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J Biol Chem, Vol. 274, Issue 52, 36876-36882, December 24, 1999
From the The vesicle-associated membrane proteins
(Vamp(s)) function as soluble N-ethylmaleimide-sensitive
factor attachment receptor proteins in the intracellular trafficking of
vesicles. The membrane attachment of Vamps requires a carboxyl-terminal
hydrophobic sequence termed an insertion sequence. Unlike other
insertion sequence-containing proteins, targeting of the highly
homologous Vamp1 and Vamp2 to the endoplasmic reticulum requires ATP
and a membrane-bound receptor. To determine if this mechanism of
targeting to the endoplasmic reticulum extends to other Vamps, we
compared the membrane binding of Vamp1 and Vamp2 with the distantly
related Vamp8. Similar to the other Vamps, Vamp8 requires both ATP and
a membrane component to target to the endoplasmic reticulum.
Furthermore, binding curves for the three Vamps overlap, suggesting a
common receptor-mediated process. We identified a minimal endoplasmic
reticulum targeting domain that is both necessary and sufficient to
confer receptor-mediated, ATP-dependent, binding of a
heterologous protein to microsomes. Surprisingly, this conserved
sequence includes four positively charged amino acids spaced along an
amphipathic sequence, which unlike the carboxyl-terminal targeting
sequence in mitochondrial Vamp isoforms, is amino-terminal to the
insertion sequence. Because Vamps do not bind to phospholipid vesicles,
it is likely that these residues mediate an interaction with a protein,
rather than bind to acidic phospholipids. Therefore, we suggest that a
bipartite motif is required for the specific targeting and integration
of Vamps into the endoplasmic reticulum with receptor-mediated
recognition of specifically configured positive residues leading to the
insertion of the hydrophobic tail into the membrane.
Despite being discovered more than 30 years ago, the targeting of
proteins bound to the cytoplasmic face of membranes by
carboxyl-terminal hydrophobic sequences (insertion sequences) remains
mysterious (1). Recently, progress has been made in the understanding of the subsequent subcellular trafficking of some of these proteins from the endoplasmic reticulum
(ER)1 to synaptic vesicles
and the Golgi. The synaptic vesicle targeting of vesicle
associated membrane proteins
(Vamps) is mediated by a region that is distal to the insertion
sequence (2). In contrast, the signal for trafficking to the Golgi of
Sed5p is related to the length of the hydrophobic segment as sequences
that are 4-5 amino acids longer exit the ER (3-5). However,
regardless of the final destination of the protein, it is postulated
that the hydrophobic sequence provides the thermodynamic driving force for initial integration into the ER membrane. Consistent with this
model, many variations are tolerated within the hydrophobic core
sequence (6).
The recent cloning of separate isoforms of cytochrome
b5 (7) and Vamp1 (8) that are targeted to either
ER or mitochondria has led to the identification of the last 9 or 5 amino acids, respectively, as critical for targeting. The results of
sequence swapping and mutagenesis experiments demonstrated that at
least two positive charges near the carboxyl terminus are essential for
insertion into the cytoplasmic face of the outer mitochondrial membrane
(7, 8). It remains to be definitively shown whether mitochondrial
targeting by these sequences is an active process (i.e. the
sequence alterations specify mitochondrial localization) or if the
endoplasmic reticulum forms contain a sequence that prevents
mitochondrial localization and that must be altered for more
promiscuous targeting.
The original studies on the mechanism of the cytochrome
b5 (Cb5) insertion sequence-mediated protein
targeting suggested that the integration step was spontaneous,
promiscuous, and nonsaturable (9-13). However, we have demonstrated
that there are at least two distinct mechanisms for integration into
membranes (14). Vamp1 is the best studied example of a protein for
which membrane integration is receptor-mediated (6, 14, 15). Vamp1 is a
member of a family of proteins that currently includes eight members
(16). Vamps 1 and 2 function as soluble
N-ethylmaleimide-sensitive factor attachment protein
receptors in the intracellular trafficking of vesicles (14, 15). Both
Vamps 1 and 2 require ATP and a membrane-bound receptor to target to
the ER (14, 15) by a process in which targeting and integration appear
to occur simultaneously. Once integrated into the ER membrane, Vamps 1 and 2 are sorted to secretory vesicles and the plasma membrane. To
elucidate the mechanism of targeting of Vamp proteins to the ER
membrane, we have compared the membrane binding characteristics of
distantly related members of the Vamp family (Vamps1 and -2 compared
with Vamp8) and identified the sequence that specifies localization to
the endoplasmic reticulum. Surprisingly the sequence necessary for
targeting in all three Vamps includes four positively charged amino
acids spaced along one side of an amphipathic sequence that is
amino-terminal to the hydrophobic region. Our results also indicate
that there is a common mechanism and probably a single receptor used to
target different Vamps to the ER.
Plasmid Construction--
Construction of a plasmid expressing
Vamp1 (GenBankTM accession no. AAA42322) was described
previously (14). Vamp2 (GenBankTM accession no. AAA42321)
and Vamp8 (GenBankTM accession no. T63214) cDNA
sequences were amplified by polymerase chain reaction (PCR) from rat
and human cDNA, respectively, and inserted into pBluescript KS-.
For in vitro transcription/translation, the protein coding
sequences were cloned into pSPUTK, a plasmid that contains both a SP6
promoter and a high efficiency 5'-untranslated region for translation
in reticulocyte lysate (17). Vamp2 and Vamp8 were subcloned into pSPUTK
by PCR. Primer sequences are available from the authors upon request.
In vitro and in vivo gPA behaves as a cytosolic
protein but can be targeted to different intracellular membranes when
fused to appropriate targeting sequences (18). Gene fusions for gPA and
either Vamp2 or Vamp8 were constructed using a unique BamHI site near the 3'-end of the coding region of gPA and a XhoI
site 3' of the gPA termination codon. The required sequences were added to the coding regions of Vamp2 and Vamp8 by PCR amplification. Amino-terminal deletions of the Vamp2 coding sequence in gPAVamp2 were
constructed using a whole plasmid PCR method as described (19) except
that PCR was performed using a 40:1 ratio of Taq (MBI,
Fermentas) and Vent (New England Biolabs) DNA polymerases. To construct
a plasmid encoding gPAVamp2-(96-116), the plasmid encoding gPAVamp2
was prepared from GM48 cells and cut with BclI (within the
Vamp2 coding region) and BamHI (near the carboxyl end of
gPA) restriction endonucleases, and then the plasmid was reclosed by
ligation. Plasmids encoding point mutations were generated by cassette
mutagenesis using internal BclI and EcoRI sites.
For random mutants the oligonucleotides were synthesized with multiple nucleotides added at the appropriate steps in the synthesis reaction (Mobix Central Facility, McMaster University, Ontario, Canada). The
relevant regions of all of the plasmids were sequenced to ensure that
additional mutations were not introduced during amplification or
cloning. Plasmids and complete construction details are available from
the authors.
Preparation of Membranes--
Canine pancreatic ER membranes
(microsomes) were prepared as described by Walter and Blobel (20). Each
batch of microsomes was tested for cotranslational translocation of
preprolactin (21). One equivalent of microsomes is defined as 100 fmol
of signal recognition particle receptor In Vitro Transcription, Translation, and Membrane
Binding--
In vitro transcription and translation were
performed as described previously (21). After a 60-min incubation at
24 °C, the translation reaction was stopped by adding cycloheximide
to a final concentration of 20 µg/ml. ATP was then removed from the translation reaction by incubation with 5 units of apyrase (Sigma) at
24 °C for 30 min. Nucleotides and other small molecules were removed
by passing the reaction though a 600 µl Sephadex G-25 (Sigma) spin
column. ATP was added to the G-25 treated translation reaction by
adding a solution containing 100 mM ATP, 10 mM
Tris-HCl, pH 7.5, to a final concentration of 0.50 mM ATP.
The membrane binding assay has been described previously (14).
Immunoblotting with calreticulin of a parallel reaction fractionated similarly demonstrated that the microsomes remained sealed throughout the procedure. Mitochondria and lysosome binding assays were performed similarly with the following modifications. Mitochondria were pelleted
at 17,000 × g in a microcentrifuge for 10 min at
4 °C, and the pellet was washed once with translation buffer (50 mM KCl, 2 mM MgCl2, 10 mM Tris-HCl, pH 7.5, and 1 mM dithiothreitol). Lysosomes were pelleted through a 0.5 M sucrose cushion by
centrifugation at 39,000 × g in a TLA100 rotor (Beckman).
Volumes of each fraction corresponding to equivalent amounts of the
original translation reaction were analyzed by SDS-polyacrylamide gel
electrophoresis using a Tris-Tricine buffer system (25). Radioactive
proteins were visualized and quantified using a PhosphorImager (Molecular Dynamics). Quantitative membrane binding assays were performed as described previously (14).
Common Mechanism of Targeting Vamps to ER Membranes--
Vamp
proteins are expressed in all tissue types, and highly conserved in
different species (26-28). Among Vamp homologues, Vamps1, -2, and -3 are the most similar (
Vamp2 and Vamp8 both bind with similar efficiency to microsomes (Fig.
1, lanes 1-3). Although the
binding efficiency (20 and 21%, respectively) was much lower than that
observed for the control protein cytochrome b5
(67%), the amount of material pelleted was significantly higher than
in control reactions without microsomes (lanes 13-15).
Specific binding of Vamps never exceeded 25% suggesting that most
Vamps do not fold into membrane binding competent conformation in our
in vitro system. However, >80% of the membrane-bound
material was resistant to extraction with sodium carbonate, pH 11.5, confirming that this material becomes integrated into the bilayer (data
not shown). When ATP was depleted from the reaction mix using apyrase or by passing the reaction mixture through a G25 column, binding was
abolished for both Vamp2 and Vamp8 (Fig. 1, lanes 4-6 and 7-9). When ATP was added to reactions from which small
molecules were depleted by G25 chromatography, membrane binding was
restored (Fig. 1, lane 10-12). Other nucleotides were
ineffective, confirming the nucleotide specificity (data not
shown).
Previously we have shown saturable binding of Vamp1 to microsomes
stripped of peripheral proteins by high salt and that this binding can
be abolished when membranes are treated with a very low concentration
of trypsin (14). Saturable binding was also observed when Vamp1, Vamp2,
and Vamp8 binding was examined to nonsalt treated microsomes (Fig.
2). Binding curves for the three proteins
are superposable with saturation occurring for 1 equivalent of
membranes at 45 fmol of protein. Furthermore, none of the proteins bound to trypsinized microsomes (data not shown). Taken together, these
results suggest that all three Vamps bind to ER membranes via an
interaction with a membrane protein. To formally demonstrate the
presence of a common receptor would require competitive binding assays.
The amount of protein required for such experiment is not attainable
using in vitro translation.
To confirm that unlike membrane binding of Cb5 membrane binding of
Vamps requires a membrane protein, Vamp1 was assayed for binding to
lipid vesicles with a composition similar to that of the ER membrane
(21). In this assay, proteins bound to liposomes float upward in the
sucrose gradients (Fig. 3A,
fraction 1). Unbound proteins remain in fraction 4, where they were
loaded originally, and aggregates pellet to the bottom (fraction 5).
Cb5 molecules were recovered from fractions 1 and 2 indicating direct
insertion into lipid bilayers. In contrast, neither Vamp1 (Fig.
3A) nor Vamp8 (data not shown) bound to the liposomes,
consistent with the presence of a Vamp receptor on the ER.
A Vamp Receptor Is Not Found on Mitochondria or
Lysosomes--
Vamp1 and -2 are known to cycle between the plasma
membrane and internal organelles (30). Therefore, it is possible that Vamp proteins may insert directly into other membranes. However, in vitro, Vamp1 and Vamp8 did not pellet with lysosomes, a
membrane system similar to that of endosomes (Fig. 3B). Cb5,
a positive control for membrane binding in vitro, bound to
lysosomes quite efficiently confirming that the membranes were intact.
Recently, a novel isoform of Vamp1 was identified that appears to
target specifically to mitochondria (8). Targeting to mitochondria is
mediated by a shortened hydrophobic domain and two positive-charged
residues at the carboxyl terminus (8). Similar to lysosomes, authentic
Vamps1 and 8 do not bind to mitochondria in vitro (data not
shown). It is not known whether the modification at the carboxyl
terminus that leads to mitochondrial binding eliminates binding to ER.
Therefore, we modified the targeting signal on Vamp2 to match the
sequence of the isoform previously shown to bind to mitochondria. This
mutant termed, Vamp2mito, bound to ER membranes as efficiently as Vamp2
(Fig. 3C) with ATP dependence (data not shown). This
suggests that mitochondrial targeting may be a result of relaxed
specificity for ER and that residues essential for ER targeting are not
found at the extreme carboxyl terminus of the Vamps.
16 Amino Acids Preceding the Transmembrane Domain Are Required for
Microsomal Binding--
The requirement for a receptor on the ER
membrane to target Vamps suggests the presence of a specific signal
that is common in the amino acid sequence of Vamp proteins but is not
shared by proteins with promiscuous insertion. A candidate sequence for targeting Vamps to ER membranes is the helix 1 sequence previously shown to be important for targeting to synaptic vesicles (2). Sequence
comparisons of the eight known Vamps revealed several other highly
conserved regions among those with well defined insertion sequences.
Because it is possible that there may also be more than one sequence
that contributes to ER-specific binding, we screened a series of Vamp2
deletion mutants fused to gPA to identify those sequences sufficient
for binding to microsomes in vitro (Fig.
4A). Fusion proteins
containing residues 80-116 of Vamp fused to gPA bound to microsomes as
efficiently as Vamp2. We therefore designated this plasmid gPAVMB
(Vamp Minimal Binding region) (Fig. 4B). The fusion protein containing residues 90-116 of Vamp
(gPAV2-(90-116)) exhibited impaired targeting. However, the deletion
of residues 1-95 (yielding gPAVamp2-(96-116)) resulted in no membrane
binding above background (Fig. 4B). As shown in Fig.
4C, gPAVMB binds to ER in an ATP-dependent
fashion (but not gPAV2-(96-116), Fig. 4B), suggesting that
the mechanism of membrane binding for the fusion protein is the same as
for the full-length Vamps.
Using several secondary structure prediction programs, three regions of
helix were predicted for the Vamps1, -2, and -8, residues 30-46 (helix
1), residues 50-72 (helix 2), and residues 76-97 (helix 3) numbering
according to Vamp2. Residues 80-96 are part of the helix 3 region of
Vamp2 encompassing the most conserved region among the three Vamps
(residues 83-96). Within helix 3, the sixteen amino acids immediately
preceding the hydrophobic sequence include five positively charged
amino acids, two tryptophans, and an asparagine that are all conserved
residues (Fig. 5A).
When helix 3 is plotted on a helical wheel, four of the five conserved
positively charged residues were on the same face, suggesting that this
region forms an amphipathic helix (Fig. 5B). These conserved
residues may contribute to a specific or may be necessary only for
correct spacing between the hydrophobic domain and the gPA passenger.
To test these hypotheses, all the positive charges in gPAVMB were
changed to hydrophilic residues (Fig. 5A). The resulting
fusion protein, gPAVMB (0+) did not bind to membranes above background
(Fig. 6A), demonstrating that
the positively charged residues are necessary for binding.
To determine if the four lysines on the same face of the helix are
sufficient to trigger membrane binding, a mutant was constructed in
which the lysine and arginine that were not on this face of the helix
(amino acids 7 and 8 in Fig. 5B) were replaced by uncharged residues. This mutant, gPAVMB(4K), bound to microsomes as efficiently as the wild-type sequence in gPAVMB (Fig. 6A). To probe the
importance of the individual lysines on the same face of the helix a
series of mutants were constructed containing various combinations of these four residues but not the lysine or arginine from the other side
of the helix. No single residue was found to be critical for membrane
binding. Instead, it appears that the four residues together form the
binding signal. Therefore, membrane binding was averaged for mutants
with the same number of positively charged amino acids. When presented
in this format (Fig. 6A), the direct relationship between
the number of positive charges and membrane binding is obvious. As
expected from this result, the corresponding region from Vamp8 fused to
gPA were also sufficient to confer membrane binding equivalent to the
full-length protein (Fig. 6B, lanes 1 and
2). When the four conserved lysine residues of Vamp8 are
replaced by noncharged hydrophilic residues (Fig. 5A),
membrane binding was greatly diminished (Fig. 6B,
lanes 2 and 3).
Thus the hydrophobic carboxyl-terminal sequence and the 16 amino acids
directly amino-terminal of this sequence constitutes a Vamp targeting
signal for ER membranes. Because this sequence is present in the Vamp1B
isoform that targets to mitochondria, we tested to see if the changes
at the extreme carboxyl terminus of the protein would alter the
mechanism of targeting, rendering targeting to the ER insensitive to
the lysines in the amphipathic helix. The signal that permits
mitochondrial binding in Vamps is unable to confer ER binding when
added to the gPAVMB (0+), the mutant that lacks the six positively
charged residues (Fig. 3C, lanes 4 and 5).
Together these experiments confirm that the four lysines on one face of
the helix are essential for binding to ER microsomes and suggest that
the changes in the carboxyl terminus that permit targeting to
mitochondria do not alter the mechanism of binding to ER. They further
suggest that the "mitochondrial" isoform of Vamp is likely to be
found at both the ER and mitochondria.
To determine if the two conserved tryptophans and asparagine also play
a role in binding of Vamps to membranes, these residues were changed to
a variety of other amino acids by cassette mutagenesis using doped
oligonucleotides. These oligonucleotides were designed to encode 1 of
10 amino acids at the sites of tryptophan and asparagine. Substitution
of the asparagine residue (with Y, T, P, H, or D) significantly reduced
membrane binding (Fig. 7). In contrast, with only one exception, membrane binding was insensitive to mutation of either of the tryptophan residues. The only mutant with decreased binding compared with wild type resulted in a marginal decrease (p = 0.02), observed when one of the tryptophans was
changed to a proline (Fig. 7) likely by disrupting local secondary
structure.
Most Vamp proteins are initially targeted to the ER membrane and
then sorted to their ultimate destinations within the secretory pathway. The membrane targeting of distantly related Vamps exhibit identical binding curves (Fig. 2) and require both ATP and a trypsin sensitive membrane component (Fig. 1 and Ref. 14). These data strongly
suggest that Vamps are targeted to pancreatic ER microsomes by a single
targeting mechanism. Consistent with Vamp proteins containing a unique
targeting signal within and around the hydrophobic transmembrane
sequence this region is 50% identical between Vamp1 and Vamp8, two
molecules with an overall identity of only 29%.
Deletion and point mutations generated in Vamps as well as fusions to
gPA were used to identify sequences other than the insertion sequence
that are important for binding Vamps to ER. Taken together, our data
suggest that the signal for ER targeting includes the 16 residues
amino-terminal to the hydrophobic transmembrane domain. This region is
predicted to form an amphipathic helix that contains four conserved
lysine residues on one face. These lysines were shown to contribute
additively to membrane binding (Fig. 6A). Thus, both the
hydrophobic region and the amphipathic sequence are necessary to direct
Vamps to the ER membrane.
Other proteins have been shown to use polar or charged residues to
mediate the initial step in membrane insertion. Several myristoylated
proteins such as Src and myristoylated alanine-rich protein kinase C
substrate require both the hydrophobic acyl chain and a sequence of
positively charged residues for membrane binding (31-33). In this case
the cluster of basic residues is required for nonspecific interaction
with acidic phospholipids, whereas the myristate is inserted into the
lipid bilayer. A synaptic vesicle-associated protein, SNAP-25, requires
an interhelical Gly-Pro-Xaa-Arg motif for membrane targeting (34).
Although this process is protein-mediated, unlike Vamp, the subsequent
membrane insertion of SNAP-25 requires the palmitoylation of cysteine
residues. Because membrane binding of Vamps requires ATP and a
trypsin-sensitive, saturable component of the ER membrane, the positive
charged residues are more likely to mediate a protein interaction than
bind nonspecifically to acidic phospholipids.
Sequence comparison with other Vamp proteins revealed that the
16-residue predicted amphipathic helix is conserved only in those Vamp
proteins with a hydrophobic putative transmembrane sequence. Comparison
of the sequences of the 16-residue amphipathic targeting sequences for
Vamps 1, 2, and 8 (Fig. 5A) revealed five additional
conserved amino acids (K, R, N, and two Ws). Of these only asparagine
contributes to binding to microsomes in vitro, suggesting
that it plays an important but as yet undefined role in membrane binding.
The evidence for separate motifs that lead to either mitochondria or ER
membrane binding suggests a novel model for the targeting of insertion
sequence proteins to the appropriate membrane. If Vamp isoforms with a
mitochondrial targeting sequence are targeted exclusively to the
mitochondria, our results indicate that there must not be a cytoplasmic
receptor present in reticulocyte lysate that prevents binding of these
proteins to ER membranes. It is also likely that a protein is needed to
insert the mitochondrial-targeted Vamps into the outer mitochondrial
membrane. Our results demonstrate that the changes at the carboxyl
terminus that lead to mitochondrial targeting do not lead to
spontaneous insertion into ER membranes (the four lysines are still
required (Fig. 3D)) or into liposomes (data not shown). It
remains to be determined if a single protein serves both functions of
targeting and insertion for the mitochondrial isoform. In principle a
cytoplasmic receptor is not essential for ER-specific targeting as
membrane binding for these Vamps requires a membrane-bound receptor
that is not found on liposomes, lysosomes, or mitochondria (Fig. 3,
A and B, and data not shown).
Our data suggesting a common receptor-mediated,
ATP-dependent mechanism used by different Vamps to target
to the ER establishes this as a bona fide targeting
mechanism for carboxyl-terminal insertion sequences. Thus there are now
at least five distinct mechanisms that have been proposed whereby
proteins containing insertion sequences attach to membranes (14).
Examples of the other mechanisms include: spontaneous insertion into
membranes and phospholipid vesicles (Cb5), insertion into multiple
membranes without insertion into liposomes (Bcl-2), insertion into
distinct intracellular membranes mediated by cytoskeletal components
(polyomavirus middle T antigen), and insertion into membranes only
after conformational changes that occur during apoptosis (Bax) (14,
35-37). Of these, the Vamp-targeting signal is the first sequence
important for integration into membranes that has been found to be
located amino-terminal of the hydrophobic transmembrane sequence.
Identification of this sequence will likely be useful in predicting the
membrane binding mechanism used by other proteins with insertion
sequences that are targeted to ER membranes. Examples include SSS1p,
Sec61 We thank Scott Gurd for assistance in the
nucleic acid sequencing of the Vamp2 mutants, and Amit Bhavsar and
Brian Allore for helpful suggestions for designing doped
oligonucleotides. We are also grateful to Helen Atkinson and Domina
Falcone for comments on the manuscript.
*
This work was supported by operating Grant MT-10490 and
senior scientist award from the Medical Research Council of Canada (to
D. W. A.).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.
The abbreviations used are:
ER, endoplasmic
reticulum;
Cb5, cytochrome b5;
PCR, polymerase
chain reaction;
Vamp, vesicle-associated membrane protein.
Identification of the Endoplasmic Reticulum Targeting Signal
in Vesicle-associated Membrane Proteins*
,,
Departments of Biochemistry and
¶ Medicine, McMaster University, Hamilton,
Ontario L8N 3Z5, Canada and the § Program in Cell
Biology, Hospital for Sick Children,
Toronto, Ontario M5G-1X5, Canada
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-subunit as measured by
Western blotting (21). Mitochondria and lysosomes were isolated from rat liver as described by Greenawalt (22) and Symons and Jonas (23),
respectively. Phospholipid vesicles (7:8:1:4
phosphatidylcholine:phosphatidylethanolamine:phosphatidylserine:cholesterol) were prepared by extrusion in 10 mM Tris-HCl buffer, pH 7.5 (24).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
71% identical). The other Vamps are 25-33%
identical to Vamp1 and to each other (16). In addition, a splice
isoform of Vamp1 has been reported that appears to target to
mitochondria (29, 30). All transmembrane Vamps contain an insertion
sequence that is expected to function as the membrane anchor domain. In
addition, both Vamps 1 and 2 require ATP and an ER receptor to bind to
ER membranes. Although it is likely that the other family members have
similar requirements for membrane targeting it is essential to
demonstrate this prior to using sequence comparisons to identify a
putative common ER targeting signal for Vamp proteins. Vamp8 was
selected as an example of a divergent Vamp because it is only 29%
identical to Vamp1 and 33% identical to Vamp2.
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Fig. 1.
Vamp2 and Vamp8 binding to ER is
ATP-dependent. Reticulocyte lysate translation
reactions of Vamp2 and Vamp8 were incubated with microsomes (untreated)
or treated as follows: apyrase, after translation for 1 h, 5 units of apyrase were added to the translation reactions and
incubation was allowed to continue for another 30 min, and microsomes
were added; G-25, after translation for 1 h small
molecules were removed from the translation reactions by centrifugation
through a column of Sephadex G-25; G-25 +ATP, after G-25
column separation, a final concentration of 0.5 mM of ATP
was added to the translation reaction before the microsomes;
-mbs, microsomes were not added to the translation reaction.
After in vitro translation of
[35S]methionine-labeled proteins, 1 equivalent of
microsomal membranes was added to 10 µl of the translation reaction
and incubated for 1 h at 24 °C. Membrane-bound proteins were
separated by pelleting through a sucrose cushion. Gradients were
divided into three fractions top (T), middle (M),
and bottom (B) or a pelleted fraction. Equivalent amounts of
each fraction were analyzed by SDS-polyacrylamide gel
electrophoresis.
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Fig. 2.
Binding of Vamps to microsomes is
saturable. Increasing amounts of individual translation reactions
containing Vamp1, Vamp2, or Vamp8 were incubated with 1 equivalent of
microsomes for 2 h at 24 °C. Translation buffer (30 µl) was
added to the reaction mixtures, and then the reaction was layered on
top of a 0.5 M sucrose cushion (110 µl). Microsomes were
separated from the reactions by centrifugation at 20 psi (110,000 × g) for 10 min at 4 °C in an A100/30 rotor in an
Airfuge (Beckman Instruments). After SDS-polyacrylamide gel
electrophoresis, radioactivity was measured using a PhosphorImager and
converted to protein concentration. A best-fit curve was plotted for
all three sets of data as there was no significant difference between
the three individual curves. Open circles, Vamp1;
solid squares, Vamp2; solid triangles,
Vamp8.
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Fig. 3.
Vamp does not bind to other membranes.
A, phospholipid vesicles were added to translation
reactions. After incubation at 24 °C for 1 h sucrose was added
to a final concentration of 0.84 M, the samples (70 µl)
were transferred to Airfuge tubes, and 110 µl of 0.34 M
sucrose in translation buffer and 40 µl of translation buffer were
sequentially layered on top of the sample. After centrifugation in a
TL-100 (Beckman Instruments) for 2 h at 55,000 rpm (120,000 × g), gradients were fractionated from the top
(T) into five fractions (55 µl each) with the solubilized
pellet as the bottom (B) fraction. Proteins are identified
above the panels. B, in vitro translated proteins
were incubated with purified lysosomes (20 µg/ml total protein) for
1 h. Membranes were separated from the reaction by centrifugation
over sucrose step gradients. Gradients were divided into three
fractions top (T), middle (M), and bottom
(B) or a pelleted fraction. Proteins are identified below
the panels. C, in vitro translated Vamp2 mutants
and fusion proteins were incubated with 1 equivalent of microsomes for
1 h at 24 °C. The microsomes were separated from the reaction
by centrifugation over sucrose step gradients as above.
gPAVMB, gPA fused to the minimal binding domain of Vamp2.
Molecules indicated by mito contain the Vamp mitochondrial
targeting sequence. (0+) indicates that the six positively charged
amino acids amino-terminal of the hydrophobic core of the insertion
sequence have been replaced with noncharged hydrophilic amino
acids.
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Fig. 4.
A 16-amino acid sequence amino-terminal of
the insertion sequence is required for Vamp1 membrane binding.
A, schematic and nomenclature of the gPAVamp2 fusion
proteins. Black bar, gPA; gray bars, regions of
Vamp2 fused to gPA. Numbers below represent the corresponding positions
in Vamp2. Enclosed box, hydrophobic putative transmembrane
domain of Vamp2 (TM). The fusion protein gPAVMB includes
residues 80-116 of Vamp2 that contain the endoplasmic reticulum
minimal binding region. B, binding assays for gPAVamp2
fusion proteins; averages from three independent experiments. The error
(one standard deviation) ranged from 1-1.6% for the different
mutants. C, membrane binding assays for gPAV2 and gPAVMB.
Apyrase, after translation for 1 h, 5 units of apyrase
were added to the translation reactions, incubation was allowed to
continue for another 30 min, and microsomes were added;
G-25, after translation for 1 h small molecules were
removed from the translation reactions by centrifugation through a
column of Sephadex G-25; -mbs, microsomes were not added to
the translation reaction. After in vitro translation of
[35S]methionine-labeled proteins, 1 equivalent of
microsomal membranes was added to 10 µl of the translation reaction
and incubated for 1 h at 24 °C. Membrane-bound proteins were
separated by pelleting through a sucrose cushion. Gradients were
divided into three fractions top (T), middle (M),
and bottom (B) or a pelleted fraction.
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Fig. 5.
The minimal binding region of Vamps modeled
as an amphipathic helix. A, amino acid sequence of the
minimal binding region from Vamps1, -2, and -8. Nonconserved residues
in Vamp2 and Vamp8 are indicated as *. Residues analyzed by mutagenesis
are boxed with positively charged residues in shaded
boxes. The four lysines indicated in B are numbered.
Substitution mutations for the charged amino acids are shown below the
sequence. B, the putative amphipathic helix from Vamp1
presented as a helical wheel. The four lysines on the same face are
numbered as in A.
View larger version (33K):
[in a new window]
Fig. 6.
Four conserved lysines within the minimal
binding region are necessary for membrane association.
A, membrane binding gPAVMB point mutants. The five lysines
and one arginine were changed to noncharged hydrophilic residues as
K83S, K85S, R86T, K87T, K91Q, and K94S, where the first amino acid
indicates the original residue, the number is the amino acid position
in Vamp2, and the final letter indicates the amino acid substitution.
0+ contains all of the mutations and therefore contains zero charged
residues. The remaining mutants all contain the K85S and K86T
mutations. Numbers beneath the bars indicate the number of
charges restored at positions 1-4 indicated in Fig. 5. Values for
membrane binding for the mutants with the designated number of charges
were averaged. 1K, four mutants K83S, K87T, K91Q, K94S;
2K, six mutants (K83S,K87T), (K83S,K91Q), (K83S,K94S),
(K87T,K91Q), (K87T,K94S), (K91Q,K94S); 3K, three mutants
(K83S,K87T,K91Q), (K83S,K87T,K94S), (K87T,K91Q,K94S); 4K,
one mutant containing all four lysines. Each mutant was analyzed in
three independent experiments. B, membrane binding by the
Vamp8 minimal binding domain requires the four lysines on the same face
of the putative amphipathic helix. Membrane binding is equivalent for
Vamp8, and gPA fused to the conserved region (sixteen amino acids) and
the transmembrane domain from Vamp8 (gPAV8MB) in single letter code
(TAQKVARKFWWKNVKMIVLICVIVFIIILFIVLFATGAFL). Substitution of the
four conserved lysines in GPAV8MB (K83S, K87T, K91Q, K94S, numbering
according to Vamp2) abolished binding to microsomes
(gPAV8MB(0+)). Results are from four independent
experiments. Error bars indicate one standard deviation.
View larger version (35K):
[in a new window]
Fig. 7.
Mutation of the conserved asparagine within
the minimal binding region reduced membrane binding. The codons
for Trp-89, Trp-90, and Asn-92 in gPV2MB were mutated to encode
different amino acids, and the resulting mutants were assayed for
binding to two equivalents of microsomes. The x axis shows
the relevant amino acid sequence for each mutant in single letter code.
Lowercase letters (wwn) indicate the wild type sequence. The
capitalized letters denote the individual mutations. Results are
averages from four to five independent experiments for each mutant.
Error bars indicate one standard deviation.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, and Sec61
, proteins that all contain a positively charged
region predicted to form an amphipathic helix amino-terminal of an
insertion sequence. Our results will also be useful for identifying the molecules that mediate membrane targeting and insertion of these proteins.
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Rm. 4H-41, Dept.
of Biochemistry, McMaster University Medical Centre, 1200 Main St.
West, Hamilton, Ontario L8N 3Z5, Canada. Tel.: 905-521-9140 (ext.
22075); Fax: 905-522-9033; E-mail: andrewsd@fhs.mcmaster.ca.
ABBREVIATIONS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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