in vitro and induced a conformational state required
for its interaction with COPI machinery (25). Because the known
conformational states for SNARE motifs are unstructured coils and
helical bundles, one suggestion was that ARF-GAP may facilitate Bet1p
hetero- or homo-oligomeric bundling and that oligomeric bundles are the
preferred substrate for coat binding and uptake into vesicles. These
speculations highlight our lack of information about the role of the
SNARE motif and SNARE protein interactions in the targeting, dynamics, and life cycle of the SNAREs themselves.
Using purified SNAREs and simple protein interaction assays and
mutagenesis, we have further investigated the organization of the
ER/Golgi quaternary complex in vitro. In contrast to results with liposome fusion, our results demonstrate that membrin and rbet1
play the structural roles of t-SNAREs, with membrin being spatially
analogous to the amino-terminal helix of SNAP-25 and rbet1 occupying a
position very similar to the carboxyl-terminal helix of SNAP-25. In
addition, we demonstrated the importance to SNARE complex assembly of a
conserved salt bridge on the surfaces of the synaptic and ER/Golgi
SNARE complexes. In a second set of experiments, we examined the
potential role of the rbet1 SNARE motif and SNARE interactions in rbet1
localization and dynamics. We found that, in contrast to what has been
observed for its binding partner syntaxin 5, the rbet1 SNARE motif was
absolutely essential for proper targeting. For a clean test of the role
of SNARE interactions in rbet1 targeting or dynamics, we created charge
repulsion mutant rbet1 K47D at the conserved salt bridge position that
rendered rbet1 defective for heteromeric SNARE interactions in
vivo. Interestingly, the steady-state localization of rbet1 K47D
was indistinguishable from wild type. Furthermore, the intracellular
dynamics of the mutant, its interactions with the COPI machinery, and
its recruitment to coated vesicles were all indistinguishable from wild
type. We conclude that the SNARE motif but not SNARE interactions is essential for the targeting and dynamics of rbet1.
Antibodies--
Monoclonal and affinity-purified polyclonal
anti-SNARE antibodies were described previously (23, 28). An
anti-hexahistidine monoclonal antibody was obtained from Sigma. Expression and Purification of ER/Golgi
SNAREs--
The following bacterially expressed protein constructs
were either described previously (9) or created for this study. GST-membrin encoded essentially the full-length protein (amino acids
2-212) including the transmembrane domain and was mutated at residue
139 with a QuikChange kit from Stratagene. GST-syntaxin 5 included the
entire cytoplasmic domain of the smaller 34-kDa syntaxin 5 isoform
(residues 55-333 of the entire syntaxin 5 open reading frame);
however, we used a natural internal thrombin cleavage site within this
construct (9) to produce a fragment containing just the SNARE motif,
residues 252-333 for binding studies. GST-rbet1 encoded the entire
cytoplasmic domain (residues 1-95) and was mutated using the
QuikChange kit to produce the rbet1 mutations employed in the
experiments. GST-SNAP-25 encoded either the entire protein (residues
1-206), the amino-terminal SNARE motif (residues 1-93), or the
carboxyl-terminal SNARE motif (residues 120-206). We also employed
three hexahistidine-tagged constructs: mouse sec22b cytoplasmic domain
(residues 2-196), yeast Sec22p cytoplasmic domain (residues 2-193),
and rbet1 cytoplasmic domain (residues 2-95). The mouse sec22b
construct was mutagenized with the QuikChange kit as indicated in the
text. All of the constructs and mutants were verified by sequencing at
the University of Michigan DNA sequencing core.
For binding studies, the SNARE proteins were purified as described
earlier (9). In short, Escherichia coli cultures were resuspended in French press buffer (50 mM Tris, pH 8.0, 0.1 M NaCl, 1 mM EDTA, 0.05% Tween 20, 1 mM dithiothreitol, 2 µg/ml leupeptin, 4 µg/ml
aprotinin, 1 µg/ml pepstatin A, and 1 mM
phenylmethylsulfonyl fluoride) (for His6-containing
constructs, we used the same medium without dithiothreitol), lysed by
French press, and then centrifuged at 20,000 × g for
20 min, and the supernatant (S1) was recovered. For
His6-sec22b, GST, GST-rbet1, and GST-SNAP-25 constructs,
the S1 was immediately centrifuged at 100,000 × g for
45 min, and the supernatant was collected (S2). For GST-syntaxin 5 (55- 333), the S1 was adjusted to 0.35% sodium sarkosyl, mixed gently for 30 min, and then supplemented with 1% Triton X-100 and 10% glycerol. After centrifugation at 100,000 × g for 45 min, the
supernatant was collected. For GST-membrin, the S1 was centrifuged at
100,000 × g for 45 min, the supernatant (S2) was
discarded, and the pellet (P2) was homogenized in the original volume
of French press buffer. This was treated with sarkosyl, glycerol, and
Triton X-100 and centrifuged as above, and the 100,000 × g supernatant was retained. All of the final 100,000 × g supernatants were then purified on columns of either
glutathione-Sepharose (GST constructs) or
Ni2+-nitrilotriacetic acid-agarose (His6
constructs). Purified fractions eluted with glutathione were cleaved
with thrombin in the case of GST fusion proteins. The purified, cleaved
proteins were dialyzed into Buffer A (20 mM Hepes, pH 7.2, 0.15 M KCL, 2 mM EDTA, 5% glycerol),
supplemented with protease inhibitors, and stored at
rbet1 employed for analytical ultracentrifugation (AU) experiments was
purified by more rigorous procedures. The first stages of purification
were identical to above. However, after elution from
glutathione-Sepharose, GST-rbet1 fusion proteins were further purified
by anion exchange chromatography on MonoQ (Amersham Biosciences). After
elution with a continuous KCl gradient, the peak of the desired protein
was pooled and cleaved with thrombin, diluted 2-fold with low salt
buffer, and passed over a column of Q-Sepharose to retain GST and
residual fusion protein. The flow-through from the Q-Sepharose,
containing cleaved rbet1, was concentrated using a YM-3 membrane on an
Amicon stirred cell concentrator and then further purified by velocity
gradient fractionation as described earlier (9). The final homogeneous
rbet1 was dialyzed into 20 mM Tris, pH 8.0, for AU analysis.
Binding Assays--
All of the binding incubations were
conducted in buffer A containing 0.1% Triton X-100. For solution
binding assays, 300-µl reactions containing ~2 µM of
each protein were incubated for varying time periods (see each figure
legend) on ice, 250 µl of which was injected onto a 24-ml Superdex
200 gel filtration column (Amersham Biosciences) run in Buffer A
containing 0.1% Triton X-100 and 30 µg/ml BSA. Individual column
fractions were either analyzed directly by SDS-PAGE and Western
blotting or were precipitated with acetone prior to gel and Western analysis.
For the binary and ternary bead binding assays, a typical binding
reaction consisted of 20 µl of 20 mg/ml BSA, 20 µl of 50% glutathione-Sepharose beads containing ~124 pmol (620 nM
final concentration in binding reaction) of the immobilized protein, and varying amounts of the soluble binding partners in a final volume
of 200 µl of Buffer A plus 0.1% Triton X-100. The binding reactions
were incubated for 1 h at 4 °C with constant agitation, and
then the beads were washed three times with Buffer A containing 0.1%
BSA in the case of binary binding reactions and Buffer A plus 0.1%
Triton X -100 in the case of ternary bead binding assays. The beads
were resuspended in denaturing sample buffer and analyzed by SDS-PAGE
followed by Western blotting, scanning, and quantitation.
Immunofluorescence Microscopy--
NRK cells seeded on
coverslips were transfected with wild type Myc-rbet1 or Myc-rbet1 K47D
using LipofectAMINE 2000 reagent (Invitrogen). In some experiments the
cells were co-transfected with a commercial CFP-tagged
galactosyltransferase localization domain construct (Living Colors
CFP-Golgi from Clontech). For brefeldin A
treatment, the cells were incubated for 60 min in growth medium
containing 10 µg/ml of brefeldin A (Calbiochem). To allow the
BFA-treated cells to recover from treatment, the BFA containing medium
was removed, and the cells were washed twice with non-BFA containing
growth medium and then incubated with fresh growth medium for the
indicated amounts of time. For the low temperature treatment, the cells
were incubated at 15 °C in precooled growth medium for the indicated
amounts of time. To fix cells for microscopy, the growth medium was
removed, and the cells were incubated with 2% paraformaldehyde in 0.1 M sodium phosphate, pH 7.0, followed by quenching twice for
10 min each with 0.1 M glycine in phosphate-buffered
saline. The cells were permeabilized by incubation with
permeabilization solution (0.4% saponin, 1% BSA, and 2% normal goat
serum in phosphate-buffered saline) for 15 min. They were then
incubated with permeabilization solution containing the primary
antibody for 1 h at room temperature and washed three times with
permeabilization solution, and the purified fluorescently labeled
secondary antibody (Jackson Immunoresearch) in permeabilization
solution was added to the cells for 30 min. The cells were finally
washed three times with permeabilization solution and mounted in
Vectashield medium (Vector Labs) on glass slides. Microscopy was
conducted on a Nikon E800 epifluorescence microscope with Texas Red and
fluorescein filter sets, a Hamamatsu Orca II camera, and Improvision
Openlab software.
Immunoprecipitation Experiments--
NRK cells were transfected
with either wild type Myc-rbet1 or Myc-rbet1 K47D constructs.
Twenty-four hours after transfection, the cells were washed twice with
cold phosphate-buffered saline and solubilized in KCl Buffer (20 mM Hepes, pH 7.2, 0.1 M KCl, 2 mM
EGTA, 2 mM EDTA) containing 1.0% Triton X-100, 1 mM dithiothreitol, 2 µg/ml leupeptin, 4 µg/ml
aprotinin, 1 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl
fluoride, 5 mM MgCl2, and 1 mM
GTP In Vitro Vesicle Generation--
Rat liver cytosol was prepared
by homogenization of a fresh liver in four volumes of 25/125 (25 mM Hepes, 125 mM potassium acetate, pH 7.2)
containing 1 mM dithiothreitol, 1 mM
4-(2-aminoethyl)benzenesulfonyl fluoride, 2 µg/ml leupeptin, 4 µg/ml aprotinin, and 2 µg/ml pepstatin, using a Potter-Elvejem
homogenizer in a drill press. After an initial centrifugation at
20,000 × g for 20 min, the supernatant was further
centrifuged at 100,000 × g for 1 h. The top lipid layer was discarded, and the remaining supernatant was desalted into
25/125 using Sephadex G-25 or dialyzed against 25/125 for 4 h at
4 °C. Cytosol aliquots were snap frozen and stored at Analytical Ultracentrifugation--
The experiments were
performed at 4 °C with a Beckman Optima XL-A analytical
ultracentrifuge equipped with 12-mm path length, six-channel,
charcoal-filled Epon cells and quartz windows, using rotor speeds of
30,000, 35,000, and 40,000 rpm. rbet1 was dialyzed into 20 mM Tris, pH 8.0; the loading concentration of rbet1 was 1 µM. Partial specific volume and solution density were
calculated using the program Sednterp (29). The data were globally fit with a single-species model with the molecular mass treated as a
fitting parameter. The WinNonLin (V1.060) program from the Analytical Ultracentrifugation Facility at the University of Connecticut (Storrs,
CT) was used for the fitting analysis.
Membrin and rbet1 Play Structural Roles Similar to the First and
Second Helices of SNAP-25, Respectively--
The purified recombinant
ER/Golgi SNAREs syntaxin 5, membrin, rbet1, and sec22b appeared to form
a quaternary complex with each protein contributing a single SNARE
motif (9). We hypothesized that the ER/Golgi complex formed a
four-helix bundle with similar structural and regulatory features to
the synaptic SNARE complex (5). Because the synaptic and late endosomal
four-helix bundles display virtually superimposable structures with the
corresponding SNARE motifs in each family (QA,
QB, QC, and R) occupying the same positions in
the two complexes, it seemed likely that the ER/Golgi quaternary
complex would also display this conserved organization. However, a
liposome fusion assay employing yeast ER/Golgi SNAREs indicated a
radically different arrangement, with the R-SNARE Sec22p acting as part
of a t-SNARE complex and the QC-SNARE Bet1p opposing the
t-SNARE in the v-SNARE position (13). We wondered whether these results
were indicative of a true difference in the structural roles of the
four classes of SNARE motifs in the ER/Golgi SNARE complex. To address
this we performed substitution experiments using purified proteins to
determine whether SNARE motifs from the synaptic complex could assemble
with ER/Golgi SNAREs and, if so, which helices in the two complexes
would be equivalent. We first asked whether SNAP-25 could replace any
of the Q-SNAREs syntaxin 5, membrin, or rbet1 in the formation of a
hybrid ER/Golgi SNARE complex. We examined the formation of complexes
in solution using gel filtration to resolve high molecular mass
complexes from monomers. Under these conditions, sec22b can only
participate in the formation of a quaternary complex and does not
exhibit binary or ternary interactions with ER/Golgi Q-SNAREs (9).
Therefore, we used the appearance of sec22b in the high molecular mass
fractions as an indicator of the formation of a four-helical complex.
As shown in Fig. 1A, SNAP-25
can substitute for both membrin and rbet1 (top two panels).
The high molecular mass complex(es) containing SNAP-25, syntaxin 5, and
sec22b appear to involve all three proteins because in control
reactions omitting SNAP-25 or syntaxin 5, sec22b eluted in the
monomeric range (Fig. 1A, third and fourth
panels). Importantly, SNAP-25 would not substitute for other
pairwise combinations, including syntaxin 5/membrin and syntaxin
5/rbet1 (Fig. 1A, bottom two panels). Note that
the gel filtration size under these conditions does not accurately predict the number of molecules in a SNARE complex, because the complexes contain an unknown quantity of bound Triton X-100, and the
aggregation state of SNARE complexes is not well understood. We do not
know why the hybrid SNAP-25 complex displays a biphasic distribution
(Fig. 1A, second panel) but speculate that the
complex may be present in two distinct aggregation states. This could be due to the two SNAP-25 SNARE motifs participating in different individual SNARE complexes (30). In summary, the results of Fig.
1A suggest that there is a high degree of structural
conservation between the two complexes and that membrin and rbet1
occupy positions in the ER/Golgi complex that approximate the positions
of SNAP-25 in the synaptic complex.
We next sought to determine which of the Q-SNAREs, membrin and rbet1,
mimics the first and which the second of the two SNAP-25 SNARE motifs
(referred to as SNAP-25N and SNAP-25C, respectively). Based upon
protein profiling techniques, membrin appears more closely related to
SNAP-25N, and rbet1 appears more closely related to SNAP-25C (11).
Indeed, as shown in Fig. 1B (top panel), a SNAP-25N fragment encoding residues 1-93 appears to substitute for
membrin in the quaternary complex, the formation of which is reflected
by the appearance of sec22b in the high molecular mass fractions. This
shift is not observed in the reaction containing syntaxin 5, sec22b,
membrin, and SNAP-25N, indicating that only membrin, and not rbet1, can
be "replaced" by SNAP-25N (Fig. 1B, second
panel). These data indicate that the structural role of membrin in
the ER/Golgi quaternary complex may be similar to that of the first
SNAP-25 helix in the synaptic SNARE complex. Curiously, the hybrid
complex containing syntaxin 5, SNAP-25N, rbet1, and sec22b had a gel
filtration size significantly smaller than the native ER/Golgi complex
(~100 kDa versus ~300 kDa; compare fraction numbers
between Fig. 1A, top panel, and Fig.
1B, top panel); this could be due to an altered
oligomerization state of this complex. However, the experiment in Fig.
1B establishes that the hybrid complex indeed contains all
four of the participant proteins, because removing rbet1 or syntaxin 5 from the incubations abolishes the high molecular mass
sec22b-containing complexes (Fig. 1B, bottom two
panels) and sec22b does not associate with syntaxin 5 and rbet1 on
its own (9). Unfortunately, we could not obtain clean substitution data
for the second SNAP-25 helical domain using this technique, because the
SNAP-25C construct displayed binary interactions with sec22b (data not
shown). In summary, the SNAP-25 and SNAP-25N substitutions indicate
that in the ER/Golgi quaternary complex, the membrin position is
similar to that of SNAP-25N and that rbet1 therefore most likely
resembles SNAP-25C.
It is important to note that although equimolar concentrations of
SNAP-25 constructs, membrin, rbet1, and sec22b were used in the
experiments, there was a substantially lower yield of the hybrid
complexes than the native ER/Golgi complex. In addition, overnight
incubations were required to produce detectable hybrid complex, whereas
the ER/Golgi complex is readily detectable in 1-2 h (not shown).
Hence, although the experiment suggests structural relatedness, it is
also consistent with some limited specificity between cognate
SNAREs in solution.
We next tested whether rbet1 could behave like a v-SNARE in its binding
characteristics. If rbet1 could behave like a v-SNARE, it might be
expected to form a complex with syntaxin 5 and the two SNAP-25 helices,
which by definition play the role of t-SNARE. Put differently, SNAP-25
would be expected to substitute for membrin and sec22b. As shown in
Fig. 1C, no detectable high molecular mass rbet1-containing
complex is formed from rbet1, SNAP-25 and syntaxin 5 (bottom
panel), but an rbet1-containing complex is readily detectable in a
reaction involving all four ER/Golgi proteins (top panel).
This experiment is consistent with the hypothesis that in the ER/Golgi
complex, rbet1 occupies the position of a SNAP-25 helix and therefore
cannot co-exist in a hybrid complex with SNAP-25. On the other hand,
sec22b readily binds to the hybrid syntaxin 5-SNAP-25 t-SNARE (Fig.
1A).
We also considered the possibility that the yeast ER/Golgi SNAREs
simply had very different binding characteristics from their mammalian
counterparts. For example, perhaps yeast Sec22p is part of a t-SNARE
complex, whereas mammalian sec22b acts in a v-SNARE mode. We tested
this possibility by preparing purified yeast Sec22p and asking whether
it behaved differently from the mammalian sec22b in binding reactions
with the mammalian Q-SNAREs syntaxin 5, membrin, and rbet1. As shown in
Fig. 1D, yeast Sec22p, just like sec22b, formed a quaternary
complex with the three Q-SNAREs (top panel). Furthermore,
like sec22b, yeast Sec22p interacted only with the combination of
syntaxin 5, membrin, and rbet1 and not with any other combination (Fig.
1D, second, third, and fourth
panels). The fact that yeast Sec22p behaves indistinguishably from
sec22b and forms a high affinity quaternary complex with syntaxin 5, rbet1, and membrin and did not form detectable t-SNARE complexes with
any subset of them is inconsistent with this yeast protein playing a
fundamentally different structural role in SNARE complexes. For
example, if Sec22p were a syntaxin light chain as suggested (13), then
it would not have been compatible with membrin or rbet1, both of which
appear to play that role (Fig. 1, A and B). To
the contrary, yeast Sec22p was entirely dependent upon the presence of
both of those proteins for a stable interaction.
Conserved Interchain Interactions between rbet1 and
sec22b--
Based upon the above substitution experiments, our working
hypothesis is that the four ER/Golgi SNARE motifs are arranged nearly
superimposably over the synaptic core complex, using these chain
correspondences: syntaxin 5 and syntaxin 1A; membrin and SNAP-25N;
rbet1 and SNAP-25C; and sec22b and VAMP 2. If corresponding chains do
indeed occupy the same space in the two complexes, then specific
interchain interactions, for example the salt bridges between the
SNAP-25C helix and VAMP, may be conserved in the ER/Golgi complex. In
the synaptic complex, there are ionic interactions on the surface of
the bundle between SNAP-25 Arg61 and VAMP 2 Glu41 and between SNAP-25 Asp86 and VAMP 2 Arg66 (5). If rbet1 and sec22b are aligned with these
chains using the zero layer Q and R to determine the location of the
layers and a superimposable backbone structure is assumed, then both salt bridges would be potentially conserved, with rbet1
Lys47 interacting with sec22b Asp144 and rbet1
Asp72 in contact with sec22b Lys169 (an
alignment is shown for reference in Fig.
2A). Based upon the protein
profiling analysis structural categorization of SNARE motifs (11), the
vast majority of SNARE complexes would have conserved salt bridges at
those positions. On the other hand, if rbet1 were modeled in the
structural role of VAMP 2, as suggested by the liposome fusion studies,
then ionic residues would not be present at those positions.
To further explore and confirm the organization of the ER/Golgi
quaternary complex, we mutated an rbet1 residue, Lys47,
that potentially interacts with sec22b Asp72 on the surface
of the bundle. We produced two mutations, K47E and K47D, to potentially
create ionic repulsion with sec22b Asp72 and tested their
effects on quaternary complex formation relative to wild type proteins
under identical conditions. As seen in Fig. 2B
(first, second, and third panels),
K47E resulted in 49% less complex formation compared with wild type,
whereas surprisingly, K47D was completely defective in quaternary SNARE
complexes. One likely explanation for the dramatic difference between
the two mutations was that because of the highly localized charge on
aspartic acid, the K47D mutation caused intolerable charge repulsion,
whereas the longer and more flexible glutamic acid side chain was able to avoid this conflict. Another possible interpretation was that K47D
would dramatically lower the intrinsic helicity of that region of the
SNARE motif. To address this possibility we created two more mutations;
K47N is the most similar possible mutation to K47D with respect to side
chain size and shape. K47G tests the inherent susceptibility of the
47th residue to helix disrupting residues. As seen in Fig.
2B (fourth and fifth panels), these
mutations reduced SNARE complex formation by 31 and 56%, respectively,
indicating that gross helicity affects are unlikely to account for the
potency of the K47D mutation. Three additional arguments against purely
helicity affects are: 1) K47D has a relatively mild effect on
rbet1-syntaxin 5 binary interactions (Fig.
3A); 2) Specific
counter-mutations display mutual complimentarity with rbet1 K47D and
can partially rescue the mutation (Fig.
4); and 3) SNARE motifs are inherently
adaptable helical domains that have been shown to adapt well to local
disruptions in structure (31). In summary, we interpret Fig. 2 to
indicate that a likely ionic interaction between rbet1
Lys47 and sec22b Asp144 contributes
significantly to the assembly of the quaternary complex, because all
mutations that would lack the ionic interaction are reduced relative to
wild type. Our results do not distinguish between effects on kinetics
of assembly as opposed to thermal stability of the complex. Secondly,
K47D causes an especially dramatic effect perhaps because of localized
charge repulsion between the rbet1 and sec22b SNARE motifs. As outlined
below, the severity of the mutation probably in part results from
repulsions that take place on intermediates in the assembly process
rather than simply between the rbet1 47 and sec22b 144 positions.
rbet1 K47D Is Defective at All Levels of Heteromeric SNARE Complex
Assembly--
If the potent effects of rbet1 K47D on quaternary
complex formation (Fig. 2B) were simply an effect of charge
repulsion between rbet1 and sec22b, then we would not expect the K47D
mutation to affect binary and ternary interactions of rbet1. However,
as illustrated in Fig. 3B, binary interactions between
bead-immobilized GST-membrin and soluble rbet1 K47D were severely
compromised, with no binary interaction detected until soluble rbet1
concentrations exceeded 2 µM. Interestingly, the other
easily detectable binary interaction that rbet1 undergoes in bead
binding studies, that between bead-immobilized GST-rbet1 and soluble
syntaxin 5 (9), displayed a much more mild defect of the mutation,
showing an ~40% reduction in binding in the 0.5-1.5
µM syntaxin 5 concentration range (Fig. 3A).
Syntaxin 5, membrin, and rbet1 are known to form a stable ternary
complex that can be detected easily by bead binding and gel filtration analysis (9). Using bead-immobilized GST-membrin and adding soluble
rbet1 to potentiate the binding of soluble syntaxin 5, we found that
rbet1 K47D was essentially entirely defective in formation of ternary
complexes (Fig. 3C). Thus, rbet1 K47D caused a severe
disruption in a subset of rbet1 binary interactions as well as rbet1
ternary and quaternary SNARE interactions.
Why would the K47D mutant affect lower order SNARE complex formation?
One possibility would be that the lower order complexes have a
fundamentally different organization than the quaternary complex and
that the aspartate could conflict with another residue besides sec22b
Asp144. From the alignment in Fig. 2A it is
apparent that membrin features an aspartic acid at position 139, which,
assuming backbone superimposability, could conflict with K47D if
membrin were to occupy the sec22b space in a four-helix bundle.
Biophysical studies have demonstrated that most SNARE helical bundles
are four-helix bundles, even lower order SNARE complexes that are not
sufficient for membrane fusion. For example, the synaptic t-SNARE
complex is a parallel four-helix bundle with two copies of syntaxin and
one of SNAP-25 (32). In addition, the binary complex between syntaxin
1A and the amino-terminal helix of SNAP-25 is also a four-helix bundle
containing two copies of each member (33). Thus, it is not unlikely
that the binary and ternary complexes we observe with ER/Golgi Q-SNAREs
could have a very different subunit organization from the ER/Golgi
quaternary complex.
To test the possibilities that a charge conflict between K47D and
sec22b Asp144 may be at the heart of the effects on
quaternary complex formation and that charge repulsion between rbet1
K47D and membrin Asp139 was involved in the effects on
lower order complex formation with rbet1 K47D, we created sec22b D144K
and membrin D139K mutations and tested these proteins in a series of
binding reactions in Fig. 4. For this experiment, binding reactions
were analyzed by gel filtration, and rbet1, rather than sec22b, was
used as the tracer to indicate complex formation. We quantified only
the high molecular mass gel filtration fractions rather than analyzing each entire gel filtration profile. As shown in Fig. 4A, a
strong high molecular mass rbet1 signal is produced by binary, ternary, and quaternary binding reactions using wild type rbet1. Note that a
limitation of this experiment is that we cannot distinguish, for
example in the quaternary mixture, how much of the high molecular mass
rbet1 is present as a quaternary complex as opposed to ternary and
binary species. However, no rbet1 signal is detected when rbet1 alone
is analyzed or in a binary mixture between rbet1 and membrin D139K,
indicating that membrin D139K is completely nonfunctional for binding
to wild type rbet1. We then examined a variety of binding reactions
utilizing rbet1 K47D and tested the ability of membrin D139K and sec22b
D144K to rescue the different levels of complex formation. As shown in
Fig. 4B, binary, ternary, and quaternary SNARE interactions
with wild type membrin and sec22b resulted in little high molecular
mass rbet1 K47D signal (lanes 6-8), consistent with the
experiments in Figs. 2 and 3. However, membrin D139K was able to
partially restore a significant signal in binary and ternary
incubations with rbet1 K47D (lane 6 versus lane 11 and
lane 7 versus lane 12). These results demonstrate mutual
complementarity between rbet1 K47D and membrin D139K (lanes 3 and 6 versus lane 11) and are
consistent with a conflict between rbet1 K47D and membrin
Asp139 being at least partially responsible for the
unexpected effects of that mutation on lower order complexes. This is
also consistent with our suggestion that in binary and ternary
complexes, membrin might occupy a position similar to that of VAMP in
the synaptic complex. One possibility is that in these complexes, there
are two copies of membrin, one in the QB position and
another in the R position. Unexpectedly, it also appeared that membrin
D139K contributed to quaternary complex formation (lane 12 versus
lane 13); however, the rbet1 signal in lane 13 is
presumably a mixture of binary, ternary, and quaternary complexes,
making it difficult to isolate the effects on the different complexes.
On the other hand, as shown in lane 8 versus lane 9, sec22b
D144K partially rescued quaternary complex formation. Because the only
sec22b interactions detectable by gel filtration are quaternary (9) (also true for yeast Sec22p in Fig. 1D of this manuscript),
it is safe in this case to assume that most of the rbet1 signal in lane 9 represents quaternary complexes. Rescue of quaternary
complex by sec22b D144K was also detectable using sec22b as the tracer in gel filtration (data not shown). In conclusion, membrin D139K partially restores lower order complex formation to rbet1 K47D, consistent with membrin occupying the R position in those complexes, and sec22b D144K partially restores quaternary complex formation to
rbet1 K47D, consistent with sec22b occupying the R position in that
complex. The finding that membrin D139K may restore some quaternary
complex to rbet1 K47D could indicate that lower order complexes may
nucleate or facilitate formation of the quaternary complex. Because we
used relatively short incubation times for these experiments, we cannot
distinguish effects of the different mutations on the kinetics of
assembly from effects on the thermodynamic stability of the final
product. Lane 14 illustrates that the combination of membrin
D139K and sec22b D144K restores quaternary complex formation to rbet1
K47D by over 40%. All of the effects of the mutations we examined are
consistent with our hypothesis about the arrangement of the ER/Golgi
quaternary complex and inconsistent with the proposal that rbet1 plays
the canonical v-SNARE role in that complex.
Determinants for rbet1 SNARE Interactions and Vesicle Coat
Interactions Differ in Vivo--
The yeast Bet1p SNARE motif is
required for its interactions with both the COPII and COPI coat systems
(24, 25). The dramatic effects of the rbet1 K47D mutation gave us the
opportunity to examine the relationship between rbet1 SNARE
interactions and other protein interactions in which the SNARE motif
participates. To examine the effects of the K47D mutation on various
protein interactions, we transfected NRK cells with either wild type
Myc-rbet1 or Myc-rbet1 K47D and then performed immunoprecipitation
experiments from detergent extracts of the cells. As shown in Fig.
5, syntaxin 5 immunoprecipitation
resulted in membrin co-precipitation in either lysate but only
Myc-rbet1 wild type co-precipitated with syntaxin 5 to a significant
extent. Interactions between syntaxin 5 and Myc-rbet1 K47D were reduced
by 91% relative to wild type. Because Myc-rbet1 and Myc-rbet1 K47D
were expressed at equivalent levels, this indicated that the K47D
mutation was equally disruptive of SNARE interactions in
vivo as it was in vitro with recombinant proteins. In
reciprocal immunoprecipitations with anti-Myc antibodies, syntaxin 5 was efficiently immunoprecipitated in the Myc-rbet1 lysates and reduced
by 93% in the Myc-rbet1 K47D lysate, again consistent with the
dramatic effects of this mutation in in vitro binding
studies. In stark contrast, immunoprecipitations with anti- rbet1 SNARE Interactions and Proper Intracellular Targeting Are
Independent Functions of the SNARE Motif--
An important question
that has not been resolved involves the relationship between SNARE
interactions and SNARE targeting in the secretory pathway. For example,
does a t-SNARE complex get targeted to its vesicles of function, or
does it form there from individually trafficked SNAREs? Because rbet1
appeared to play the role of QC-SNARE in a putative t-SNARE
complex, because rbet1 is known to undergo rapid constitutive cycling
between the ER and Golgi, and because we had powerful and specific
mutations that affected its ability to form SNARE complexes, we had a
good opportunity to examine whether SNARE interactions are important for any aspect of proper rbet1 targeting. First, we wanted to establish
whether the rbet1 SNARE motif itself was even important for rbet1
targeting. As demonstrated in Fig. 6A in
the top row, expression of Myc-rbet1 in NRK cells resulted
in a juxtanuclear, Golgi-like concentration of staining similar to the
cis-Golgi marker GM130 but with a significant number of
peripheral spots representing VTCs and/or ER exit sites (23, 34). This
distribution of recombinant protein was similar to the endogenous rbet1
(Fig. 6A, third row), except that the balance
between Golgi area staining and peripheral spots is noticeably shifted
toward Golgi in the recombinant case, perhaps because of higher
expression levels. On the other hand, when the rbet1 cytoplasmic domain
was removed and replaced with GFP, the hybrid construct, GFP-rbet1TM,
did not localize properly. As shown in Fig. 6B (left
panel), GFP-rbet1TM was mislocalized primarily to ER tubules,
demonstrating that unlike syntaxin 5 and several others, the rbet1
transmembrane domain is not sufficient for proper targeting and that
essential targeting information resides in the cytoplasmic domain.
Unlike many SNAREs, rbet1 lacks an independent amino-terminal domain
and contains only an ~25 residue amino-terminal peptide prior to the
SNARE motif. Removal of the first 25 amino acids of the rbet1
cytoplasmic domain did not significantly perturb rbet1 localization
(Fig. 6B, right panel), indicating that like
syntaxin 6, another QC-SNARE with a cyclical itinerary,
essential targeting determinants reside within the SNARE motif itself
(18, 19).
We next compared the subcellular distributions of wild type Myc-rbet1
and Myc-rbet1 K47D in transfected NRK cells. As seen in Fig.
6A (top two rows), the steady-state distribution
of the mutant was indistinguishable from the wild type construct,
indicating that SNARE interactions do not influence steady-state
targeting of rbet1. However, we also wanted to determine whether, like
endogenous rbet1, the mutant construct was able to engage in identical
dynamics and constitutive cycling between the ER and Golgi. We
investigated the dynamics of the recombinant constructs using
experimental perturbations that preferentially affect one leg of the
recycling pathway. As shown in Fig.
7A (second and
third rows), extensive brefeldin A treatment of NRK cells
resulted in dispersion of both Myc-rbet1 constructs into large,
punctate, "frustrated" ER exit sites or VTCs (35-37). This is in
contrast to typical Golgi resident proteins, which are diluted into a
fine, reticular, ER pattern by this treatment (not shown), and
trans-Golgi network proteins, such as syntaxin 6 (Fig.
6A, top row), which are barely affected by BFA.
The concentration of the rbet1 constructs in the ER exit structures is
indicative of strong determinants for a cyclical vesicle localization,
as opposed to a static localization. When brefeldin A was removed from
the cells, both constructs transitioned with equal time courses out of
ER exit sites to the central juxtanuclear localization seen at steady
state. We interpret the BFA results to indicate that heteromeric SNARE
interactions have no impact on the strong forward recruitment of rbet1
from the ER to Golgi. We also incubated transfected NRK cells at
15 °C, a treatment known to trap rapidly recycling proteins in newly
formed peripheral VTCs (38). We used the rate of redistribution from
the Golgi area to peripheral VTCs as an indication of rbet1 recycling
rate in the early secretory pathway. As seen in Fig. 7B
(left panel of each pair), both wild type Myc-rbet1 and
Myc-rbet1 K47D peripheral staining began to intensify after as little
as 10 min at 15 °C and was further dispersed after 60 min. To
demonstrate specificity, we co-transfected the cell with a CFP-tagged
galactosyltransferase construct that localizes to Golgi stacks and did
not redistribute significantly over this time course (Fig.
7B, right panel of each pair). In summary, from
the experiments of Figs. 6 and 7, we conclude that rbet1 SNARE
interactions are entirely dispensable to rbet1 steady-state targeting
or dynamic cycling in the early secretory pathway. Although we cannot
eliminate the possibility that SNARE complexes containing rbet1 are
trafficked along this itinerary under the wild type condition, it does
appear that heteromeric SNARE bundles are not a required or strongly
favored mode of rbet1 targeting.
SNARE Interactions Are Dispensable for rbet1 Recruitment onto
Budding Transport Vesicles--
A recent study reported that ARF-GAP
induced a conformational change in the Bet1p SNARE motif that primed
this domain for direct interactions with the COPI and even the COPII
coat machinery (25). Although the nature of the conformational change
was not investigated, one suggestion was that it may be the induction of SNARE bundling that increased its affinity for the coats.
Furthermore, it was suggested that priming of SNARE motifs by ARF-GAP
may be essential for recruitment of the SNAREs into transport vesicles. Because the Myc-rbet1 K47D mutant is defective for SNARE interactions, we asked whether the lack of SNARE interactions influenced the efficiency of recruitment of this SNARE onto coated vesicles. We
developed a simple coated vesicle generation assay that employs scrape-permeabilized NRK cells as a source of donor ER and Golgi. No
attempt is made to separate nor discriminate in this assay between
recruitment to COPI and COPII vesicles. As seen in Fig. 8A, after incubation of the
washed permeabilized cells with cytosol and an ATP-regenerating system
followed by pelleting of the cells, the supernatant contained slowly
sedimenting membranes that could be resolved by floatation on an
Optiprep gradient. Coated transport vesicles appeared to migrate to
fractions 7 and 8 on the Optiprep gradients, as indicated by the
convergence of COPI, COPII, SNAREs, and the cycling vesicle constituent
p24 in those fractions. A less dense peak of SNAREs appeared in
fractions 3 and 4 but did not co-fractionate with coat subunits. The
relative size of this less dense peak varied considerably between
experiments, whereas the putative coated vesicle peak remained stable.
Although we have not identified the less dense membranes, we speculate
that they may represent uncoated vesicles, VTCs, or a fusion product of
vesicles from the incubation. These early fractions were not well
separated from apparently nonspecific ER fragments containing calnexin
that were released without regard to temperature, cytosol, or energy.
In the experiment in Fig. 8B, we used the Optiprep gradients
to isolate the coated vesicle fraction following budding incubations
and examined the biochemical requirements for budding and the vesicle
recruitment efficiencies of different proteins. The coated vesicle
fraction from budding reactions contained the expected array of
ER/Golgi SNAREs (syntaxin 5, membrin, rbet1, and sec22b), vesicle
machinery (p24, COPI, and COPII coat subunits
Importantly, both transfected wild type Myc-rbet1 and Myc-rbet1 K47D
were packaged into diffusible transport vesicles with the same
biochemical requirements and overall efficiency (Fig. 8B,
fifth and sixth rows). Because we wanted to
determine whether SNARE interactions were required for optimal
packaging, we also examined the time course of recruitment/budding of
the Myc-rbet1 constructs relative to each other and relative to
endogenous rbet1. As shown in Fig. 9,
both constructs exhibited very similar time courses of budding,
reaching a value of about 10% incorporation of the total cellular pool
by 90 min of incubation, as did the endogenous rbet1. Our
interpretation of these results is that rbet1 SNARE interactions are
largely dispensable for recruitment to transport vesicles in the early
secretory pathway. It seems likely that interactions between the SNARE
motif and the coat machinery are at least partly responsible for
recruitment into vesicles and by extension that these interactions
involve conformations/structural determinants that are independent of
the SNARE binding determinants.
Evidence against rbet1 Homo-oligomeric SNARE Bundles--
Our data
indicate that heteromeric SNARE interactions, e.g. involving
t-SNARE complexes, are not essential for rbet1 targeting or recruitment
to vesicles. However, it does not necessarily address whether
homo-oligomeric SNARE interactions play an essential role. Some SNAREs
apparently form homodimers in vitro and/or in
vivo, and these interactions may involve distinct determinants
from heteromeric SNARE interactions (39). Yeast Bet1p
homo-oligomerization was suggested as a possible ARF-GAP-induced
trigger for SNARE-coat interactions (25). We were thus interested in
whether homomeric interactions of the rbet1 SNARE motif occur and could
be important for its targeting function. We first investigated the
oligomeric state of purified rbet1 using gel filtration on
Superdex 75. rbet1 cytoplasmic domain eluted with a calculated
molecular mass of ~25 kDa (Fig.
10A). rbet1 K47D cytoplasmic
domain behaved similarly (Fig. 10A). The rbet1 constructs
employed in these studies had a calculated monomer molecular mass of
~11 kDa, significantly less than their elution volume suggested and
approximately half the size of the sec22b cytoplasmic domain, despite
the fact that rbet1 and sec22b co-eluted. Because this rbet1 gel
filtration behavior was consistent with a stable rbet1 dimer, we
performed AU on isolated wild type rbet1 cytoplasmic domain to
rigorously determine its oligomeric state. The calculated molecular
mass of our rbet1 construct based upon amino acid composition is 11,072 Da. Single species analysis provides an apparent molecular mass of
9,830 ± 950 Da (Table I). Although
this molecular mass is lower than that expected for a monomer, if we
take account of the presence and abundance of rbet1 degradation
products present in our AU preparation (Fig. 10B), the
calculated average theoretical molecular mass drops to 10,632 Da, which
is then in good agreement with that determined by AU. There appears to
be a small speed dependence in the molecular masses (Table I), which is
consistent with the heterogeneity in the sample (Fig. 10B).
In summary, the AU analysis indicates that the rbet1 SNARE motif is
essentially monomeric and that the unexpectedly small gel filtration
volume must be due to unusual properties of rbet1 rather than
homo-oligomerization. Although the AU was carried out with relatively
dilute protein (1 µM), gel filtrations over a range of
concentrations indicated that the oligomeric state of rbet1 was stable
up to 10 µM. We speculate that the aberrant gel
filtration volume may be due to the rbet1 SNARE motif existing in an
unstructured, nonglobular state. Several SNARE motifs have been shown
to be unstructured when unbound (31, 40).
To test whether rbet1 forms homo-oligomers in vivo, we
investigated whether interactions between wild type Myc-rbet1 and
endogenous untagged rbet1 were detectable in cell lysates. As shown in
Fig. 10C, efficient immunoprecipitation of Myc-rbet1 from
NRK cell extracts did not result in detectable co-precipitation of
endogenous rbet1, using the same conditions in which heteromeric
interactions with syntaxin 5 were easily detectable (Fig. 5). In
summary, although we cannot rule out low level or transient
homo-oligomeric SNARE interactions contributing to rbet1 targeting and
vesicle recruitment, our data do not support a role for rbet1
homo-oligomers as a significant species in cells. Instead, our data are
most consistent with the targeting machinery operating independently of
SNARE interactions altogether. Further studies will be needed to
delineate the structural features of the SNARE motif required for rbet1 targeting.
How Is the ER/Golgi SNARE Complex Organized?--
Our
biochemical substitution experiments (Fig. 1) and interchain
interaction data (Figs. 2-4) support a conserved organization of the
ER/Golgi complex relative to the synaptic and endosomal four-helix
bundles, with membrin and rbet1 playing roles analogous to those of
SNAP-25N and SNAP-25C, respectively, and sec22b corresponding spatially
to VAMP 2. Thus, the simplest interpretation of our data would be that
in vivo sec22b opposes a t-SNARE complex comprised of
syntaxin 5, membrin, and rbet1. In further support of this model,
sec22b appears to bind strongly only to the combination of all three
Q-SNAREs, making its binding a logical membrane-bridging step (9). How
can we reconcile our data with the well established fact that this
expected topology does not support liposome fusion in vitro
(13)? Reconciliation may not actually be necessary, because the lack of
fusion with opposing sec22b is actually predicted from our
data. An unexpected feature of the ER/Golgi SNAREs is that syntaxin 5, membrin, and rbet1 form a kinetically trapped SNARE complex that cannot
accept a molecule of sec22b and thus cannot advance to form a
quaternary complex (9). In vitro, the quaternary complex
forms only when all four proteins are added simultaneously. The
unexpected topology found to mediate liposome fusion, with Sed5p
(syntaxin 5), Bos1p (membrin), and Sec22p on one membrane is unique in
that this combination of SNAREs do not interact significantly and would
thus "postpone" SNARE complex formation until all four SNAREs came
together simultaneously in the presumed docking event. The important
issue is whether the kinetically trapped SNARE complex is in fact an
in vitro artifact because of the lack of SNARE regulatory
factors. If, in vivo, other factors prevent formation of or
otherwise remediate this presumably off-pathway intermediate, then
membrane fusion could conceivably proceed via the conserved topology at
least as rapidly as in any other topology. Consistent with this, Sec22p
did catalyze liposome fusion in the v-SNARE topology when opposed to
the functional t-SNARE comprised of Sso1p and Sec9p (3). A direct test
of this hypothesis would require identification of the presumed factors that regulate ER/Golgi t-SNARE assembly and activity.
If, on the other hand, ER/Golgi SNARE complex formation in
vivo proceeds in the nonconserved topology suggested by liposome fusion studies, then how could we interpret our substitution data? One
possibility is that the SNARE motif positions in the complex are
conserved, as suggested by the substitutions and by the crystal structure of the endosomal complex (7), but that membrane topology is
not. That is, perhaps rbet1 could in fact be a SNAP-25C homolog and
hold that position in the complex and at the same time be anchored in
the opposite membrane from syntaxin 5, membrin, and sec22b. This would
represent a functional dissociation of the SNARE complex structure from
the topology of the proteins and could have important consequences for
the intermediate steps and route of SNARE complex assembly. Further
work in more physiological contexts will be required to determine
whether this kind of flexibility exists in vivo.
The mechanistic basis of the kinetically trapped ternary complex has
not been investigated. However, new data in this manuscript suggest
that the syntaxin 5-membrin-rbet1 complex contains a membrin aspartate
139 in contact with rbet1 lysine 47. In other words, this ternary
complex most likely contains a membrin molecule positioned like VAMP or
sec22b. This membrin molecule could prevent entry of sec22b and
quaternary complex formation, thus potentially explaining the
"locked" t-SNARE phenomenon (9). This could either represent a
"misplaced" membrin molecule in a 1:1:1 ternary complex or an "extra" copy of membrin in a 1:1:2 ternary complex. We previously reported a 1:1:1 subunit stoichiometry of the ternary complex (9) after
isolation of this complex using an anti-rbet1 monoclonal antibody. We
were unable to produce sufficiently pure quantities of the ternary
complex for a stoichiometric analysis without immunoprecipitation. It
is possible that the rbet1 immunoprecipitation affected the stoichiometry and that the 1:1:1 stoichiometry we obtained was incorrect. Greater quantities of purified complex will be required for
a more precise analysis.
The rbet1 SNARE Motif Mediates Proper rbet1 Targeting and Dynamic
Cycling in the Absence of Heteromeric SNARE Interactions--
Unlike
syntaxins 3, 4, and 5, whose transmembrane domains were reported to be
sufficient to specify their steady-state localizations (14-16), rbet1
requires its SNARE motif for proper targeting (Fig. 6). This finding is
reminiscent of the trans-Golgi network/endosomal SNARE
syntaxin 6, which is actually not a syntaxin but a
QC-SNARE, like rbet1 (19). Because the SNARE motif plays a
required role in targeting, one wonders whether its interactions with
other SNAREs, e.g. syntaxin 5 and membrin, could be key
determinants in this process. However, our data clearly refute this
conjecture and demonstrate that the rbet1 SNARE motif can specify all
aspects of the dynamic targeting of rbet1 independently of SNARE
complexes. Thus, targeting appears to represent a truly autonomous
function of the rbet1 SNARE motif. This does not rule out the
possibility that t-SNARE complexes are trafficked between the ER and
Golgi but does rule out the possibility that they are a favored
substrate of the targeting machinery. We also cannot rule out that
other SNAREs are dependent upon SNARE interactions with rbet1 for their proper localizations. It is also formally possible that very low levels
or transient SNARE interactions that persist in the rbet1 K47D mutant
play an essential role, although reduced by 92% relative to wild type
(Fig. 5).
What protein interactions are required for dynamic rbet1 targeting? The
best candidate interactions are between the rbet1 SNARE motif and the
coat machinery. Although we cannot rule out interactions with other
types of proteins, strong interactions with both coat systems could
conceivably be sufficient to impart rbet1 with constitutive cycling and
proper steady-state localization. Direct interactions between the Bet1p
SNARE motif and COPII (24) as well as COPI (25) subunits have been
documented, although the precise structural determinants required and
their relationship to SNARE complex assembly are unknown.
Interestingly, ARF-GAP primed SNAREs for binding to both coat systems
(25), possibly indicating a conserved mode of interaction with both
coats and similar conformational requirements. One suggestion was that
ARF-GAP may prepare Bet1p for coat interactions by inducing SNARE
bundling (25). Our results argue strongly against heteromeric SNARE
bundling as the prevalent mode of interactions with the vesicle
machinery in vivo, because rbet1 K47D was efficiently
recruited and packaged (Figs. 8 and 9). Although homodimeric bundling
of Bet1p could have been the ARF-GAP-dependent event in the
previous study, we did not find evidence of a prevalent rbet1
homo-oligomer in solution or in detergent extracts of cells (Fig. 10).
Although rbet1 lacks a significant amino-terminal domain, yeast Bet1p
contains a 52-amino acid amino-terminal extension that, according to
the PSIPRED algorithm (19), contains at least one significant length of
Because SNARE bundles were not required for rbet1 targeting and because
the rbet1 SNARE motif may be unstructured on its own, one possibility
is that the coat machinery binds the rbet1 SNARE motif in a fully
extended conformation. This would be reminiscent of the
binding of botulinum toxin B to the extended VAMP 2 SNARE motif (41).
If the targeting machinery does in fact bind to rbet1 in an extended
conformation, then only nonbundled SNARE motifs would be included, and
the active trafficking of SNARE complexes would be prohibited. Although
we did not observe an increase in the rate of trafficking or
recruitment to vesicles of rbet1 K47D relative to wild type rbet1, it
is possible that the half-life of rbet1 SNARE complexes in
vivo is too short to cause a noticeable lag in trafficking.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP
antiserum was raised in a rabbit by injection of the peptide
CKKEAGELKPEEEITVGPVQK conjugated to keyhole limpet hemocyanin. p24
2
antiserum was raised in a rabbit by injection of the peptide
CQMRHLKSFFEAKKLV conjugated to keyhole limpet hemocyanin. An
anti-rsec23 antibody (which recognizes all isoforms) was raised in a
rabbit against the peptide CQQNEERDGVRFSWNVWPSSR conjugated to keyhole
limpet hemocyanin and then affinity-purified using the peptide.
Glucosidase II
and protein disulfide isomerase antibodies
were purchased from Stressgen (catalog numbers VAP-PT034 and SPA-890,
respectively). Anti-calnexin was from Dr. Ari Helenius, and anti-GM130
was from Dr. Martin Lowe. Anti-syntaxin 6 monoclonal antibody (clone
3A10) was from Drs. Richard Scheller and Jason Bock.
80 °C. The
proteins were quantified by comparison with BSA standards on
Coomassie-stained SDS gels, employing an Agfa Arcus 1200 flatbed scanner and Kodak 1D Image gel analysis software.
S. The extract obtained was centrifuged at 100,000 × g for 30 min. The clarified supernatants were processed for
immunoprecipitation using specific antibodies and protein A beads.
After a 2-h incubation with the extracts, the beads were washed with
Buffer A containing 0.1% Triton X-100, and the proteins were
solubilized with SDS-PAGE sample buffer. The immunoprecipitates and the
starting extracts were then analyzed by SDS-PAGE and immunoblotting.
80 °C.
For budding of vesicles, one 10-cm plate of just-confluent NRK cells
were scraped from the plate with a rubber policeman into buffer 50/90
(50 mM Hepes, pH 7.2, 90 mM potassium acetate). The cells were then washed once with 50/90, resuspended in a final volume of 100 µl of 50/90, and added to a budding reaction in a total
volume of 800 µl containing buffer 25/125 supplemented with 2.5 mM MgOAc, 5 mM EGTA, 1.8 mM
CaCl2, 1 mM ATP, 5 mM creatine phosphate, 5 units/ml creatine phosphokinase, and 200 µl of rat liver
cytosol. Budding reactions were incubated at 32 °C for 90 min and
stopped on ice for 5 min, and the cells were removed with a 4,000 × g centrifugation for 1 min. Then either the
supernatant was directly subjected to a 100,000 × g
centrifugation and the pellet was subjected to immunoblotting, or the
supernatant was fractionated by iodixanol gradient prior to immunoblot
analysis. For gradient analysis, the 4,000 × g
supernatants from budding reactions (800 µl) were layered on top of
150 µl of 7% iodixanol (diluted with Optiprep diluent: 0.25 M sorbitol, 10 mM Hepes, 1 mM EDTA,
pH 7.4) layered on top of 300 µl of 50% iodixanol and centrifuged at
100,000 × g for 40 min. The top 800 µl was then aspirated, and the remaining 450 µl was mixed and bottom-loaded under
a 5-25% continuous iodixanol gradient. After a 90-min 100,000 × g centrifugation, the gradient was fractionated from the
top, diluted with Optiprep diluent, and subjected to a 120,000 × g centrifugation for 1 h. The pellets were then
analyzed by immunoblotting.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Substitutions of SNAP-25 helices delineate
corresponding helices in the ER/Golgi SNARE complex. Purified,
bacterially produced recombinant SNAREs were mixed in the indicated
combinations and incubated overnight at 4 °C. Each protein had a
final concentration of ~2 µM and was used at an
identical concentration in every reaction. Incubated binding reactions
were gel-filtered on Superdex 200, and the individual fractions were
immunoblotted for the protein indicated to the left of each
panel. In B and D, the sample labeled
28 is a pool of 28, 30, and 32; the sample labeled
34 is a pool of 34, 36, and 38. In A and
C, fractions were analyzed individually. The elution
positions of globular marker proteins are indicated above
the fraction numbers.
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Fig. 2.
A specific charge reversal at a conserved
salt bridge eliminates ER/Golgi quaternary complex formation.
A, alignments of each ER/Golgi SNARE domain with the most
similar SNARE domain from the synaptic complex. Layers of contacting
residues on the inner surface of the synaptic complex are numbered
above and tracked by vertical lines. Bold
black residue letters on sequences highlight the ionic zero layer
position. Residue letters colored red are identical, and
blue letters are similar, with the following groupings
considered similar: R and K; Q and N; T and S; E and D; and V, I, L, F,
and M. Ionic residues that participate in surface salt bridges in the
synaptic complex between SNAP-25C and VAMP 2 are highlighted with
yellow. B, ER/Golgi quaternary complex
formation as assayed in Fig. 1, except that binding reactions were
carried out for 4 h at 4 °C. The only difference between the
five reactions is the residue at position 47 of rbet1. Each rbet1
protein was purified with the same procedures and utilized at 2 µM in the binding reactions.
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Fig. 3.
rbet1 K47D is defective for binary and
ternary SNARE interactions. A, binary bead binding
reactions testing syntaxin 5 binding to GST-rbet1 beads
(syn5/GST-rbet1), syntaxin 5 binding to GST-rbet1
K47D beads (syn5/GST-rbet1 K47D), or syntaxin 5 binding to GST beads (syn5/GST). B,
binary bead binding reactions testing rbet1 binding to GST-membrin
beads (rbet1/GST-membrin), rbet1 K47D binding to
GST-membrin beads (rbet1 K47D/GST-membrin), and
rbet1 binding to GST beads (rbet1/GST).
C, ternary bead binding reactions testing syntaxin 5 binding
to GST-membrin beads in the presence of varying concentrations of rbet1
(rbet1/GST-membrin), syntaxin 5 binding to
GST-membrin beads in the presence of varying concentrations of rbet1
K47D (rbet1 K47D/GST-membrin), or syntaxin 5 binding to GST beads in the presence of varying concentrations of rbet1
(rbet1/GST).
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Fig. 4.
Charge reversal mutations on sec22b and
membrin partially restore SNARE complex assembly with rbet1 K47D.
Purified, bacterially produced recombinant SNAREs were mixed in the
combinations indicated below and incubated 2 h at 4 °C. Each
protein had a final concentration of ~2 µM and was used
at an identical concentration in every reaction. For analysis, the
binding reactions were gel-filtered on Superdex 200, and a pool of
fractions 16-18 was immunoblotted for the presence of rbet1 and
quantitated. The rbet1 signal in each condition was normalized to that
in condition 5. A, binding reactions employing wild type
rbet1. The values plotted are representative single
determinations. B, binding reactions employing rbet1 K47D.
The values plotted are the means of three independent determinations,
with the range of values indicated. WT, wild type;
K/D, rbet K47D; D/K, membrin D139K (conditions 3 and 11-14) and sec 22b D144K (conditions 9, 10, and 14).
-COP
antiserum resulted in equal co-immunoprecipitation of both Myc-rbet1
constructs. Our interpretation of this result was that the interaction
between the rbet1 SNARE motif and the COPI machinery is entirely
independent of heteromeric SNARE interactions. Whatever structural
properties of the SNARE motif are required for COPI interactions are
completely preserved in the K47D construct that is largely incapable of
participating in ER/Golgi SNARE bundles.
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Fig. 5.
rbet1 K47D exhibits dramatically reduced
SNARE interactions but unaffected coatomer interactions in
vivo. NRK cells were transfected with either wild type
Myc-rbet1 (myc-WT) or Myc-rbet1 K47D (myc-K47D).
Whole cell Triton X-100 lysates were prepared, subjected to
immunoprecipitation (I.P.) with the antisera shown on the
left, and subjected to immunoblotting with the antisera
indicated on the right. The columns marked
extract represent 1% of the extract subjected to
immunoprecipitation and displayed in the columns labeled
I.P. The three immunoprecipitations were carried out in
three different transfection experiments. The box labeled
C was a mock immunoprecipitation carried out with protein A
beads lacking primary antibody. Asterisks denote the
positions of antibody light chains.
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Fig. 6.
The SNARE motif of rbet1 directs proper
steady-state localization independently of SNARE interactions.
A, the top two rows show immunofluorescence
images of NRK cells transfected with wild type Myc-rbet1
(myc-WT) or Myc-rbet1 K47D (myc-K47D) stained
with anti-Myc (red) or GM130 (green) antibody.
Individual red and green stainings are shown as
grayscale as well as the colored merge. The areas inside white
boxes are expanded to the right of each larger panel
for greater detail. The arrows indicate peripheral staining
exhibited by rbet1 constructs but not by GM130. The third
row shows the same features of endogenous (endog.)
rbet1 staining in nontransfected NRK cells. B, steady-state
localization of the indicated constructs in transfected NRK
cells.
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Fig. 7.
The intracellular dynamics and constitutive
recycling of rbet1 are independent of SNARE interactions.
A, NRK cells were transfected with wild type Myc-rbet1
(myc-WT) or Myc-rbet1 K47D (myc-K47D) and
subjected to a 1-h brefeldin A treatment. Brefeldin A was removed, and
the cells were allowed to recover for the indicated lengths of time
prior to fixation and staining with anti-Myc (bottom two
rows) or anti-syntaxin 6 (top row) antibodies. The
left column illustrates steady-state staining prior to BFA
addition. B, NRK cells were co-transfected with either wild
type Myc-rbet1 (myc-WT) or Myc-rbet1 K47D
(myc-K47D) and the localization domain of
galactosyltransferase linked to CFP (GT-CFP). The cells were
shifted from 37 to 15 °C and incubated for 0, 10, or 60 min prior to
fixation and staining with anti-Myc antibody. For each time point, a
pair of images are displayed showing anti-Myc (left) and CFP
(right) fluorescence. The peripheral rbet1 staining became
progressively more intense relative to rbet1 juxtanuclear staining and
CFP fluorescence and the peripheral spots became larger with increased
time at 15 °C (examples are marked by arrows).
COP and rsec13,
respectively), and cargo (not shown; vesicular stomatitus virus G
protein), in a temperature-, cytosol-, and nucleotide-dependent fashion. On the other hand, ER
resident proteins (protein disulfide isomerase, calnexin, and
glucosidase) that were abundant in the permeabilized cells were
specifically de-enriched in the vesicle fraction. We found that SNAREs,
especially sec22b, were very efficiently incorporated into these
vesicles. Interestingly, rbet1 and membrin recruitment was efficient
but less energy-dependent than other vesicle constituents
(Fig. 8B, second and fourth rows versus first, third,
eighth, and ninth rows). This may have to do with
the observation in yeast that Bet1p and Bos1p seem to interact most
strongly with the coat machinery (24). In fact, ARF-GAP was able to
induce interactions between these SNAREs and both coats even in the
absence of the GTPases Arf1p and Sar1p (25). Hence, it is conceivable
that in the absence of nucleotides, membrin and rbet1 are packaged at a
reduced efficiency into artifactual vesicles that lack other SNAREs,
vesicle machinery, and cargo.
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Fig. 8.
Efficient recruitment of rbet1 onto transport
vesicles is independent of SNARE interactions. A,
equilibrium density gradient analysis of diffusible vesicles released
from permeabilized cells in the presence of cytosol and ATP at
32 °C. The permeabilized cells were incubated as detailed under
"Experimental Procedures," and diffusible vesicles were isolated by
differential centrifugation followed by floatation into continuous
iodixanol gradients. The fractions were unloaded from the top and
analyzed by immunoblotting and quantitation of the indicated proteins.
B, rbet1 K47D budding requirements and efficiency are
indistinguishable from wild type (WT). NRK cells were
transfected with wild type Myc-rbet1 or Myc-rbet1 K47D, permeabilized,
and subjected to budding reactions with or without MgATP, cytosol, or
elevated temperature as indicated. The coated vesicle population was
isolated by differential centrifugation and floatation in iodixanol
gradients as in A. Fractions from the peak of coated
vesicles (fractions 6-8) were immunoblotted for the proteins listed on
the left. The right column shows 2% of each
protein in the starting permeabilized cells as an indication of
relative budding efficiency. rbet1 refers to endogenous
rbet1 detected with anti-rbet1 antisera, whereas the Myc constructs
were detected with anti-Myc antibodies.
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Fig. 9.
Disruption of SNARE interactions does not
affect the rate of rbet1 incorporation into coated vesicles. NRK
cells were transfected with either wild type Myc-rbet1 or Myc-rbet1
K47D, permeabilized and subjected to budding reactions containing
cytosol and MgATP for the indicated times at 32 °C. Released
diffusible vesicles were then isolated by differential centrifugation
and immunoblotted for the presence of the indicated proteins.
A shows quantitation of the immunoblots, which are shown in
B. The right column in B shows the
amount of each protein in 10% of the starting permeabilized cells.
This quantity was used to calculate the percentage of budding
efficiency plotted on the y axis in A. WT and wt, wild type.
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Fig. 10.
rbet1 does not engage in stable
self-associations in vitro or in
vivo. A, gel filtration analysis of purified
rbet1 and sec22b cytoplasmic domains on Superdex 75. Elution positions
of globular markers are indicated above. B, SDS-PAGE of the
rbet1 preparation employed for analytical ultracentrifugation, stained
with Coomassie Blue. C, immunoprecipitation
(I.P.) of wild type Myc-rbet1 from NRK cell detergent
extracts did not co-precipitate endogenous rbet1. NRK cells were
transfected with wild type Myc-rbet1, subjected to Triton X-100
extraction, and immunoprecipitated with anti-Myc antisera.
Immunoblotting was carried out with anti-rbet1 antisera that recognizes
both endogenous (endog.) rbet1 and transfected
Myc-rbet1.
Single species analysis of sedimentation equilibrium data for rbet1
cytoplasmic domain
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-helix. One possibility is that ARF-GAP primed Bet1p for coat
interactions not by bundling the SNARE motif but by altering a
potential interaction between the amino-terminal extension and the
SNARE motif. This type of regulation might not be relevant to rbet1,
which lacks a significant amino-terminal extension.
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ACKNOWLEDGEMENT |
---|
We are indebted to Dr. Fred Hughson (Princeton) for very helpful discussions.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Grant GM59378 (to J. C. H.).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.
¶ Supported by National Science Foundation Grant MCB-0211754.
To whom correspondence should be addressed: Dept. of
Molecular, Cellular, and Developmental Biology, University of Michigan, 830 North University Ave., Ann Arbor, MI 48109. Tel.:
734-647-6662; Fax: 734-647-0884; E-mail: jessehay@umich.edu.
Published, JBC Papers in Press, February 2, 2003, DOI 10.1074/jbc.M300659200
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ABBREVIATIONS |
---|
The abbreviations used are:
SNARE, soluble
N-ethylmaleimide-sensitive factor attachment protein
receptor;
v-, vesicle;
t-, target membrane;
ER, endoplasmic reticulum;
GST, glutathione S-transferase;
BSA, bovine serum albumin;
AU, analytical ultracentrifugation;
BFA, brefeldin A;
GTPS, guanosine 5'-3-O-(thio)triphosphate;
SNAP, soluble
N-ethylmaleimide-sensitive factor attachment protein;
NRK, normal rat kidney;
CFP, cyan fluorescent protein;
VTC, vesicular
tubular cluster;
ARF, ADP ribosylation factor;
GAP, GTPase-activating
protein.
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REFERENCES |
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